Using deep gravel and bacteria to control nitrogen

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dw1305
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 20 Aug 2017, 22:22Darrel- When you say "fluctuating redox" do you mean any given zone fluctuates between aerobic and anaerobic or do you mean there are multiple zones which alternate between anaerobic and aerobic as described in the paper I referenced? That paper suggests that the zone around the roots will be aerobic and that there will be anaerobic zones above and below, i.e. alternating zones rather than alternation within a zone.
I think it will be a mixture of both, there will be zones that fluctuate between being oxygenated and de-oxygenated, as you suggest dependent upon a wide range of factors (http://journals.plos.org/plosone/articl ... ne.0143428), and there will also be a zonation within the substrate.

The roots are the important bit, they are "deliberately" leaky structures and will be releasing both oxygen and carbohydrates into the substrate in the rhizosphere.

Symbiosis between legumes and nitrogen fixing bacteria (https://en.wikipedia.org/wiki/Rhizobia) and Azolla and Anabaena(http://theazollafoundation.org/azolla/t ... mbiosis-2/) etc are well documentedhttps://www.ukaps.org/forum/threads/wha ... ost-430969, but nearly all plants are mycorrhizial to some degree and will alter the conditions in the substrate to favour the microbial assemblage (https://en.wikipedia.org/wiki/Root_microbiome) that offer them some evolutionary benefit.

One thing that you have to bear in mind with Lobelia dortmanna is that it is a small plant, with very slow growth, that occurs in cold oligotrophic lakes (http://www.plant-identification.co.uk/s ... tmanna.htm).

If you looked at a plant with a higher potential growth rate (like a Spartina x anglica, Rhizophora mangle, Cyperus papyrus or Phragmites australis) etc. they would have a much larger zone of influence around the root (https://www.srs.fs.usda.gov/pubs/20165/).

There is quite a lot of work on Phragmites etc. for use in constructed wetlands.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by TwoTankAmin »

TY Darrel
@dw1305

You made me read a whole lot :-B

I am not sure when was the last time I tested for nitrate in any of my tanks. I think it was several years ago because I was doing a dry season on a zebra pleco tank (unlit and no plants) which meant fewer and smaller water changes. I wanted the TDS to rise but not due to nitrate which I wanted to keep low. So I tested once or twice for nitrate levels which were not a problem.

The one thing I know about denitrification, besides it needs an anaerobic environment, is that it requires a slow flow to be effective. A deep bed would definitely be very slow flow, but one's filter can also be slow. This means any need for additional circulation, aeration or current has to be provided by other devices than the filter. Nitrification also works best with lower flow rates than many filters have. To encourage denitrification via a filter requires using large amounts of media which makes it similar to a deep bed in some respects.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 22 Aug 2017, 22:16 I am not sure when was the last time I tested for nitrate in any of my tanks. I think it was several years ago because I was doing a dry season on a zebra pleco tank (unlit and no plants) which meant fewer and smaller water changes. I wanted the TDS to rise but not due to nitrate which I wanted to keep low. So I tested once or twice for nitrate levels which were not a problem.
I don't test for nitrates either, we have the capability (we use the NO3 Ion Selective Electrodes a lot), but even with ISE there are still issues, and heavy planting removes nitrate accumulation as an issue. I'm still a regular water changer, and if I was forced to forgo plants I would increase the volume of the water changes.
TwoTankAmin wrote: 22 Aug 2017, 22:16The one thing I know about denitrification, besides it needs an anaerobic environment, is that it requires a slow flow to be effective. A deep bed would definitely be very slow flow, but one's filter can also be slow. This means any need for additional circulation, aeration or current has to be provided by other devices than the filter. Nitrification also works best with lower flow rates than many filters have. To encourage denitrification via a filter requires using large amounts of media which makes it similar to a deep bed in some respects.
The slow flow in denitrification coils etc. is really just to ensure that the water is de-oxygenated.
TwoTankAmin wrote: 22 Aug 2017, 22:16Nitrification also works best with lower flow rates than many filters have.
I'm not sure about that, I think it would depend upon the nature of the biofilm. I like high flow rates because they ensure oxygenation. In activated sludge sewage treatment (https://en.wikipedia.org/wiki/Activated_sludge) you have a huge amount of churn (to get enough oxygen into the system to deal with the large bio-load), and the microbes are aggregated into free floating flocs in the filtration system.

Potential oxygen issues are why I don't like de-nitrication in a canister filter, you are always balanced on a knife edge between aerobic nitrification and anaerobic de-nitrification. This is because there is a finite amount of oxygen that enter the filter and that oxygen can't be replenished while the water is inside the filter.

If you ensure that all your filter media is aerobic the potential for ammonia levels to build up (where there is insufficient oxygen for nitrification) isn't an issue.

If you have a substrate (or an HMF), these have access to oxygenated water and there will always be areas (near the interface with the tank water) where nitrification will occur.

Having rooted (ideally emergent) plants offers belt and braces, because you have the increased (synergistic) nitrification capacity of plant/microbe bio-filtration.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by Bas Pels »

Potential oxygen issues are why I don't like de-nitrication in a canister filter, you are always balanced on a knife edge between aerobic nitrification and anaerobic de-nitrification. This is because there is a finite amount of oxygen that enter the filter and that oxygen can't be replenished while the water is inside the filter.
this is precisely why I don´t like canister filters: the amount of oxytgen entering is finite, and therefore you risk flushing in water with nitrite or ammonium. Water which, further, is virtually free of oxygen.

This can be, partially, compensated by flushing the water along the surface, enabling it to take up oxygen, but still - this is not what one wants.

All other filters have acces to new oxygen, and are, therefore, better.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
Bas Pels wrote: 24 Aug 2017, 14:49 this is precisely why I don´t like canister filters: the amount of oxygen entering is finite, and therefore you risk flushing in water with nitrite or ammonium. Water which, further, is virtually free of oxygen.

This can be, partially, compensated by flushing the water along the surface, enabling it to take up oxygen, but still - this is not what one wants.

All other filters have access to new oxygen, and are, therefore, better.
That is it.

I use canister filters, partially for ease of use, and because they are quiet, but I'm definitely a fan of trickle filters, HOBs and HMFs.

The one point I'd really like to make for people is please, please don't use your filter as a syphon. You want oxygen and ammonia in the filter, you don't want anything else.

There is a good web-site, aimed at aquaculturists, on bio-filtration at (http://biofilters.com).

It hasn't been updated recently, but it contains a lot of useful information that can be scaled down for the aquarist.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by Lycosid »

I hate to play necromancer and raise this thread from the dead but I recently discovered that I had (accidentally) replicated some of the conditions that spawned this thread in the first place. I present this tale for your edification.

First, what did I replicate?
A heavily planted tank with deep substrate, a decent number of (small) fish, no water changes (only top-offs) for about a nine months, and a final reading of 0 across all nitrogenous waste products.
planted tank.jpg
This happened through a series of bizarre accidents. Several years ago when I took my current job there were a number of fish tanks in a teaching lab that were, frankly, filthy cesspools. I drained these and slowly set them up as teaching display tanks. A 55-gallon at one end of the room became my largest attempt at an ecosystem tank ever. I've done these before in 2-liter soda bottles. The basic idea is to create a self-sustaining ecosystem in a closed system (normally literally sealed) by adding water and mud from a local still-water ecosystem and letting things sprout and hatch. (Current record: eight years. Largest animal: copepods.) So I took this tank, stripped everything down, and then split the substrate into a rocky side and a muddy side. The substrate included some potting soil that was in lab and a lot of mud from a local wetland and the water ended up being multiple samples from every natural body of water I stuck my containers into for a span of two years. Fast forward a few years. At this point I've dumped dozens of plant and invertebrate species into this tank and it's green with plants and the water seethes with tiny animals. It's also got a tank light, sits in front off a window, and has a bubble wall and an overflow-spray bar water circulation system through a small sump. No technical filtration, though, and since it's an ecosystem tank no maintenance beyond replacing evaporated water.

At this point I begin to need a ready supply of feeder fish for some of my research on predatory strategies in larger fish. My guppy breeding experiment needs to be scaled up and the fry are being eaten, so I throw the guppies into the ecosystem tank without changing anything. It works brilliantly, and I soon have guppies everywhere. Which creates a problem, because every time I try to siphon water out I siphon guppy fry out. I promise myself I'll work out a way to siphon out water without siphoning out guppies "soon". Nine months later I realize that it's been a while since I moved guppies into the tank without a single actual water change and make sure my RA tests the guppy tank along with a test on another tank that we are concerned about. The test shows nitrates in that tank, so it works, but everything reads zero in the guppy tank.

So, do I believe that deep substrate is magical? No. (Although I did use a lot of powdered unicorn horn in the original tank layout.) I think this is what happened (and what may be happening in the original LFS).

1. The plants, obviously.
2. The deep substrate....as a support for so many plants. Not only does it hold a lot of roots but it also started with a lot of nutrients and once I started feeding a breeding colony of guppies it would trap extra nutrients. It's totally impossible to vacuum this substrate.
3. Algae. Not surprisingly, with this much light there's a problem with green hair algae growing, sometimes in big tangles, on the back of the tank. I occasionally just pull it out because it's unsightly (as I suspect an LFS owner would), but it also tears loose in tangles and sticks to the overflow teeth, which can cause issues if I let too much of it sit there, so I pull that out. As it happens, I also keep telling myself that I'm going to use the biomass of the algae for something and so I've been dumping it all into a bucket next to the tank, and so I know that in the past nine month I've removed enough algae that when dried it takes up about as much room as five of my own closed fists would. I suspect that this is the missing nitrate right here.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
Lycosid wrote: 04 Jan 2018, 03:11 ........1. The plants, obviously.
2. The deep substrate....as a support for so many plants. Not only does it hold a lot of roots but it also started with a lot of nutrients and once I started feeding a breeding colony of guppies it would trap extra nutrients. It's totally impossible to vacuum this substrate.
3. Algae. Not surprisingly, with this much light there's a problem with green hair algae growing, sometimes in big tangles, on the back of the tank. I occasionally just pull it out because it's unsightly (as I suspect an LFS owner would), but it also tears loose in tangles and sticks to the overflow teeth, which can cause issues if I let too much of it sit there, so I pull that out. As it happens, I also keep telling myself that I'm going to use the biomass of the algae for something and so I've been dumping it all into a bucket next to the tank, and so I know that in the past nine month I've removed enough algae that when dried it takes up about as much room as five of my own closed fists would. I suspect that this is the missing nitrate right here.
I'm sure you are right, it doesn't matter what the plant is, when you remove it, you remove fixed nitrogen.

I've been looking at Dr Kevin Novak's blog on anoxic filters. I don't think he is right about how his filters work, but they use a similar combination of plants and and substrate with fluctuating REDOX values (biocensosis bucket) to maintain water quality.

This is from <http://anoxicfiltrationsystem.blogspot. ... -tony.html>.

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cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
I thought that reviving this thread might be the most suitable place to put this reference in.

I recently, serendipitously, found an interesting open source paper: "Myriophyllum aquaticum Constructed Wetland Effectively Removes Nitrogen in Swine Wastewater" (https://www.frontiersin.org/articles/10 ... 01932/full).

Although it is dealing with, somewhat, more polluted water than we are it quantifies a lot of the variables mentioned in this thread.

This is from the abstract, but the whole paper is worth reading.
.....stands of Myriophyllum aquaticum to treat swine wastewater. Steady-state conditions were achieved throughout the 600-day operating period, and a high (98.3%) average ammonia removal efficiency under a N loading rate of 9 kg ha-1 d-1 was observed. To determine whether this high efficiency was associated with the performance of active microbes, the abundance, structure, and interactions of microbial community were compared in the un-vegetated and vegetated samples. Real-time quantitative polymerase chain reactions showed the abundances of nitrifying genes (archaeal and bacterial amoA) and denitrifying genes (nirS, nirK, and nosZ) were increased significantly by M. aquaticum in the sediments, and the strongest effects were observed for the archaeal amoA (218-fold) and nirS genes (4620-fold). High-throughput sequencing of microbial 16S rRNA gene amplicons showed that M. aquaticum greatly changed the microbial community, and ammonium oxidizers (Nitrosospira and Nitrososphaera), nitrite-oxidizing bacteria (Nitrospira), and abundant denitrifiers including Rhodoplanes, Bradyrhizobium, and Hyphomicrobium, were enriched significantly in the sediments.
cheers Darrel
Last edited by dw1305 on 14 May 2019, 16:38, edited 3 times in total.
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Re: Using deep gravel and bacteria to control nitrogen

Post by TwoTankAmin »

I think this thread is a pretty good one to keep going over time. I am not sure if I posted it here, but there is an old paper which first appeared in Limnology and Oceanography, 1997. I found it via the site of Dr. Timothy A. Hovanec in his article, "Aquatic Plants and the Nitrogen Cycle."
http://www.drtimsaquatics.com/resources ... ogen-cycle

Here is the info on the paper.

"Nitrification and denitrification in the rhizosphere of the aquatic macrophyte Lobelia dortmanna L."
Nils Risgaard‐ Petersen, Kim Jensen
https://aslopubs.onlinelibrary.wiley.co ... .42.3.0529
Abstract

Nitrogen and O2 transformations were studied in sediments covered by Lobelia dortmanna L.; a combination of 15N isotope pairing and microsensor (O2, NO3−, and NH4+) techniques were used. Transformation rates and microprofiles were compared with data obtained in bare sediments. The two types of sediment were incubated in doublecompartment chambers connected to a continuous flow‐through system.

The presence of L. dortmanna profoundly influenced both the nitrification‐denitrification activity and porewater profiles of O2, NO3−, and NH4+ within the sediment. The rate of coupled nitrification‐denitrification was greater than sixfold higher in L. dortmnanna‐vegetated sediment than in bare sediment throughout the light–dark cycle. Illumination of the Lobelia sediment reduced denitrification activity by ∼30%. In contrast, this process was unaffected by light–dark shifts in the bare sediment. Oxygen microprofiles showed that O2 was released from the L. dortmanna roots to the surrounding sediment both during illumination and in darkness. This release of O2 expanded the oxic sediment volume and stimulated nitrification, shown by the high concentrations of NO3− (∼30 µM) that accumulated within the rhizosphere. Both 15N2 isotope and microsensor data showed that the root‐associated nitrification site was surrounded by two sites of denitrification above and below, and this led to a more efficient coupling between nitrification and denitrification in the Lobelia sediment than in the bare sediment.
Also, to clarify my statement that nitrification (and denitrification) work best with slower flow rates. I should have made it clearer. Slower is relative in that there are two considerations. One is contact time. The organisms in the biofilm can only extract what they need from the water when they are in contact with it. But more important is the potential effect of shearing which is caused by an excessive flow rate. This refers to causing parts or all of the biofilm being "ripped" off of the surface to which it is attached.

When I set up my first 3 inch thick, 20 ppi Mattenfilter on a 33 long using a pump, I was advised by Dr. Tanner to shoot for flow rate between 150 and 200 gph. Considering the oft stated rule of needing a 10 times/hour turnover, that 33 would have used closer to a 330 gph flow. Since I began using Mattens and the Poret cubes, I have not tested nitrate levels in any tanks where I use these filters as i have seen no reason for doing so.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 14 May 2019, 16:27 I think this thread is a pretty good one to keep going over time. I am not sure if I posted it here, but there is an old paper which first appeared in Limnology and Oceanography, 1997. I found it via the site of Dr. Timothy A. Hovanec in his article, "Aquatic Plants and the Nitrogen Cycle."
http://www.drtimsaquatics.com/resources ... ogen-cycle
I'd like to think the thread is of some interest to people, other than you and me, as well.

Anything that helps people have healthy vibrant aquariums, that they can enjoy, rather than staggering from disaster to disaster must be a good thing.

You linked in the Petersen & Jensen Lobelia dortmanna paper on page 3. It is also referenced in the Sun et al M. aquaticum paper.

I haven't been all the way through them yet, but I have some other papers looking at intermittent aeration of constructed wetlands. The aim is to increase total nitrogen removal by extending the zones of fluctuating REDOX, where both aerobic nitrification and anaerobic denitrification will occur.
TwoTankAmin wrote: 14 May 2019, 16:27 Also, to clarify my statement that nitrification (and denitrification) work best with slower flow rates. I should have made it clearer. Slower is relative in that there are two considerations. One is contact time. The organisms in the biofilm can only extract what they need from the water when they are in contact with it. But more important is the potential effect of shearing which is caused by an excessive flow rate. This refers to causing parts or all of the biofilm being "ripped" off of the surface to which it is attached.
There is an optimal depth of biofilm, the Kaldnes "K" type floating cell media were actually designed to be self-cleaning, but to retain this optimal depth of biofilm. I think they were developed for commercial Salmon Aquaculture, but have found a wider application in waste water treatment (http://www.veoliawatertech.com/news-res ... /46021.htm).
TwoTankAmin wrote: 14 May 2019, 16:27When I set up my first 3 inch thick, 20 ppi Mattenfilter on a 33 long using a pump, I was advised by Dr. Tanner to shoot for flow rate between 150 and 200 gph. Considering the oft stated rule of needing a 10 times/hour turnover, that 33 would have used closer to a 330 gph flow.
The 10x rule only really applies to Hi-tech planted tanks where aquascapers have added CO2, it is to distribute the dissolved CO2, it isn't really anything to do with biological filtration. Often the filter will be augmented with a Koralia type powerhead to increase the flow. If you don't add CO2 (I don't and I never will) you only need much lower turn-over rates.
TwoTankAmin wrote: 14 May 2019, 16:27 Since I began using Mattens and the Poret cubes.
I look on HMF filters as a special case, they combine the role of filter and substrate. I'm a great advocate of using them.

I think it is linked in earlier in the thread, but for any-one who hasn't read it I'd recommend Dr Stephan Tanner's article on "aquarium biofiltration" (http://www.swisstropicals.com/library/a ... iltration/).
TwoTankAmin wrote: 14 May 2019, 16:27I have not tested nitrate levels in any tanks where I use these filters as i have seen no reason for doing so
I very occasionally use a lab. ISE to measure NO3 from the tanks, but usually I just use plant health (via the Duckweed Index (http://www.planetcatfish.com/forum/view ... hp?t=36231)) and an even more occasional conductivity measurement.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by bekateen »

You guys get way more technical than I sometimes understand, but I do get lots out of this and it is fascinating to learn the crossover between aquariums, aquaculture and wastewater management.

The irony of this thread is that now I'm 100% in the opposite direction from where I started. Instead of using deep gravel, I now limit my tanks to a thin film of sand, just a few mm deep. I've had too many experiences in the meantime with sulfur gas deaths that I work hard to avoid its accumulation.

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Re: Using deep gravel and bacteria to control nitrogen

Post by TwoTankAmin »

I ran a 50 gal. high tech planted tank for over a decade. Because of space considerations I opted to push my pressurized co2 into the intake of an Eheim Pro II 2026. I chose Eheim as it seemed the best choice to deal with gas passing though the impeller well. This filter has a max. flow rating of 950 l/h (247 gph). Moreover, I ran the spray bar vertically in the back right corner. The way the tank was scaped the actual volume of water was more like 35 gal. So my max turnover rate was about 7x/h. My best guess is with my media load it was more like 6x/h or even lower.

My plants did fine. The fish did so as well. It was the tank in which my Farlos spawned. The problem was the tank took up way to much of my weekly maint. time compared to its share of gals. and fish. I finally sold the co2 system and ultimately replaced the tank with a 75 which is now filled with L236s and no plants. But that Eheim is still the primary filter :)

I must be getting old, repeating myself in a thread. It must be that OBS and CRS again.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
bekateen wrote: 15 May 2019, 14:03 ......and it is fascinating to learn the cross-over between aquariums, aquaculture and wastewater management.
It is really go with what you know.

I'm a pretty shoddy fish keeper, but I can mitigate for this by providing the fish with a high quality environment.
bekateen wrote: 15 May 2019, 14:03I now limit my tanks to a thin film of sand, just a few mm deep.
If I kept non-planted tanks that is all I would use. I've had a couple of high flow tanks (with lower planting levels) and I used a mix of sand and fine gravel in those, and let the flow sort it. The layer was pretty thin.

If you had an HMF (matten) filter that could perform a lot of the roles that would occur in the substrate (documented in Stephan Tanner's article).

It is the plant roots that allow you to have a deeper substrate, the rhizosphere is much more important factor than many people realize (which is documented in Myriophyllum aquaticum Constructed Wetland Effectively Removes Nitrogen in Swine Wastewater" (https://www.frontiersin.org/articles/10 ... 01932/full).)

Even with planting I don't have a huge depth of substrate.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
A couple of papers have come my way that maybe of interest to some.

There is some further work on the oxygen dynamics within the rhizosphere of Lobelia dortmanna:

"The effect of Lobelia dortmanna L. on the structure and bacterial activity of the rhizosphere". K Lewicka-Rataj, A Świątecki, D Górniak - Aquatic botany, 2018. That one is behind a pay-wall, but I can email it if any-one really wants a copy?

There is also a really good, open source, review article on ammonia oxidation:

Laura E Lehtovirta-Morley "Ammonia oxidation: Ecology, physiology, biochemistry and why they must all come together:" <https://doi.org/10.1093/femsle/fny058> FEMS Microbiology Letters, Volume 365, Issue 9, May 2018. The author works for UEA in Norwich UK <https://people.uea.ac.uk/l_lehtovirta-m ... hinterests>.
AOA vastly outnumber AOB in most soil and aquatic environments, often by orders of magnitude (Leininger et al.2006). It is estimated that there are 1 × 10^28 AOA cells in the Earth's oceans and they are some of the most numerous living organisms on Earth, accounting for up to 40% of all prokaryotes in marine ecosystems and 1%–5% in terrestrial ecosystems (Karner, DeLong and Karl 2001; Leininger et al.2006). In contrast, AOB usually dominate numerically over AOA in wastewater treatment plants and occasionally in fertilised soils (Bates et al.2011; Mussmann et al.2011). Little is known about the abundance and diversity of comammox Nitrospira, although their abundance has been reported to be comparable to, or higher than the abundance of other ammonia oxidisers.......

....The notion that ammonia oxidation is problematic at low pH was overturned by the discovery of the first obligately acidophilic ammonia oxidiser, N. devanaterra (Lehtovirta-Morley et al.2011). Originally isolated from an acidic agricultural soil, this archaeon grows autotrophically in the pH range of 4–5.5 in laboratory culture with ammonium chloride as its sole energy source. Although Nitrosotalea is the only obligately acidophilic ammonia oxidiser described to date, it is unlikely to be the only microorganism performing nitrification......

.........The kinetic theory of the optimal pathway length suggested that the comammox process would lead to a higher yield but lower growth rate than incomplete ammonia oxidation, giving comammox organisms a competitive advantage when ammonia concentration is low (Costa, Pérez and Kreft 2006). It is interesting to note that the half-saturation constants of many AOA and comammox are roughly within the same range.
cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
Honestly the last one for the moment, but there is also an update to Myriophyllum remediation of swine waste water paper (https://www.ncbi.nlm.nih.gov/pubmed/29056931).

It is "Enhancement of facultative anaerobic denitrifying communities by oxygen release from roots of the macrophyte in constructed wetlands (CW)" (https://www.ncbi.nlm.nih.gov/pubmed/29056931) It looks at Radial Oxygen Loss (ROL) in the rhizoplane (the section of the rhizosphere nearest the plant root).
......that the abundance of facultative anaerobic denitrifiers in the rhizoplane was the most of the three niches, that in the water (5–10 cm) was the less and that in the sediment was the least. .......Denitrifiers in the rhizoplane were mainly dominated by enriched Pseudomonas,Aeromonas, and Acinetobacter. The theoretical calculation of oxygen sources and consumption indicated that water re-aeration should support the oxygen demands for nitrification in the aerobic layer (0–5 cm), and the ROL could stimulate the growth of facultative anaerobic denitrifiers in the rhizoplane and water (5–10 cm) to achieve denitrification within CW systems.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
Another interesting (to me anyway) paper has come my way, it isn't a plant based one, but looks in more detail at the microbial diversity in freshwater aquarium filters, and also provides some figures.

The paper is Sauder, L. et al. (2018) "“Candidatus Nitrosotenuis aquarius,” an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter" Appl. Environ. Microbiol. 84:e01430-18. (https://aem.asm.org/content/84/19/e01430-18).

I'm not sure what the access arrangement is for it, but if any-one wants a copy PM me.
Conclusions.

We report the cultivation and complete genome sequence of “Ca. Nitrosotenuis aquarius,” a mesophilic, autotrophic, ammonia-oxidizing archaeon that originates from a freshwater aquarium biofilter and belongs to the “Ca. Nitrosotenuis” lineage of the Thaumarchaeota.

“Ca. Nitrosotenuis aquarius” cells grow up to 3 µm, which is the longest cell length reported for AOA representatives. Cells are consistently slender (0.4 µm), maintaining a high surface area-to-volume ratio, which is characteristic of oligotrophic cells. “Ca. Nitrosotenuis aquarius” possesses several genes for protein glycosylation, which could modify flagella or S-layer proteins, and may promote enhanced surface adhesion.

Incubations of biomass from the freshwater aquarium biofilter where “Ca. Nitrosotenuis aquarius” originates demonstrate that AOA contribute to ammonia-oxidizing activity and support the previously reported numerical dominance of AOA in freshwater aquarium biofilters (1, 2).

This work suggests that, contrary to common belief in the aquarium industry, AOB are likely not primarily responsible for ammonia oxidation in aquarium biofilters. Laboratory cultures of AOA originating from freshwater aquaria, such as “Ca. Nitrosotenuis aquarius” cultures, are useful for future investigations of the ecology and physiology of freshwater AOA, with potential commercial applications in aquarium and aquaculture operations.
cheers Darrel
Last edited by dw1305 on 04 Sep 2019, 17:17, edited 1 time in total.
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Re: Using deep gravel and bacteria to control nitrogen

Post by bekateen »

Thanks, Darrel, for continuing to provide more info on this topic.

There are so many different (AMD similar) prokaryotes... I'm confident the discoveries will be coming for a long time.

Cheers, Eric
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Re: Using deep gravel and bacteria to control nitrogen

Post by Bas Pels »

As I am a fan of panted tanks - even with cichlids I try to keep plants - I use faily thick layers of sediment.

My largest tanks have a some 5 cm thick layer on average, even if both of them are unplanted, due to the herbivourous nature of the fishes involved (headstanders are quite similar to cows).

However, in spring ?I changed the soil in another tank, from 5 cm of sand into 10 cm of a clay- sand mixture. I wanted to have a field of Echionodorus argentinensis in full bloom. And this is a species needing a lot of food.

Plants are still a bit young, but are doing OK. However, after I entered some fishes (Tanichthys) I never saw them again. It could have been the soil, it could have been another cause, such as the temperature. The water was cold, early in spring.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
bekateen wrote: 04 Sep 2019, 16:55.....There are so many different (AMD similar) prokaryotes... I'm confident the discoveries will be coming for a long time.
I'd definitely expect that they will find more. I'm particularly interested in whether they find a universal "core" assemblage of microbes, or whether that assemblage changes dramatically with differing ammonia loadings.

There is some interesting research in the UK by Laura E Lehtovirta-Morley (at UEA (https://people.uea.ac.uk/l_lehtovirta-m ... hinterests)):

It is summarised in "Ammonia oxidation: Ecology, physiology, biochemistry and why they must all come together" FEMS Microbiology Letters, Volume 365, Issue 9, May 2018 (https://academic.oup.com/femsle/article ... 58/4931719).
....AOA vastly outnumber AOB in most soil and aquatic environments, often by orders of magnitude (Leininger et al.2006). It is estimated that there are 1 × 10^28 AOA cells in the Earth's oceans and they are some of the most numerous living organisms on Earth, accounting for up to 40% of all prokaryotes in marine ecosystems and 1%–5% in terrestrial ecosystems (Karner, DeLong and Karl 2001; Leininger et al.2006). In contrast, AOB usually dominate numerically over AOA in wastewater treatment plants and occasionally in fertilised soils (Bates et al.2011; Mussmann et al.2011). Little is known about the abundance and diversity of comammox Nitrospira, although their abundance has been reported to be comparable to, or higher than the abundance of other ammonia oxidisers.......

....The notion that ammonia oxidation is problematic at low pH was overturned by the discovery of the first obligately acidophilic ammonia oxidiser, N. devanaterra (Lehtovirta-Morley et al.2011). Originally isolated from an acidic agricultural soil, this archaeon grows autotrophically in the pH range of 4–5.5 in laboratory culture with ammonium chloride as its sole energy source. Although Nitrosotalea is the only obligately acidophilic ammonia oxidiser described to date, it is unlikely to be the only microorganism performing nitrification in acidic soils......

.........The kinetic theory of the optimal pathway length suggested that the comammox process would lead to a higher yield but lower growth rate than incomplete ammonia oxidation, giving comammox organisms a competitive advantage when ammonia concentration is low (Costa, Pérez and Kreft 2006). It is interesting to note that the half-saturation constants of many AOA and comammox are roughly within the same range.
cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
Bas Pels wrote: 04 Sep 2019, 20:04 As I am a fan of planted tanks - even with cichlids I try to keep plants ...........
I'd be very reluctant to have a system without plants, even if I had to have a spatial separation between fish and plants.

I don't have any proof, but I would guess that the planted over-tank trickle filters, that I used to use, would be more efficient than any of the other type of filter by an order of magnitude.
Bas Pels wrote: 04 Sep 2019, 20:04However, in spring ?I changed the soil in another tank, from 5 cm of sand into 10 cm of a clay- sand mixture. I wanted to have a field of Echionodorus argentinensis in full bloom. And this is a species needing a lot of food.

Plants are still a bit young, but are doing OK. However, after I entered some fishes (Tanichthys) I never saw them again. It could have been the soil, it could have been another cause, such as the temperature. The water was cold, early in spring.
I'm not sure, Tanichthys albonubes are pretty tolerant of cooler water. The clay shouldn't have added much BOD (it hasn't got any organic matter to decompose), but it could still potentially be an oxygen issue if the REDOX value of the substrate dropped far enough. There is also the possibility of metals being solubilised (by the reducing conditions in the substrate), but I think that is unlikely.

Once the plants have root development there shouldn't be any issues, as the roots would act as "oxygen conduits" into the substrate.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by MarcW »

Thanks for your input Darrel.

As someone that routinely kills java fern, it'd be great if you could let me know a few plants which could survive with low light and not planted in the substrate?

Anubias do OK, but don't really seem to grow in my tanks, java fern is OK in some tanks but dies in others, maybe that's to do with temperature rather than lighting, it does OK in my lower tanks but not at the top of the rack where the water is 2-4c higher.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
MarcW wrote: 05 Sep 2019, 15:02 .......As someone that routinely kills java fern, it'd be great if you could let me know a few plants which could survive with low light and not planted in the substrate?

Anubias do OK, but don't really seem to grow in my tanks, java fern is OK in some tanks but dies in others........
Have you still got the moss that came with the Asellus etc.? That should grow OK low light.

The best low light plant for me is Bolbitis heudelotii, it does much better in low nutrients and soft water than Java Fern (Microsorum pteropus).

I've also found that some of the selected cultivars of Java Fern (like "Needle Leaf" and "Trident") aren't anything like as good growers low tech as the ordinary variety.

Bolbitis isn't a quick grower, but it is a bit like Anubias, you don't really notice it growing but eventually it forms a reasonable clump (below).

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It isn't cheap to buy, and even more expensive as a "mother plant", (presumably because it isn't a quick grower even emersed), but your more than welcome to some of mine.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by MarcW »

Hi Darrel, yes I still have the moss it's in a tank with the Asellus, hopefully that'll grow to a decent sized clump.

Your comments about the java fern variants match with my experience, now I've thought about it, the narrow leaved version has all died, the few I have remaining are all the normal type and growing OK.

I've ordered some Bolbitis heudelotii to try out thanks for the tip.
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Re: Using deep gravel and bacteria to control nitrogen

Post by TwoTankAmin »

Here is what Tropica says about the heudelotti:
Bolbitis comes from West Africa, a fern with very beautiful transparent green leaves, 15-40 cm tall and wide. When planting do not cover the rhizome because it will rot, and it is best to plant Bolbitis heudelotii on a root or stone. Keep the plant in position with fishing line until it has gained a hold. Easy to propagate by splitting the horizontal rhizome. Growth can be increased considerably by supplying CO2, and is only optimal in soft, slightly acidic water.
from https://tropica.com/en/plants/plantdeta ... WS%29/4407

I seem to recall I tried to plant it where there was decent flow/circulation. I am not sure if that was an urban aquarium myth or good advice.

As for the AOA and AOB. I am not ready to accept the conclusions of Sauder. L. whose name appears on all of the studies I have read so far which conclude AOA are the dominant ammonia oxidizers in aquariums. Science has no idea how much ammonia a single cell of either organism is able to oxidize. The current science usually counts the number of of archaeal and bacterial amoA genes to determine numbers. I believe some AOB have more than one amoA gene. Further, the most recent research appears to indicate that there is an amoA gene in Nitrospira and this organism can oxidize ammonia to nitrate directly.
Although, AOA were numerically dominant over AOB, a presumed third ammonia-oxidizer was also present in the biofilter sand matrix. Identification of Nitrospira-like amoA (Figure 7B) in the biofilter and the strong correlation between the abundance of the Nitrospira nxrB uwm-2 gene and this Nitrospira amoA, suggests a complete ammonia-oxidizing Nitrospira spp. resides in the UWM biofilter. In fact, we found that the comammox amoA was the most abundant ammonia-oxidizing gene in the biofilter (on average 1.9X that of AOA amoA).
(Sorry I am not great at proper crediting, but the complete paper can be read here https://www.frontiersin.org/articles/10 ... 00101/full )

Original Research ARTICLE
Front. Microbiol., 30 January 2017 | https://doi.org/10.3389/fmicb.2017.00101

Freshwater Recirculating Aquaculture System Operations Drive Biofilter Bacterial Community Shifts around a Stable Nitrifying Consortium of Ammonia-Oxidizing Archaea and Comammox Nitrospira

Ryan P. Bartelme, Sandra L. McLellan and Ryan J. Newton*
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 06 Sep 2019, 19:14I seem to recall I tried to plant it where there was decent flow/circulation. I am not sure if that was an urban aquarium myth or good advice.
I'm not sure. None of my plants are fixed to anything, so they tend to end up out of the direct flow from the filter.

The photo above is in my kitchen tank, rain-water and low nutrients and that plant grew from a single rhizome with three or four leaves in ~five or six years.
TwoTankAmin wrote: 06 Sep 2019, 19:14As for the AOA and AOB. I am not ready to accept the conclusions of Sauder. L. whose name appears on all of the studies I have read so far which conclude AOA are the dominant ammonia oxidizers in aquariums. Science has no idea how much ammonia a single cell of either organism is able to oxidize. The current science usually counts the number of of archaeal and bacterial amoA genes to determine numbers. I believe some AOB have more than one amoA gene.
There is a fair bit of research from waste water treatment. "Unraveling the active microbial populations involved in nitrogen utilization in a vertical subsurface flow constructed wetland treating urban wastewater" (https://www.sciencedirect.com/science/a ... 9717301006). This is "The more important role of archaea than bacteria in nitrification of wastewater treatment plants in cold season despite their numerical relationships" (https://www.sciencedirect.com/science/a ... 5418306961)
TwoTankAmin wrote: 06 Sep 2019, 19:14......Freshwater Recirculating Aquaculture System Operations Drive Biofilter Bacterial Community Shifts around a Stable The more important role of archaea than bacteria in nitrification of wastewater treatment plants in cold season despite their numerical relationships Consortium of Ammonia-Oxidizing Archaea and Comammox Nitrospira

Ryan P. Bartelme, Sandra L. McLellan and Ryan J. Newton*
I'm pretty sure that people will carry on finding the complete nitrifier "COMAMMOX Nitrospira" wherever they look. My guess is also that people will keep on finding novel nitrifying micro-organisms, whether they are bacteria or archaea.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by TwoTankAmin »

I have one problem generalizing from waste water studies which find Nitrobacter is the dominant nitrite oxidizer. The one thing I think has been established beyond any doubt is that Nitrobacter is not found in any meaningful level, if found at all, in aquariums. It is suited to oxidizing higher levels of nitrite than is found in tanks. Nitrospira is what is regularly detected.

Combine this with the discovery of Nitrospira capable comammox and there is a whole host of microorganisms that are likely at work dealing with ammonia in our tanks. I have come to believe there is probably no single dominant bacteria or archaea across the universe of tanks. Rather each tank will have its own particular balance. I am also pretty sure the exact complement of nitrifying organisms in a tank can change over time as the ammonia loading and other parameters change.
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 07 Sep 2019, 19:34 ......I have come to believe there is probably no single dominant bacteria or archaea across the universe of tanks. Rather each tank will have its own particular balance. I am also pretty sure the exact complement of nitrifying organisms in a tank can change over time as the ammonia loading and other parameters change......
I think that is where we are now.

I'll attempt to keep up with the scientific literature, but I probably won't have a proper trawl through it again before Christmas.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
dw1305 wrote: 07 Sep 2019, 20:19 ..........I'll attempt to keep up with the scientific literature, but I probably won't have a proper trawl through it again before Christmas........
Another paper has come my way:

Zhao et al. (2015) "Microbial community and removal of nitrogen via the addition of a carrier in a pilot-scale duckweed-based wastewater treatment system" (https://www.sciencedirect.com/science/a ... 241401774X).

It is a plant/microbe bio-filtration paper, and it quantifies the net contributions, to nitrogen removal, of a floating plant (Duckweed - Lemna japonica) and microbial biofilm in a Duckweed based waste water treatment. It also investigates why the nitrogen balance might be slightly different when you use Water Hycanth (Eichornia crassipes) as a floating plant.

They attribute these differences in nitrification to the much larger root area of Eichornia, when compared to Lemna, with this expanded rhizosphere supporting a much larger volume of microbial biofilm.

What is really interesting is that when they added a plastic carrier to the effluent flow (to act as a surrogate "root surface" in the Lemna treatment), to give a comparable area for microbial biofilm adhesion.

They found that the addition of a carrier improves nitrogen (N) removal in a duckweed system and that abundant N-removal microbes on the carrier biofilm contribute to improved N removal.

cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by dw1305 »

Hi all,
TwoTankAmin wrote: 06 Sep 2019, 19:14.....As for the AOA and AOB. I am not ready to accept the conclusions of Sauder. L. whose name appears on all of the studies I have read so far which conclude AOA are the dominant ammonia oxidizers in aquariums.........
This is a paper where they didn't find any AOA, but they found a range of novel AOB.

Hüpeden, J. et al (2020) "Taxonomic and functional profiling of nitrifying biofilms in freshwater, brackish and marine RAS biofilters" Aquacultural Engineering 90.
In the freshwater biofilters >99 % of the total AOB reads were assigned to uncultured ammonia oxidizing bacteria with Nitrosomonas communis Nm2 as the closest relative (96 % sequence similarity to the 16S rRNA gene). .....................

In the freshwater biofilters >99 % of the total NOB reads were classified to the genus Nitrospira, which had Ns. defluvii as closest known relative (99 % similarity to the 16S rRNA gene)........

At each RAS, a site-specific nitrifying community was identified. The majority of reads was affiliated with unknown members of Nitrosomonas and Nitrospira, representing several potentially novel species within these genera, which were currently not possible to culture by traditional techniques. The comprehensive phylogenetic analysis to determine the closest taxonomically described species revealed a co-existence of different, Nitrosomonas-like AOB and Nitrospira-like NOB in all biofilters with one representative being the predominant ammonia or nitrite oxidizer at each RAS, respectively.......

The presence and in some cases dominance of AOA over AOB in aquaria and RAS biofilters was reported previously (Bartelme et al., 2017; Brown et al., 2013; Sakami et al., 2012; Sauder et al., 2011) and growth of AOA at low ammonia concentrations has been demonstrated for enriched and isolated representatives (Nicol et al., 2011; Sauder et al., 2012). In other studies, the quantity of AOA in RAS was reported negligible or zero (Foesel et al., 2008; Keuter et al., 2017). The load of ammonia was discussed as important factor for the coexistence of AOA and AOB in RAS, since AOA have a higher affinity towards ammonia than AOB and could be outcompeting AOB at lower concentrations (Brown et al., 2013; Martens-Habbena et al., 2009; Roalkvam et al., 2020). Mostly, AOA dominate in niches characterized by low nutrients, low pH and low DO (Erguder et al., 2009) as well as elevated temperature (Taylor et al., 2017). These parameters are not valid here and in our study, archaeal reads were not present in the 16S rRNA amplicon sequencing data set. In addition, no archaeal amoA sequences were found by specific PCR and no typical cells of AOA were detected via electron microscopic investigation of the biofilms
cheers Darrel
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Re: Using deep gravel and bacteria to control nitrogen

Post by bekateen »

Thanks Darrel, for keeping this up to date. I'm learning a lot.

Cheers, Eric
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