Does depleted KH stop the nitrogen cycle?

[Bradders]

I would still be interested to understand the difference between initially cycling at a 7PH level and a 4PH level, and how a low PH cycle differs in time to establishment. I can’t seem to find how fast these newly discovered acidic growing bacteria establish.

But maybe that is for a different thread and a different day!!

 

[dw1305]

Hi all,

does the Nitrifying bacteria which is happy to form in very acidic water take longer to produce and therefore make a cycle impracticable (months more than weeks) in the home Aquarium hobby?

It definitely isn't impractical in real time, it is what many of us do. It is the question we don't have a direct answer to, but I'd be very surprised if after 6 weeks of growing in if a tank wasn't "cycled", where ever you've started from.

I actually think that <"Secret Ingredient Soup"> is a viable commercial option, and wouldn't be at all surprised if some-one goes down that route.

If you had an aquarium without plants it would take longer, but I don't know the time-scale. This is for marine aquariums (so high dKH), but I'm guessing that it is pretty close to the situation for non-planted freshwater aquariums <"Tim Hovanec's "Nitrification in marine aquarium" article">.

I think the issue there is the cycling concept as a <"binary switch"> from "unsafe" to "safe", rather than the time scale.

The best over view we have are probably Dr Ryan Newton's comments in <"Correspondence with Dr Ryan Newton - School of Freshwater Sciences, University of Wisconsin—Milwaukee"> (quoted below)

...... 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.......

I think the questions now are:

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

 

[dw1305]

Hi all,

An additional option could be the <carbonic anhydrase> that could help the bacteria/archaea to produce bicarbonate from CO2 intracellularly with reasonable speed.

Brilliant, that is why we need proper scientists.

"Carbonic anhydrase", (which I wouldn't have known about without @hax47 post), and "comammox Nitrospira" as a search term takes us to: <"Comammox Nitrospira and Ammonia-Oxidizing Archaea Are Dominant Ammonia Oxidizers in Sediments of an Acid Mine Lake Containing High Ammonium Concentrations">

Li D, Ren Z, Zhou Y, Jiang L, Zheng M, Liu G. (2023) Comammox Nitrospira and Ammonia-Oxidizing Archaea Are Dominant Ammonia Oxidizers in Sediments of an Acid Mine Lake Containing High Ammonium Concentrations. Appl Environ Microbiol. 2023 Mar 29;89(3):e0004723. doi: 10.1128/aem.00047-23. Epub 2023 Mar 13. PMID: 36912626; PMCID: PMC10056971.

Which I think helps with @Bradders question.

..... The lake water was characterized by acidic pH below 5 with a high ammonium concentration of 175 mg-N/liter, which is rare on the earth. Nitrification was active in sediments with a maximum nitrate production potential of 70.5 μg-N/(g-dry weight [dw] day) for mixed sediments. Quantitative PCR assays determined that in AML sediments, comammox Nitrospira and AOA amoA genes had relative abundances of 52% and 41%, respectively, among the total amoA genes. Further assays with 16S rRNA and amoA gene amplicon sequencing and metagenomics confirmed their dominance and revealed that the comammox Nitrospira found in sediments belonged to comammox Nitrospira clade A.2. Metagenomic binning retrieved a metagenome-assembled genome (MAG) of the comammox Nitrospira from sediments (completeness = 96.76%), and phylogenomic analysis suggested that it was a novel comammox Nitrospira. Comparative genomic investigation revealed that this comammox Nitrospira contained diverse metal resistance genes and an acidophile-affiliated F-type ATPase. Moreover, it had a more diverse genomic characteristic on nitrogen metabolism than the AOA in sediments and canonical AOB did. The results suggest that comammox Nitrospira is a versatile nitrifier that can adapt to acidic environments even with high ammonium concentrations.......

cheers Darrel

 

[LMuhlen]

@Bradders Take a look at @_Maq_ 's thread where he tests different substrates.

Thread 'Maq's Substrate Experiment'

24 Sep 2023

I'm considering a new experiment, and there are some points I'd like to get your advice about.

I want to check behaviour of a "typical" aquasoil compared to sandy substrate, and a sandy substrate with and without "typical" root tabs.

Some of you already know my experimental workstation. I imagine I'll establish these four tanks completely anew, lay down the substrate, run a dark cycling for four weeks without WC, then a big WC and introducing some plants. As always, without CO2 injection, without filters, only power head. Water changes 50 per cent weekly (or so), always adding...

• _Maq_
• aquasoil root tabs substrate
• Replies: 178
• Forum: Substrates

• 

He gets some low pH levels there and doses ammonia as a nitrogen source, so you can infer the cycling speed of the tanks.

 

[hax47]

I'll add this here at the moment. <"https://journals.asm.org/doi/full/10.1128/aem.00104-24">. It is quite an interesting paper and worth a read for those interested in this sort of thing.

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">

One thing that caught my attention in this paper is that nitrification-related microbes (mainly Nitrospira) appear to make up only a small fraction (1-2%) of the total microbial community. What are the other 98-99% doing? Could it be that the dominant process in the aquarium filter is the degradation of organic compounds?

 

[dw1305]

Hi all,

One thing that caught my attention in this paper is that nitrification-related microbes (mainly Nitrospira) appear to make up only a small fraction (1-2%) of the total microbial community. What are the other 98-99% doing? Could it be that the dominant process in the aquarium filter is the degradation of organic compounds?

I'm guessing that the dominant process in the aquarium filter is the microbial oxidation of organic compounds, but I think that you can take this into account and <"mitigate for it">.

It is one of the reasons that I've been very keen to exclude bulky organic wastes from the filter body. My thinking has been that organic wastes will supply substrate for heterotrophic bacteria, and they will deplete the dissolved oxygen "unnecessarily". I want <"as much dissolved oxygen as possible"> to be available for nitrification.*

Even if they are relatively inert (like <"wood fibres">) they could impede flow and stop aerobic nitrification being as efficient as it should be.

I just want dissolved gases etc entering the filter, I don't want it <"to act as a syphon">.

Edit: I'll link this paper in <"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

 

[Zeus.]

So in a nutshell - Low pH levels in water without any kH doesnt shop the cycling/nitrogen cycle 'per se'. Details are present, but should not effect the tank or user.

Please advise it this generalision is incorrect

 

[dw1305]

Hi all,

Low pH levels in water without any kH doesnt shop the cycling/nitrogen cycle 'per se'.

Yes, I think that is <"where we are">.

cheers Darrel.

 

[dw1305]

Hi all,

The paper mentions that some of the aquariums were planted, but doesn't use this as a variable in the analysis. I'll try contacting Josh Neufeld and see if that is an area that interests them.

I've emailed him, I've no idea if he will reply, but if he does, and is OK with it, I'll add his response to this thread.

cheers Darrel

 

[dw1305]

Hi all,

but if he does, and is OK with it, I'll add his response to this thread.

I've actually had, very informative, replies from both Josh Neufeld <"Josh D. Neufeld | Biology | University of Waterloo"> and Michelle McKnight <"Michelle McKnight | Neufeld Research Group">, and I'd just like to thank them for their time and effort in replying to a random email..

I'll start a new thread and link it here once I've done.

Link <"Correspondence with the Neufeld lab. University of Waterloo">

cheers Darrel

 

[dw1305]

Hi all,

One thing that caught my attention in this paper is that nitrification-related microbes (mainly Nitrospira) appear to make up only a small fraction (1-2%) of the total microbial community. What are the other 98-99% doing? Could it be that the dominant process in the aquarium filter is the degradation of organic compounds?

The paper, linked in to <"Oase Biomaster Pro 2 - Comming Soon">, gives some figures for oxygen consumption by the varying types of microbe.

Wanhe Qi, Peter Vilhelm Skov, Kim João de Jesus Gregersen, Lars-Flemming Pedersen, (2022) <"Estimation of nitrifying and heterotrophic bacterial activity in biofilm formed on RAS biofilter carriers by respirometry,"> Aquaculture, 561,

cheers Darrel

 

[hax47]

Hi all,

The paper, linked in to <"Oase Biomaster Pro 2 - Comming Soon">, gives some figures for oxygen consumption by the varying types of microbe.

Wanhe Qi, Peter Vilhelm Skov, Kim João de Jesus Gregersen, Lars-Flemming Pedersen, (2022) <"Estimation of nitrifying and heterotrophic bacterial activity in biofilm formed on RAS biofilter carriers by respirometry,"> Aquaculture, 561,

cheers Darrel

Interesting. I am surprised by the relatively low oxygen consumption of heterotrophic bacteria, even though they seem to compose most of the biomass in the filter, especially if we compare their activity to the endogenous/background respiration, which is only a little bit less.

They also discuss this, and their discussion gives some explanation and suggests to me that the activity of the heterotrophic bacteria cannot be directly compared to that of the nitrifying ones with this method.

.... Another reason might be that the heterotrophs in the biofilm collected were not acclimated to external carbon source of acetate. In RAS, organic matter particularly dissolved organic matter (DOM) accumulates in culture water due to fecal waste and feed spill, which could promote the growth of heterotrophs as they obtained their energy by degrading DOM (Aguilar-Alarcón et al., 2020; Rojas-Tirado et al., 2019; Timmons et al., 2018). Unlike acetate, DOM as an indigenous carbon source is a complex mixture of chemical compounds, including chemical groups of CHNO, CHNOS, CHO, CHOS and halogens (Aguilar-Alarcón et al., 2020). Although acetate is widely used as a readily biodegradable carbon source to characterize heterotrophic biomass in wastewater (Li et al., 2019; Majewsky et al., 2011), it only represents as fraction of the complex DOM, and therefore not accurately reflect the activities of native heterotrophic bacteria in the biofilm of this study. Further studies should take the impact of carbon sources into consideration when characterizing activities of heterotrophic biofilm in RAS with respirometry.

However, I like the idea of estimating AOB/NOB/HB activity by measuring the oxygen consumption rate instead of measuring the substrate concentration.

 

[dw1305]

Hi all,

Interesting. I am surprised by the relatively low oxygen consumption of heterotrophic bacteria, even though they seem to compose most of the biomass in the filter, especially if we compare their activity to the endogenous/background respiration, which is only a little bit less.

I wondered about that, it does seems unlikely. You can find BOD values for carbohydrate rich pollutants, <"like silage effluent">, and they are huge numbers.

They also discuss this, and their discussion gives some explanation and suggests to me that the activity of the heterotrophic bacteria cannot be directly compared to that of the nitrifying ones with this method.

Which would make sense.

However, I like the idea of estimating AOB/NOB/HB activity by measuring the oxygen consumption rate instead of measuring the substrate concentration.

That also makes sense.

Cheers Darrel

 

[Bradders]

I tried to read this, and then my head exploded! Sorry.

Is there any way that an expert can summarise this? i.e. some form of understanding of "under these test circumstances, you can expect 100% DO going into the test environment and X% was used by X bacteria".

 

[dw1305]

Hi all,

under these test circumstances, you can expect 100% DO going into the test environment and X% was used by X bacteria

Unfortunately we still don't have that, mainly because of the <"unknown unknowns">, but that was exactly the idea behind using respirometry. It measures the oxygen usage, rather than trying to measure how much of the TAN (NH3 / NH4+) addition is converted to nitrite (NO2-) & nitrate (NO3-) <"Nitrification not beginning in new tank, and PH/hardness mystery">.

Optional fixed nitrogen measuring bit

.... I think it would have to be via <"stable nitrogen isotopes">, ideally supplied in the initial ammonia (NH3) addition.

This is a plant one from phytoremediation <"Use of stable nitrogen isotopes to track plant uptake of nitrogen in a nature-based treatment system">, but there are plenty of wastewater ones that look at the complete nitrification / denitrification arc.
<"Using stable isotopes to identify nitrogen transformations and estimate denitrification in a semi-constructed wetland">.

Back to oxygen
Qi et al. review the biological oxygen consumption methodology in <"their paper">.

... The methods are based on the concept that biological oxygen consumption due to substrate turnover can serve as a proxy for biological activity (Ginestet et al., 1998; Lu et al., 2020). In a conventional respirometric test, activated sludge, detached biofilm or biofilm media samples are submerged in water within respirometric chambers, and are aerated to ensure oxygen saturation, then followed specific substrate spike, changes in dissolved oxygen (DO) are monitored by a DO probe (Ginestet et al., 1998; Surmacz-Gorska et al., 1996). The oxygen consumption rate (OCR), calculated as the rate of changes in oxygen mass over time, acts as an indicator of bacterial activities responsible for biological processes of substrate turnover (Ginestet et al., 1998; Surmacz-Gorska et al., 1996). A similar approach was proposed by Moussa et al. (2003) who used respirometry to measure the activities of AOB and NOB in mixed bacterial cultures by consecutive addition of nitrite and ammonium.

This next section is the interesting bit, but it is also where things start to unravel.

However, the activity of heterotrophs is not included in the above-mentioned method.........

In their study they measured the oxygen consumption of the heterotrophic bacteria as well, but found that they used very little oxygen

..... The highest activities normalized to volumetric oxygen consumption rates, were found in polymeric foam with values of 677 ± 125, 764 ± 156 and 166 ± 36 g O2·m−3·d−1 for NOB, AOB and HB, respectively.

and that is unlikely, when we have the known (huge) BOD values for organic wastes.

Hundred percent oxygen saturation is about 10 mg / L (at 20oC), so you can see some of the issues straight away and <"why dissolved oxygen is the metric that matters"> in biological filtration and everything else is just froth.

I'll see if I can find a Salmonid Aquaculture paper with figures, it is likely to more closely replicate our tanks. For comparison clean stream water has less than 1 mg / L BOD, and "moderately polluted" water somewhere in between 2 - 8 mg / L BOD - <"Biochemical oxygen demand in rivers (sdg_06_30).">

cheers Darrel

 

[Bradders]

Thanks for helping me understand this @dw1305 - really appreciate you making it consumable.

 

[dw1305]

Hi all,

really appreciate you making it consumable.

I struggle a bit with <"that aspect">, but I know it is <"really important">.

That is one reason why I dislike <"the advertising for filter media">, buffers etc, it seems the vendors aim is always to <"obscure what their product does">. If you had a <"good product">, and you had <"scientific evidence for why it is a good product">, surely you would want to use that as your advertising?

Stephan Tanner <"Library - SWISSTROPICALS"> and Tim Hovanec <"Bacteria Revealed"> <"have a product to sell">, but they haven't embraced the <"post truth world">.

cheers Darrel

 

[dw1305]

Hi all,

I'll see if I can find a Salmonid Aquaculture paper with figures, it is likely to more closely replicate our tanks.

Unfortunately this is probably as good as we've got at the moment. I'll follow the paper and see who cites it.

They also discuss this, and their discussion gives some explanation and suggests to me that the activity of the heterotrophic bacteria cannot be directly compared to that of the nitrifying ones with this method.

Just conjecture but the section (immediately before the text you quoted) may offer a reason why pre-filter sponges work so efficiently.

..... A relatively low HB activity in biofilm was found in this study in contrast to the study of Leonard et al. (2000) where majority of heterotrophic bacteria was found in RAS biofilter. The low HB activity in the present study might be due to the removal of particulate organic matter by the drum filter in the experimental RAS platform where biocarriers were collected, resulting in a low concentration of organic matter in effluent that limited the growth of heterotrophs in biofilm.......

Which may be why the idea that <"a pre-filter sponge">, that keeps bulky organic debris out of the filter (<"so not using the filter as a syphon for mechanical filtration">), is a good idea.

cheers Darrel

 

[hax47]

Which the idea that <"a pre-filter sponge">, and keeping bulky organic debris out of the filter (<"so not using it as a syphon for mechanical filtration">), is a good idea.

You are probably right, in the other paper:

So far only the bits in <"Microbial community analysis of biofilters reveals a dominance of either comammox Nitrospira or archaea as ammonia oxidizers in freshwater aquaria">*.

The filters were regular, I assume without any prefiltering, so the organic debris went directly into the sponge.

 

[dw1305]

Hi all,

The filters were regular, I assume without any prefiltering, so the organic debris went directly into the sponge.

Almost certainly.

I had initially (naively, I now realise) expected that everyone <"would use a pre-filter">, and was really surprised that most people <"used their canister filters"> to vacuum faeces, waste food, dead leaves etc. and often even saw this as their primary function.

I've tried to tell people that <"filter as syphon"> is a really bad idea, but it often hasn't gone down well.

cheers Darrel