The nitrifying microbes in aquariums and cycling

[dw1305]

Hi all,
This review paper - <"Ammonia-oxidizing archaea and complete ammonia-oxidizing Nitrospira in water treatment systems"> has recently come my way.

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. <Ammonia-oxidizing archaea and complete ammonia-oxidizing Nitrospira in water treatment systems>.

Because we have a continual trickle of threads on the <"best way to start a tank"> and <"cycling"> - <"Dr Timothy Hovanec's comments about Bacterial supplements"> I thought I'd start a new thread.

I'm not sure how much is available to everyone, I downloaded it at work, but I can email the pdf to any-one who wants a copy?

The reason it is such an interesting paper is that it discusses both water, wastewater and ammonia loading, in the wider context, and also specifically in aquarium filters.

If any-one wants to "cut to the chase", this is the <"Aquarium Filter section">.

...... Nitrification, the oxidation of ammonia to nitrate via nitrite, is important for many engineered water treatment systems. The sequential steps of this respiratory process are carried out by distinct microbial guilds, including ammonia-oxidizing bacteria (AOB) and archaea (AOA), nitrite-oxidizing bacteria (NOB), and newly discovered members of the genus Nitrospira that conduct complete ammonia oxidation (comammox).

Even though all of these nitrifiers have been identified within water treatment systems, their relative contributions to nitrogen cycling are poorly understood. Although AOA contribute to nitrification in many wastewater treatment plants, they are generally outnumbered by AOB. In contrast, AOA and comammox Nitrospira typically dominate relatively low ammonia environments such as drinking water treatment, tertiary wastewater treatment systems, and aquaculture/aquarium filtration..........

cheers Darrel

 

[dw1305]

Hi all,
I'll continue in a new post, so that the original isn't too text dense

...... An important part of this treatment is nitrification, which is the oxidation of ammonia to nitrate, a key step in the biogeochemical cycling of nitrogen (Fig. 1). Ammonia is toxic to aquatic life in relatively low concentrations, and although ammonia and nitrate both contribute to eutrophication in nitrogen-limited waters, nitrate is generally preferable because it has no direct oxygen demand, can be further converted to nitrogen gas by anaerobic respiration (e.g., denitrification and anammox), and has comparatively low toxicity for aquatic organisms.

Fig. 1. The nitrogen cycle, with an emphasis on nitrification-mediating microorganisms.
Ammonia (NH3/NH4+) and nitrite (NO2−) oxidation are the first and second step of nitrification, respectively. Complete ammonia oxidation (comammox) involves both steps of nitrification within a single organism. Oxidation reactions are shown with green arrows, reduction reactions are shown with blue arrows, and non-redox reactions are shown with gray arrows. DNRA is dissimilatory nitrite reduction to ammonia, anammox is anaerobic ammonia oxidation. R-NH2 indicates organic molecules containing nitrogen as amine groups (i.e., biomass), NO is nitric oxide, NO3− is nitrate, N2O is nitrous oxide, and N2 is dinitrogen. This figure is adapted from Stein and Klotz (2016).

...... For over 100 years, only chemolithoautotrophic bacteria (ammonia-oxidizing bacteria, AOB) were thought to catalyze ammonia oxidation. These characterized nitrifiers belonged to the phylum Proteobacteria, ........... i.e., Nitrosomonas, Nitrosospira), and others belonging to the Chromatiales (i.e., Nitrosococcus), all now classified within the class Gammaproteobacteria (Purkhold et al., 2000) (Fig. 2). ............ members of the Thermoproteota (formerly Thaumarchaeota) can also oxidize ammonia (ammonia-oxidizing archaea, AOA). An anaerobic counterpart, anaerobic ammonia oxidation (anammox), is carried out by six bacterial genera associated with the Planktomycetota (formerly Planctomycetes) (Strous et al., 1999). The second step of nitrification, nitrite oxidation, is performed by nitrite-oxidizing bacteria (NOB) that are taxonomically diverse and belong to four different phyla (i.e., Proteobacteria, Nitrospinota, Nitrospirota, and Chloroflexota) (Daims et al., 2016).

Fig. 2. Phylogenetic relationships of AOA, AOB, and comammox Nitrospira based on AmoA amino acid sequences.

Environments of origin for each sequence are grouped into major categories and shown to the right of each sequence entry. Comammox Nitrospira sequences originate from metagenomic and cultivation studies, with cultivated species shown in red. The sequence alignment is inferred using the Le Gascuel evolutionary model (Le and Gascuel, 2008), and the maximum likelihood method was used to construct the tree, using a discrete Gamma distribution to model evolutionary rate differences among sites (4 categories (+G, parameter = 2.9096)). The AmoA sequences of AOA were used to root the tree.

cheers Darrel

 

[Oldguy]

I'm not sure how much is available to everyone

Many thanks for the post. Tried some random downloads from within you post. All worked.
Interesting & informative read.

 

[dw1305]

Hi all,

Interesting & informative read.

I'll carry on abstracting and posting some more chunks from the original paper in this thread. I might try an "edited highlights post" on a couple of other, less plant orientated, forums as well and see what the general response is.

I think it strongly reinforces my comments in <"90x45x45, new project on the track !">, but I'm guessing that won't be the universal view.

Some non-essential conjecture.
First the bit <"we know">, that the nature of that <"microbial assemblage"> is <"fine-tuned over time"> to reflect the levels of ammonia (NH3) and dissolved oxygen in the water.

This would be conjecture, but I visualise the microbial assemblage in a filter in the same way that I think about the <"benthic invertebrate assemblage in a stream">. In clean water (water with a lot of dissolved oxygen and a low Biochemical Oxygen Demand (BOD)) you have a diverse assemblage of invertebrates, including <"Mayflies (Ephemeroptera), Stoneflies (Plecoptera), Caseless Caddis (Trichoptera) etc."> with Tubificid worms (Naididae) and "Bloodworms" (Chronomidae) etc present, but as a minor component of the assemblage.

As pollution (BOD) increases dissolved oxygen levels fall and you lose the more sensitive species from the assemblage. At the same time the number of Blood worm and "Tubifex" increases. As pollution continues to increase eventually only the haemoglobin containing Blood worms and Tubifex are left, and these often <"build up to huge numbers">.

The "Tubifex and Blood-worm" scenario is the traditional view of "cycling", with Nitrobacter winogradskyi etc representing Tubifex etc. If you only ever look at sewage treatment works? You never find the Mayflies.

cheers Darrel

 

[dw1305]

Hi all,
COMAMMOX

....Although the two steps of aerobic nitrification were long thought to be carried out by distinct microorganisms, theoretical predictions indicated that these steps could be conducted by individual microorganisms capable of complete ammonia oxidation (comammox), and that such microorganisms were likely to exist in the environment (Costa et al., 2006). Comammox bacteria were hypothesized to have high growth yields, low growth rates, and be competitive in biofilm habitats where ammonia mass transfer is low. A decade after the prediction of the existence of microorganisms mediating comammox, bacteria of the genus Nitrospira (Fig. 2) capable of carrying out both ammonia and nitrite oxidation were reported simultaneously by two research groups (Daims et al., 2015; van Kessel et al., 2015). ....

.......... Given their recent discovery, less is known about the distribution and activity of comammox Nitrospira. Many engineered systems are specifically designed to reduce ammonia loads by promoting nitrification, such as domestic and industrial wastewater treatment plants (WWTPs), aquarium filters, aquaculture operations, and drinking water treatment systems. To enhance nitrogen removal and system operations, a more fundamental understanding of the community ecology and activity of ammonia oxidizers is essential. Exploring factors involved in niche differentiation of the three nitrifying guilds provides a basis for controlling nitrification. Recent reviews have focused on the presence and role of Nitrospira species in engineered systems (Mehrani et al., 2020), applied “biotechnological” potential of comammox (Lawson and Lücker, 2018), or the ecophysiology of comammox Nitrospira in various ecosystems (Koch et al., 2018). Given the relatively recent discoveries of AOA and comammox Nitrospira in engineered systems, this review focuses on summarizing recent literature examining the abundance, distribution, and activity of nitrifiers in water treatment systems. ........

....These aquatic habitats provide a surface for attached growth and are relatively oligotrophic, consistent with the predicted niche for comammox Nitrospira (Costa et al., 2006). Subsequently, comammox Nitrospira were discovered via metagenomics in a drinking water treatment plant, again processing water with relatively low ammonia concentrations (Pinto et al., 2015).....

cheers Darrel

 

[dw1305]

Hi all,
Ammonia-oxidizing archaea (AOA)

The AOA are broadly distributed in soil (Tourna et al., 2011), marine (Qin et al., 2014), and freshwater (French et al., 2012) environments, in addition to WWTPs (Li et al., 2016; Sauder et al., 2017). AOA can be classified into five main clusters based on phylogeny of the amoA gene, which encodes the active subunit of the ammonia monooxygenase (AMO) enzyme that catalyzes the first step of ammonia oxidation (Pester et al., 2012)......All cultivated AOA are thus far considered chemolithoautotrophs because they gain energy from ammonia oxidation and fix carbon dioxide into biomass (e.g., Könneke et al., 2014; Tourna et al., 2011; Walker et al., 2010)......

.....Indeed, such high ratios could help explain a dominance of some AOA (and potentially comammox Nitrospira) in low ammonia environments, such as oligotrophic waters. The reported affinities of AOA for ammonia vary widely depending on species, with NH3 half-saturation constant (Km) values from ∼ 3 nM for Nitrosopumilus maritimus (Martens-Habbena et al., 2009) to 4.4 µM for Candidatus Nitrosotenuis uzonensis (Kits et al., 2017). In comparison, AOB typically have Km values of 6 to 11 µM, but oligotrophic AOB have been reported to have NH3 Km values in the range of 0.3 to 4.0 µM, putting them in a similar range as known AOA (Hu and He, 2017; Kits et al., 2017; Lehtovirta-Morley, 2018; Prosser and Nicol, 2012). This complicates a view of niche partitioning of ammonia oxidizing microorganisms based on ammonia affinity alone......

cheers Darrel

 

[dw1305]

Hi all,
Wastewater

.....Despite the importance of nitrifying microorganisms in wastewater treatment, the relative contributions of AOA, comammox Nitrospira, and AOB to wastewater nitrification remain unclear. In general, wastewater contains relatively high concentrations of ammonia, which should favor AOB over AOA and comammox Nitrospira (Costa et al., 2006; Martens-Habbena et al., 2009; Schleper, 2010). Indeed, many studies have reported the numerical dominance of AOB in municipal and industrial WWTPs (e.g., Mussmann et al., 2011; Wells et al., 2009; Zheng et al., 2021) and a high abundance of comammox Nitrospira have been detected in tertiary treatment systems, which have relatively low ammonia concentrations (Spasov et al., 2020).

Wastewater AOA were first identified based on archaeal amoA genes in DNA extracts from activated sludge of five municipal WWTPs,.....

........ Although ammonia concentrations appear to influence ammonia-oxidizing community composition, additional factors are likely important. For example, temperature may play a role in the nitrifying communities of wastewater treatment systems. Studies have demonstrated that AOA were the dominant active ammonia oxidizers in WWTPs during cold months, as measured by labelled heavy carbon incorporation during autotrophic ammonia oxidation (Fan et al., 2018; Pan et al., 2018b). Fan et al. (2018) found that more AOA amoA genes were labelled than those of AOB, despite AOB outnumbering AOA in seed sludge.....

cheers Darrel

 

[dw1305]

Hi all,
Drinking water

AOA and comammox Nitrospira in drinking water systems
Ammonia removal is important for drinking water systems because of strict regulatory guidelines for drinking water (Tatari et al., 2017b). AOA have been detected in several nitrifying engineered biofilter environments related to drinking water treatment, including granular activated carbon of drinking water treatment plants (DWTPs) (Kasuga et al., 2010) and groundwater treatment systems for drinking water production (Albers et al., 2015; de Vet et al., 2009, 2011; Nagymáté et al., 2016; van der Wielen et al., 2009). van der Wielen et al. (2009) used qPCR and sequencing to examine the distribution and the abundance of AOB and AOA in three groundwater source drinking water treatment plants. In this system, AOA dominated certain stages of the drinking water systems, whereas AOB dominate other stages. The overall abundance of AOA and AOB correlated positively with ammonia removal and either negatively or positively associated with dissolved organic carbon concentrations in the treatment trains, respectively. These results suggest that a high dissolved organic carbon concentrations in the water might inhibit the growth of AOA (van der Wielen et al., 2009)...........

cheers Darrel

 

[dw1305]

Hi all,
This is probably the bit that will interest most people.

AOA and comammox Nitrospira in aquarium biofilters and aquaculture operations

Ammonia is a metabolic waste product excreted by fish and other aquatic organisms. Ammonia toxicity is of particular concern for relatively closed ecosystems, such as aquaculture operations and home and commercial aquaria, where ammonia can accumulate quickly to lethal concentrations in the absence of active nitrification. The un-ionized form of ammonia (NH3) is particularly toxic to fish, with chronic stress and disease associated with concentrations exceeding 0.1 mg L−1 in aquarium and aquaculture systems (Andrews et al., 1988; Parker, 2002). In order to convert toxic ammonia to nitrate, aquarium biofilters are designed to promote the growth of nitrifying populations using the high surface area of filter support material (e.g., sponge, ceramic, or polymer) and rapid flow rates of aerated water. Despite their importance to fish health within many industrial biofilters, including aquaculture, little is known of the microorganisms catalyzing nitrification in association with aquarium biofilter support material.

Traditionally, AOB such as Nitrosomonas spp., were thought to be solely responsible for ammonia oxidation in aquatic environments, including aquaria. The use of AOB-containing aquarium supplements is widespread for promoting aquarium nitrification. Indeed, evidence suggests that AOB may play a role in aquarium nitrification. .......... although they only detected AOB in 2 of 38 freshwater aquaria, despite observing vigorous nitrification rates. This led the authors to conclude that an unknown group of microorganisms was performing ammonia-oxidation in freshwater aquarium biofilters. Subsequent studies determined that Nitrosomonas spp. could be enriched from freshwater aquarium biofilters (Burrell et al., 2001) indicating that they may exist in these environments at abundances too low for detection.

AOA in aquarium filters

Since the discovery of AOA, several studies have investigated AOA in aquarium biofilters. ........... Ca. Nitrosotenuis aquarius, cultivated from a freshwater aquarium biofilter, demonstrates in situ ammonia oxidation activity, indicating that AOA contribute to ammonia oxidation in aquarium biofilters (Sauder et al., 2018). Other studies have analyzed the diversity of AOA in aquarium biofilters. Urakawa et al. (2008) identified the presence of amoA genes from AOB and AOA in marine aquarium biofilters from a public aquarium in Japan and suggested that the diversity of AOA and AOB decreased in low temperature marine aquaria. These studies highlight the overlooked importance of AOA in aquaria biofilters. Additional studies have detected AOA in marine and freshwater aquaria, and have provided evidence for the numerical dominance of AOA in freshwater aquarium biofilters (Bagchi et al., 2014; Bik et al., 2019; Sauder et al., 2011). Sauder and colleagues examined saltwater and freshwater aquaria, and highlighted that most AOA amoA sequences derived from freshwater and saltwater cluster separately, suggesting niche adaptation of AOA to saltwater or freshwater environments. Additionally, the ratio of AOB to AOA appeared to be largely governed by aquarium ammonia concentrations (Sauder et al., 2011).

COMAMMOX Nitrospira

However, due to their recent discovery, comammox Nitrospira were not considered in any of these studies. A recent survey of aquarium biofilters discovered that both comammox and AOA are dominant ammonia oxidizers within freshwater biofilters (McKnight and Neufeld, 2021). Clade A comammox Nitrospira were detected in all 38 freshwater biofilters sampled during the survey, indicating the prevalence of comammox Nitrospira in this environment was previously overlooked prior to their discovery. In contrast, there were no comammox Nitrospira detected in the saltwater aquaria where their biofilters were either dominated by AOA or AOB. Although the ubiquitous presence of comammox Nitrospira across freshwater biofilters in this survey suggests an important role in aquarium nitrification, further research is needed to confirm their activity in situ.

RAS

Recirculating aquaculture system (RAS) biofilters use similar strategies to WWTPs to regulate water quality and share many characteristics with aquaria. Several studies have examined ammonia-oxidizing microbial communities in aquaculture systems......... Similarly, the abundance of archaeal amoA genes was 12-fold higher than those of bacterial amoA in a freshwater RAS (Khangembam et al., 2017). In addition to AOA, recent studies have identified the presence of comammox Nitrospira within RAS biofilters. One study reported that nitrite-oxidizing Nitrospira and comammox Nitrospira co-exist in a RAS filter at similar abundances (Bartelme et al., 2017). In the filter, comammox Nitrospira were approximately two-fold more abundant than AOA, and AOA were, in turn, more abundant than Nitrosomonas AOB. Because Nitrosomonas and Nitrobacter are commonly used to model RAS capacities, existing models to determine capacities may be inaccurate given their exclusion of abundant nitrifiers.

Aquaponics

Similar to RAS biofilters, aquaponics systems also implement a recirculating system that combines both fish and vegetable growth, with potential roles for nitrifying microbial guilds. Comammox Nitrospira have recently been identified as the dominant ammonia oxidizers within a backyard aquaponics system, based on 16S rRNA gene sequencing and metagenomic data (Heise et al., 2021). The system operated at a low steady-state ammonia concentration, which corresponds well to the low-ammonia predicted niche of comammox Nitrospira. Interestingly, the presence of traditional AOB and AOA was negligible, which suggested that the Nitrospira present were responsible for nitrification in the sampled sediments (Heise et al., 2021). However, the study did not assess microbial communities present in the grow beds of the system, where nitrification also occurs.

Given the low ammonia concentrations imposed by design constraints and the predicted niche of nitrifying guilds, it is unsurprising that aquaculture systems can favor AOA and comammox Nitrospira over AOB (Bartelme et al., 2019). Overall, an improved knowledge of these nitrifiers offers an opportunity to fine-tune these systems by providing optimal conditions for the growth of the microorganisms that can achieve the desired nitrification rate to optimize animal production......

cheers Darrel

 

[dw1305]

Hi all,
Aquaponics continued.

....... Comammox Nitrospira have recently been identified as the dominant ammonia oxidizers within a backyard aquaponics system, based on 16S rRNA gene sequencing and metagenomic data (Heise et al., 2021). The system operated at a low steady-state ammonia concentration, which corresponds well to the low-ammonia predicted niche of comammox Nitrospira. Interestingly, the presence of traditional AOB and AOA was negligible, which suggested that the Nitrospira present were responsible for nitrification in the sampled sediments (Heise et al., 2021). However, the study did not assess microbial communities present in the grow beds of the system, where nitrification also occurs.

Given the low ammonia concentrations imposed by design constraints and the predicted niche of nitrifying guilds, it is unsurprising that aquaculture systems can favor AOA and comammox Nitrospira over AOB (Bartelme et al., 2019). Overall, an improved knowledge of these nitrifiers offers an opportunity to fine-tune these systems by providing optimal conditions for the growth of the microorganisms that can achieve the desired nitrification rate to optimize animal production.......

cheers Darrel

 

[Cédric]

Hi Darrel,
Thanks for the thread. Very interesting. I heard about Nitrospira but Archaea were unknown to me.

 

[MichaelJ]

Thanks Darrel! For me at least, this is going to take some effort to read, let alone understand.

The Al-Ajeel et al. paper downloads fine on my end as well.

Cheers,
Michael

 

[dw1305]

Hi all,

I heard about Nitrospira but Archaea were unknown to me.

It looks like COMAMMOX Nitrospira are really important players in all situations with relatively low ammonia loadings and they've only found them within the last ten years. People are discovering new Ammonia Oxidising Microbes (AOM) all the time (by looking for the genes that code for ammonia oxidation) and I'd guess we are nowhere near the end of it yet.

<"Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities - The ISME Journal">
<"Ammonium Removal in Aquaponics Indicates Participation of Comammox Nitrospira - Current Microbiology">

cheers Darrel

 

[dw1305]

Hi all,
This is from <"https://journals.asm.org/doi/pdf/10.1128/mSphere.00143-19">*

It is well worth a read and relatively accessible for a paper with a lot of D(R)NA work in it. Based on the comments in the conclusion I'll see if I can contact Ryan Newton* and see if he has other aquarium related bits he can offer. It is probably a long shot, but I'll give it a go.

...... We noted previously that AOA and Nitrospira genotype abundance patterns in a single RAS biological filter were correlated across a fish rearing cycle and that both groups of nitrifiers were present at 1 x 10^8 nitrifying marker genes per gram of sand (22). AOA are favored over ammonia-oxidizing bacteria (AOB) under conditions of low ammonia substrate concentrations (54). Since ammonia oligotrophy is a design constraint in RAS and aquaponic systems, high abundances of AOA should be present in these environments. We found our results to be consistent with this idea, as AOA were the dominant ammonia-oxidizing taxa across nearly all RAS and aquaponic systems (Fig. 4). Going forward, AOA physiology rather than AOB physiology should form the basis of future nitrifying biofilter design and nitrogen flux modeling. It is unknown whether AOA arise as the dominant ammonia-oxidizing taxa in these systems at biofilter initiation or do so after substrate concentrations decrease and stabilize following the occurrences of the high ammonia concentrations typically seen during the start-up phase.........

From the conclusion.

......... Having conducted our survey of nitrogen cycle amplicon markers, it is apparent that the AOA-plus-NOB Nitrospira nitrifying guild is the most common across freshwater aquaria and RAS (22, 47, 62). It is also worth noting that, although the aquaculture practitioners from our survey were knowledgeable about nitrification as a system process, many believed that Nitrosomonas and Nitrobacter species were the sole nitrifying taxa present. The results from this study and others (63) indicate this is not the nitrification schema present in operational RAS, and it is our recommendation that aquacultural organizations incorporate new nitrogen cycle findings into stakeholder outreach plans to better inform system operators when they select starter cultures or substrates for a biological filter ......

* Bartelme RP, Smith MC, Sepulveda-Villet OJ, Newton RJ. (2019). "Component microenvironments and system biogeography structure microorganism distributions in recirculating aquaculture and aquaponic systems". mSphere 4:e00143-19. <"https://doi.org/ 10.1128/mSphere.00143-19">.

* I've emailed him - 28/11/2022

cheers Darrel

 

[dw1305]

Hi all,
*I'm pleased to say Dr Ryan Newton has replied. I'm just waiting for him to get back to me, but as soon as he has OK'ed it, I'll post his email in a new thread and <"link it here">.

cheers Darrel

 

[Guest]

I might try an "edited highlights post" on a couple of other, less plant orientated, forums as well and see what the general response is.

Did you get around to posting the edited highlights on less plant orientated forums? And if so, how did it go down?

 

[dw1305]

Hi all,

Did you get around to posting the edited highlights on less plant orientated forums? And if so, how did it go down?

I haven't done it yet. Now I've got Dr Newton's comments for back-up I'll try <"PlanetCatfish"> and / or <"Apistogramma forums">. I'd guess I'll get more of hearing there than I would on Facebook etc.

cheers Darrel

 

[dw1305]

Hi all,
Thread with Ryan Newton's reply: <"Correspondence with Dr Ryan Newton - School of Freshwater Sciences, University of Wisconsin—Milwaukee">.

cheers Darrel

 

[dw1305]

Hi all,

Did you get around to posting the edited highlights on less plant orientated forums? And if so, how did it go down?

I've added it onto an existing PlanetCatfish thread <"Using deep gravel and bacteria to control nitrogen - Page 6">. I'd say the reception has been both mixed and underwhelming.

cheers Darrel

 

[dw1305]

Hi all,
Another "Aquaponics" one (<"Nitrogen transformations across compartments of an aquaponic system">*) has come my way. It has figures for dissolved oxygen, REDOX etc. for the different components of the system (below), which maybe of interest to @jaypeecee etc?

One thing of note is that the water would be a lot more nutrient rich then we would get in our tanks, even if we kept <"insane stocking levels">.

It doesn't really add anything new, but it is quite an interesting read and supports the idea that plants, low ammonia loadings <"and high oxygen levels"> lead to a diverse and resilient microbial community.

....Bacterial diversity was highest in the biofilm from the hydroponic table. This effect on the microbial biodiversity can be caused by the potential influence of various herbs planted in the system, as each plant species enriches its unique root microbiome..........

*Zala Schmautz, Carlos A. Espinal, Theo H.M. Smits, Emmanuel Frossard, Ranka Junge, (2021) "Nitrogen transformations across compartments of an aquaponic system", Aquacultural Engineering, 92.

cheers Darrel