Nitrogen Kinetic Isotope Effects of Nitrification by the Complete Ammonia Oxidizer Nitrospira inopinata

Nitrification is an important nitrogen cycle process in terrestrial and aquatic environments. The discovery of comammox has changed the view that canonical AOA, AOB, and NOB are the only chemolithoautotrophic organisms catalyzing nitrification.

Microbial community analysis of biofilters reveals a dominance of either comammox Nitrospira or archaea as ammonia oxidizers in freshwater aquaria

Nitrification by aquarium biofilters transforms toxic ammonia waste (NH3/NH4+) to less toxic nitrate (NO3-) via nitrite (NO2-). Ammonia oxidation is mediated by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and the recently discovered complete ammonia oxidizing (comammox) Nitrospira. Prior to the discovery of comammox Nitrospira, previous research revealed that AOA dominate among ammonia oxidizers in freshwater biofilters. Here, we characterized the composition of aquarium filter microbial communities and quantified the abundance of all three known groups of ammonia oxidizers. Aquarium biofilter and water samples were collected from representative freshwater and saltwater systems in Southwestern Ontario, Canada. Using extracted DNA, we performed 16S rRNA gene sequencing and quantitative PCR (qPCR) to assess community composition and quantify the abundance of amoA genes, respectively. Our results show that aquarium biofilter microbial communities were consistently represented by putative heterotrophs of the Proteobacteria and Bacteroides phyla, with distinct profiles associated with fresh versus saltwater biofilters. Among nitrifiers, comammox Nitrospira amoA genes were detected in all 38 freshwater aquarium biofilter samples and were the most abundant ammonia oxidizer in 30 of these samples, with the remaining biofilters dominated by AOA, based on amoA gene abundances. In saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. These results demonstrate that comammox Nitrospira play an important role in biofilter nitrification that has been previously overlooked and such microcosms are useful for exploring the ecology of nitrification for future research.

Chitosan-Urea Nanocomposite for Improved Fertilizer Applications: The Effect on the Soil Enzymatic Activities and Microflora Dynamics in N Cycle of Potatoes (Solanum tuberosum L.)

The impact of polymer-based slow-release urea formulations on soil microbial N dynamics in potatoes has been sparingly deciphered. The present study investigated the effect of a biodegradable nano-polymer urea formulation on soil enzymatic activities and microflora involved in the N cycling of potato (Solanum tuberosum L.). The nano-chitosan-urea composite (NCUC) treatment significantly increased the soil dehydrogenase activity, organic carbon content and available potassium compared to the conventional urea (CU) treatment. The soil ammonical nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) contents and urease activity were significantly decreased in the NCUC-amended soil. The slow urea hydrolysis rate led to low concentrations of NH4+-N and NO3−-N in the tested potato soil. Furthermore, these results corroborate the low count of ammonia oxidizer and nitrate reducer populations. Quantitative PCR (q-PCR) studies revealed that the relative abundance of eubacterial (AOB) and archaeal ammonia-oxidizing (AOA) populations was reduced in the NCUC-treated soil compared to CU. The abundance of AOA was particularly lower than AOB, probably due to the more neutral and alkaline conditions of the tested soil. Our results suggest that the biodegradable polymer urea composite had a significant effect on the microbiota associated with soil N dynamics. Therefore, the developed NCUC could be used as a slow N-release fertilizer for enhanced growth and crop yields of potato.

Competition of Ammonia-Oxidizing Archaea and Bacteria from Freshwater Environments

In the environment, nutrients are rarely available in constant supply. Therefore, microorganisms require strategies to compete for limiting nutrients. In freshwater systems, ammonia-oxidizing archaea (AOA) and bacteria (AOB) compete with heterotrophic bacteria, photosynthetic microorganisms, and each other for ammonium, which AOA and AOB utilize as their sole source of energy and nitrogen. We investigated the competition between highly enriched cultures of an AOA (AOA-AC1) and an AOB (AOB-G5-7) for ammonium. Based on the amoA gene, the newly enriched archaeal ammonia oxidizer in AOA-AC1 was closely related to Nitrosotenuis spp. and the bacterial ammonia oxidizer in AOB-G5-7, Nitrosomonas sp. Is79, belonged to the Nitrosomonas oligotropha group ( Nitrosomonas cluster 6a). Growth experiments in batch cultures showed that AOB-G5-7 had higher growth rates than AOA-AC1 at higher ammonium concentrations. During chemostat competition experiments under ammonium-limiting conditions, AOA-AC1 dominated the cultures, while AOB-G5-7 decreased in abundance. In batch cultures, the outcome of the competition between AOA and AOB was determined by the initial ammonium concentrations. AOA-AC1 was the dominant ammonia oxidizer at an initial ammonium concentration of 50 μM and AOB-G5-7 at 500 μM. These findings indicate that, during direct competition, AOA-AC1 was able to use ammonium that was unavailable to AOB-G5-7, while AOB-G5-7 dominated at higher ammonium concentrations. The results are in strong accordance with environmental survey data suggesting that AOA are mainly responsible for ammonia oxidation under more oligotrophic conditions, whereas AOB dominate under eutrophic conditions. Importance Nitrification is an important process in the global nitrogen cycle. The first step - ammonia oxidation to nitrite – can be carried out by Ammonia-oxidizing Archaea (AOA) and Ammonia-oxidizing Bacteria (AOB). In many natural environments, these ammonia oxidizers coexist. Therefore, it is important to understand the population dynamics in response to increasing ammonium concentrations. Here, we study the competition between AOA and AOB enriched from freshwater systems. The results demonstrate that AOA are more abundant in systems with low ammonium availabilities and AOB when the ammonium availability increases. These results will help to predict potential shifts in community composition of ammonia oxidizers in the environment due to changes in ammonium availability.

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that—like for AOB—ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

Cultivation and Molecular Studies to Reveal the Microbial Communities of Groundwaters Discharge Located in Hungary

In the present study 12 water samples of five sampling sites (Tatabánya, Dandár, Szentendre, Szent Flórián and Ciprián groundwaters) known as nutrient-depleted aquatic environments were studied using amplicon sequencing (NGS) and cultivation techniques. Diversity indices and cell counts were determined to assess the species richness in relation to the cell counts within the samples, and the oligocarbophile growth capability of the isolated bacteria was tested in microtiter plates. Altogether, 55 bacterial phyla were identified from the samples by amplicon sequencing. The microbial communities of the different sampling times of the same sites did not differ significantly. Patescibacteria and Proteobacteria were present in all samples. Ciprián sample was dominated by Bacteroidetes, while in Dandár sample a high ratio of Chloroflexi was detected. Rokubacteria and WOR-1 dominated Szent Flórián sample and Tatabánya had a high number of Epsilonbacteraeota. Nine archaeal phyla were also detected; the samples were characterized by the presence of unclassified archaea and Nanoarchaeota, among them Woesearchaeia, as the most dominant. Crenarchaeota and Altiarchaeota were detected in high ratios in Dandár water samples. Among Thaumarchaeota the family Nitrosopumilaceae, and orders of Nitrosotaleales and Nitrososphaerales appeared in Szent Flórián and Tatabánya samples. Key organisms of the different biogeochemical cycles were discovered in these nutrient-depleted environments: methanogenic archaea, methanotrophic bacteria, ammonia oxidizer, nitrate reducers, diazotrophs, sulfate reducers, and sulfur oxidizer. Diversity indices and cell counts of the samples show negative correlation in case of bacteria and positive in case of archaea in Ciprián sample. The high diversity indices in Szentendre samples are connected to low cell counts, most probably due to the vulnerability of the groundwaters to the external environment factors which lead to the infiltration of soil microbes and contaminants to the water. The isolated bacteria were affiliated into four phyla, most of them belonging to Proteobacteria (59%) followed by Actinobacteria (21%), Firmicutes (17%) and Verrucomicrobia (1%). The members of the facultative chemolithotrophic genera of Sphingobium, Sphingomonas, Sphingopyxis were characterizing only Szentendre, Szent Flórián and Tatabánya samples. Only 10% of the isolated species showed an obligate oligocarbophile character. From the samples, a high number of novel bacterial taxa were cultivated. As a conclusion, our results confirmed the predominance of unclassified and unknown taxa in subsurface water, pointing to the importance and necessity of further studies to characterize these microbial populations.

Biogeography of ammonia oxidizers in New England and Gulf of Mexico salt marshes and the potential importance of comammox

Few studies have focused on broad scale biogeographic patterns of ammonia oxidizers in coastal systems, yet understanding the processes that govern them is paramount to understanding the mechanisms that drive biodiversity, and ultimately impact ecosystem processes. Here we present a meta-analysis of 16 years of data of ammonia oxidizer abundance, diversity, and activity in New England (NE) salt marshes and 5 years of data from marshes in the Gulf of Mexico (GoM). Potential nitrification rates were more than 80x higher in GoM compared to NE marshes. However, nitrifier abundances varied between regions, with ammonia-oxidizing archaea (AOA) and comammox bacteria significantly greater in GoM, while ammonia-oxidizing bacteria (AOB) were more than 20x higher in NE than GoM. Total bacterial 16S rRNA genes were also significantly greater in GoM marshes. Correlation analyses of rates and abundance suggest that AOA and comammox are more important in GoM marshes, whereas AOB are more important in NE marshes. Furthermore, ratios of nitrifiers to total bacteria in NE were as much as 80x higher than in the GoM, suggesting differences in the relative importance of nitrifiers between these systems. Communities of AOA and AOB were also significantly different between the two regions, based on amoA sequences and DNA fingerprints (terminal restriction fragment length polymorphism). Differences in rates and abundances may be due to differences in salinity, temperature, and N loading between the regions, and suggest significantly different N cycling dynamics in GoM and NE marshes that are likely driven by strong environmental differences between the regions.

Efficient nitrification and low N2O emission in a weakly acidic bioreactor at low dissolved oxygen levels are due to comammox

Nitrification is an essential process for nutrient removal from wastewater and an important emission source of nitrous-oxide (N2O), which is a powerful greenhouse gas and a dominant ozone-depleting substance. In this study, nitrification and N2O emissions were tested in two weakly acidic (pH = 6.3–6.8) reactors: one with dissolved oxygen (DO) over 2.0 mg/L and the other with DO approximately 0.5 mg/L. Efficient nitrification was achieved in both reactors. Compared to the high-DO reactor, N2O emission in the low-DO reactor decreased slightly by 20% and had insignificant correlation with the fluctuations of DO (P = 0.935) and nitrite (P = 0.713), indicating that N2O might not be mainly produced via nitrifier denitrification. Based on qPCR, qFISH, functional gene amplicon and metagenome sequencing, it was found that complete ammonia oxidizer (comammox) Nitrospira significantly outnumbered canonical ammonia-oxidizing bacteria (AOB) in both weakly acidic reactors, especially in the low DO reactor with the comammox/AOB amoA gene ratio increasing from 6.6 to 17.1. Therefore, it was speculated that the enriched comammox was the primary cause for the slightly decreased N2O emission under long-term low DO in weakly acidic reactor. This study demonstrated that comammox Nitrospira can survive well under the weakly acidic and low-DO conditions, implying that achieving efficient nitrification with low N2O emission as well as low energy and alkalinity consumption is feasible for wastewater treatment. Importance Nitrification in wastewater treatment is an important process for eutrophication control and an emission source for greenhouse gas of N2O. The nitrifying process is usually operated at a slightly alkaline pH and high DO (>2 mg/L) to ensure efficient nitrification. However, it consumes a large amount of energy and chemicals especially for wastewater without sufficient alkalinity. This manuscript demonstrated that comammox can adapt well to the weakly acidic and low-DO bioreactors, with a result of efficient nitrification and low N2O emission. These findings indicate that comammox are significant for sustainable wastewater treatment, which provides an opportunity to achieve efficient nitrification with low N2O production as well as low energy and chemical consumption simultaneously.