Dataset Information

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

ABSTRACT: Recirculating aquaculture systems (RAS) are unique engineered ecosystems that minimize environmental perturbation by reducing nutrient pollution discharge. RAS typically employ a biofilter to control ammonia levels produced as a byproduct of fish protein catabolism. Nitrosomonas (ammonia-oxidizing), Nitrospira, and Nitrobacter (nitrite-oxidizing) species are thought to be the primary nitrifiers present in RAS biofilters. We explored this assertion by characterizing the biofilter bacterial and archaeal community of a commercial scale freshwater RAS that has been in operation for >15 years. We found the biofilter community harbored a diverse array of bacterial taxa (>1000 genus-level taxon assignments) dominated by Chitinophagaceae (~12%) and Acidobacteria (~9%). The bacterial community exhibited significant composition shifts with changes in biofilter depth and in conjunction with operational changes across a fish rearing cycle. Archaea also were abundant, and were comprised solely of a low diversity assemblage of Thaumarchaeota (>95%), thought to be ammonia-oxidizing archaea (AOA) from the presence of AOA ammonia monooxygenase genes. Nitrosomonas were present at all depths and time points. However, their abundance was >3 orders of magnitude less than AOA and exhibited significant depth-time variability not observed for AOA. Phylogenetic analysis of the nitrite oxidoreductase beta subunit (nxrB) gene indicated two distinct Nitrospira populations were present, while Nitrobacter were not detected. Subsequent identification of Nitrospira ammonia monooxygenase alpha subunit genes in conjunction with the phylogenetic placement and quantification of the nxrB genotypes suggests complete ammonia-oxidizing (comammox) and nitrite-oxidizing Nitrospira populations co-exist with relatively equivalent and stable abundances in this system. It appears RAS biofilters harbor complex microbial communities whose composition can be affected directly by typical system operations while supporting multiple ammonia oxidation lifestyles within the nitrifying consortium.

SUBMITTER: Bartelme RP

PROVIDER: S-EPMC5276851 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Bartelme Ryan P RP McLellan Sandra L SL Newton Ryan J RJ

Frontiers in microbiology 20170130

Recirculating aquaculture systems (RAS) are unique engineered ecosystems that minimize environmental perturbation by reducing nutrient pollution discharge. RAS typically employ a biofilter to control ammonia levels produced as a byproduct of fish protein catabolism. Nitrosomonas (ammonia-oxidizing), Nitrospira, and Nitrobacter (nitrite-oxidizing) species are thought to be the primary nitrifiers present in RAS biofilters. We explored this assertion by characterizing the biofi ...[more]

PMID: 28194147

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Project description:Nitrification by aquarium biofilters transforms ammonia waste (NH3/NH4+) to less toxic nitrate (NO3-) via nitrite (NO2-). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) Nitrospira, previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 103 ± 1.5 × 103 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 103 ± 2.7 × 103 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 101 ± 1.1 × 102 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification.ImportanceNitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira, and highlights the value of aquaria as microcosms for studying nitrifier ecology.

Project description:Exploring nitrifiers in extreme environments is vital to expanding our understanding of nitrogen cycle and microbial diversity. This study presents that complete ammonia oxidation (comammox) Nitrospira, together with acidophilic ammonia-oxidizing archaea (AOA), dominate in the nitrifying guild in sediments of an acid mine lake (AML). 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. IMPORTANCE Ammonia-oxidizing archaea (AOA) was previously considered the sole dominant ammonia oxidizer in acidic environments. This study, however, found that complete ammonia oxidation (comammox) Nitrospira was also a dominant ammonia oxidizer in the sediments of an acidic mine lake, which had an acidic pH < 5 and a high ammonium concentration of 175 mg-N/liter. In combination with average nucleotide identity analysis, phylogenomic analysis suggested it is a novel strain of comammox Nitrospira. Moreover, the adaption of comammox Nitrospira to the acidic lake had been comprehensively investigated based on genome-centric metagenomic approaches. The outcomes of this study significantly expand our understanding of the diversity and adaptability of ammonia oxidizers in the acidic environments.

Project description:In agricultural soils fertilized with a high amount of ammonium nitrogen, the pH decreases because of the oxidation of ammonia by nitrifiers. Molecular-based analyses have revealed that members of the genus Nitrospira dominate over other nitrifiers in some acidic soils. However, terrestrial Nitrospira are rarely cultivated and little is known about their ecophysiology. In addition, recent studies discovered a single microbe with the potential to oxidize both ammonia and nitrite (complete ammonia oxidizer; comammox) within Nitrospira, which had been previously recognized as a nitrite oxidizer. Despite their broad distribution, there are no enrichment samples of comammox from terrestrial or acidic environments. Here, we report the selective enrichment of both comammox and nitrite-oxidizing Nitrospira from the acidic soil of a heavily fertilized tea field. Long-term enrichment was performed with two individual continuous-feeding bioreactors capable of controlling ammonia or nitrite concentration and pH. We found that excessive ammonium supply was a key factor to enhance the growth of comammox Nitrospira under acidic conditions. Additionally, a low concentration of nitrite was fed to prevent the accumulation of free nitrous acid and inhibition of cell growth under low pH, resulting in the selective enrichment of nitrite-oxidizing Nitrospira. Based on 16S rRNA gene analysis, Nitrospira accounting for only 1.2% in an initial soil increased to approximately 80% of the total microorganisms in both ammonia- and nitrite-fed bioreactors. Furthermore, amoA amplicon sequencing revealed that two phylotypes belonging to comammox clade A were enriched in an ammonia-fed bioreactor. One group was closely related to previously cultivated strains, and the other was classified into a different cluster consisting of only uncultivated representatives. These two groups coexisted in the bioreactor controlled at pH 6.0, but the latter became dominant after the pH decreased to 5.5. Additionally, a physiological experiment revealed that the enrichment sample oxidizes ammonia at pH <4, which is in accordance with the strongly acidic tea field soil; this value is lower than the active pH range of isolated acid-adapted nitrifiers. In conclusion, we successfully enriched multiple phylotypes of comammox and nitrite-oxidizing Nitrospira and revealed that the pH and concentrations of protonated N-compounds were potential niche determinants.

Project description:Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems.IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Dataset Information

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

Publications

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

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