Insights into the Variations of Hao-Dependent Nitrifying and Nir-Dependent Denitrifying Microbial Communities in Ammonium-Graduated Lake Environments

Biological nitrification and denitrification play significant roles in nitrogen-associated biogeochemical cycles. However, our understanding of the spatial scales at which microbial communities act and vary is limited. We used gene-specific metagenomic PCR to explore changes in nitrifying and denitrifying microbial communities within pristine lake and its branches, where the ammonium and dissolved organic carbon (DOC) concentrations form a gradient. The biomarkers hydroxylamine oxidoreductase and nitrite reductase genes indicated that strong relationships exist between the diversities and community structures of denitrifiers and ammonium gradients. It showed that the Nitrosomonas oligotropha cluster dominates the nitrifying bacteria in low-nutrition environments, while a new Nitrosomonas ureae cluster accounted for nearly 80% of the nitrifying bacteria in high-nitrogen environments. The distribution and diversity of nirS/K-dependent denitrifiers in the various habitats were similar, but predominantly affiliated with unknown clusters. Moreover, the abundance of all the hao genes dramatically outnumbered that of nir genes. The relative abundance of hao was clearly higher during eutrophication (13.60%) than during oligotrophy (5.23%), whereas that of nirS showed opposite tendencies. Overall, this study provides valuable comparative insights into the shifts in nitrifying and denitrifying microbial populations in lake environments with ammonium gradients, suggesting that unique dominant denitrifiers probably play an important role in the nitrogen cycle.

Draft Genome Sequence of Nitrosomonas sp. Strain APG5, a Betaproteobacterial Ammonia-Oxidizing Bacterium Isolated from Beach Sand

Nitrosomonas sp. strain APG5 (=NCIMB 14870 = ATCC TSA-116) was isolated from dry beach sand collected from a supralittoral zone of the northwest coast of the United States. The draft genome sequence revealed that it represents a new species of the cluster 6 Nitrosomonas spp. that is closely related to Nitrosomonas ureae and Nitrosomonas oligotropha.

New Methods for Analysis of Spatial Distribution and Coaggregation of Microbial Populations in Complex Biofilms

ABSTRACTIn biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescencein situhybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generatingin silicobiofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizersNitrosomonas oligotrophasubclusters I and II and the nitrite oxidizerNitrospirasublineage I in three different types of wastewater biofilms, suggesting niche differentiation between theN. oligotrophasubclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed thatN. oligotrophasubcluster II aggregated closer toNitrospirathan didN. oligotrophasubcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.

Ecophysiological Characterization of Ammonia-Oxidizing Archaea and Bacteria from Freshwater

ABSTRACTAerobic biological ammonia oxidation is carried out by two groups of microorganisms, ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). Here we present a study using cultivation-based methods to investigate the differences in growth of three AOA cultures and one AOB culture enriched from freshwater environments. The strain in the enriched AOA culture belong to thaumarchaeal group I.1a, with the strain in one enrichment culture having the highest identity with “CandidatusNitrosoarchaeum koreensis” and the strains in the other two representing a new genus of AOA. The AOB strain in the enrichment culture was also obtained from freshwater and had the highest identity to AOB from theNitrosomonas oligotrophagroup (Nitrosomonascluster 6a). We investigated the influence of ammonium, oxygen, pH, and light on the growth of AOA and AOB. The growth rates of the AOB increased with increasing ammonium concentrations, while the growth rates of the AOA decreased slightly. Increasing oxygen concentrations led to an increase in the growth rate of the AOB, while the growth rates of AOA were almost oxygen insensitive. Light exposure (white and blue wavelengths) inhibited the growth of AOA completely, and the AOA did not recover when transferred to the dark. AOB were also inhibited by blue light; however, growth recovered immediately after transfer to the dark. Our results show that the tested AOB have a competitive advantage over the tested AOA under most conditions investigated. Further experiments will elucidate the niches of AOA and AOB in more detail.

Active Autotrophic Ammonia-Oxidizing Bacteria in Biofilm Enrichments from Simulated Creek Ecosystems at Two Ammonium Concentrations Respond to Temperature Manipulation

ABSTRACTThe first step of nitrification, the oxidation of ammonia to nitrite, is important for reducing eutrophication in freshwater environments when coupled with anammox (anaerobic ammonium oxidation) or denitrification. We analyzed active formerly biofilm-associated aerobic ammonia-oxidizing communities originating from Ammerbach (AS) and Leutra South (LS) stream water (683 ± 550 [mean ± standard deviation] and 16 ± 7 μM NH4+, respectively) that were developed in a flow-channel experiment and incubated under three temperature regimens. By stable-isotope probing using13CO2, we found that members of theBacteriaand notArchaeawere the functionally dominant autotrophic ammonia oxidizers at all temperatures under relatively high ammonium loads. The copy numbers of bacterialamoAgenes in13C-labeled DNA were lower at 30°C than at 13°C in both stream enrichment cultures. However, the community composition of the ammonia-oxidizing bacteria (AOB) in the13C-labeled DNA responded differently to temperature manipulation at two ammonium concentrations. In LS enrichments incubated at thein situtemperature (13°C),Nitrosomonas oligotropha-like sequences were retrieved with sequences fromNitrosospiraAmoA cluster 4, while the proportion ofNitrosospirasequences increased at higher temperatures. In AS enrichments incubated at 13°C and 20°C, AmoA cluster 4 sequences were dominant;Nitrosomonas nitrosa-like sequences dominated at 30°C. Biofilm-associated AOB communities were affected differentially by temperature at two relatively high ammonium concentrations, implicating them in a potential role in governing contaminated freshwater AOB distributions.

Ammonia-Oxidizing Bacteria in Biofilters Removing Trihalomethanes Are Related toNitrosomonas oligotropha

ABSTRACTAmmonia-oxidizing bacteria (AOB) in nitrifying biofilters degrading four regulated trihalomethanes—trichloromethane, bromodichloromethane, dibromochloromethane, and tribromomethane—were related toNitrosomonas oligotropha. N. oligotrophais associated with chloraminated drinking water systems, and its presence in the biofilters might indicate that trihalomethane tolerance is another reason that this bacterium is dominant in chloraminated systems.

Diversity, Abundance, and Spatial Distribution of Sediment Ammonia-Oxidizing Betaproteobacteria in Response to Environmental Gradients and Coastal Eutrophication in Jiaozhou Bay, China

ABSTRACT Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, the amoA gene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizing Betaproteobacteria (Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced from amoA gene sequences grouped within four distinct clusters in the Nitrosomonas lineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in the Nitrosospira lineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. Both amoA clone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in the Nitrosomonas oligotropha lineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.

Epiphyton as a Niche for Ammonia-Oxidizing Bacteria: Detailed Comparison with Benthic and Pelagic Compartments in Shallow Freshwater Lakes

ABSTRACT Next to the benthic and pelagic compartments, the epiphyton of submerged macrophytes may offer an additional niche for ammonia-oxidizing bacteria in shallow freshwater lakes. In this study, we explored the potential activities and community compositions of ammonia-oxidizing bacteria of the epiphytic, benthic, and pelagic compartments of seven shallow freshwater lakes which differed in their trophic status, distribution of submerged macrophytes, and restoration history. PCR-denaturing gradient gel electrophoresis analyses demonstrated that the epiphytic compartment was inhabited by species belonging to cluster 3 of the Nitrosospira lineage and to the Nitrosomonas oligotropha lineage. Both the ammonia-oxidizing bacterial community compositions and the potential activities differed significantly between compartments. Interestingly, both the ammonia-oxidizing bacterial community composition and potential activity were influenced by the restoration status of the different lakes investigated.

Changes in ammonia oxidiser population during transition to low pH in a biofilm reactor starting with Nitrosomonas europaea

Recent experiments in our laboratory using both biofilm and suspended biomass reactors have demonstrated high rate nitrification at low pH with known autotrophic nitrifying bacteria originating from wastewater treatment plants refuting previous assumptions that nitrification is significantly inhibited at low pH. Since much of the earlier microbiological work regarding ammonia oxidising bacteria (AOB) physiology was carried out using Nitrosomonas europaea, this model bacterium's capability for high rate nitrification at low pH in a continuous biofilm reactor was tested. A biofilm reactor filled with sintered glass particles was inoculated with a pure culture of N. europaea. The reactor was first operated to high nitrification rates under conditions favourable to N. europaea (pH > 7; high ammonium concentrations). To eliminate inhibitory concentrations of nitrite at low pH, an enriched culture of Nitrospira (a nitrite oxidising bacterium) was then added. The transition from neutral to acidic conditions was attempted by sharply lowering the nitrification rate and by using a feeding solution containing insufficient buffer for complete nitrification. As opposed to other successful transitions, the pH in the N. europaea/Nitrospira reactor initially dropped only slightly and maintained pH > 6 for over two weeks. The reactor reached pH 4.5 only after four weeks. FISH results showed that while the percent of AOB and Nitrospira to eubacteria remained relatively constant at 51.1±8.2% and 40.8±6.4%, respectively, the AOB community changed completely in 60 days from 100% N. europaea to 100% Nitrosomonas oligotropha. Even though N. oligotropha was not intentionally introduced into the reactor, it is apparently much better adapted to conditions of low pH.

Distribution of ammonia-oxidizing bacteria in sewage activated sludge: analysis based on 16S rDNA sequence

This study carried out analysis of ammonia-oxidizing bacteria (AOB) communities in 12 sewage activated sludge systems standing in eight sewage treatment plants located in Tokyo. The systems were different in the treatment process configuration: anaerobic/anoxic/aerobic (A2O), anaerobic/aerobic (AO), and conventional activated sludge (AS) processes. AOB communities were analyzed by sequences of 16S rDNA amplicons, which were separated by denaturing gradient gel eletrophoresis (DGGE) after specific polymerase chain reaction (PCR) amplification. The results demonstrated that low ammonium concentrations in the influents of the 12 sewage activated sludge systems resulted in the dominance of Nitrosomonas oligotropha-like sequences. Further, Nitrosomonas europaea- and Nitrosomonas cryotolerans-like sequences were recovered from only one A2O system of which the influent contained higher ammonium and chloride concentrations than those of other systems. Nitrosomonas communis-like sequences were found in every A2O and AO system, but mostly not found in every AS system. In summary, influent characteristics and treatment process configuration affected the AOB communities in the 12 sewage activated sludge systems.