Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full-scale wastewater treatment plants

Numerous past studies have shown members of the genus Nitrospira to be the predominant nitrite-oxidizing bacteria (NOB) in nitrifying wastewater treatment plants (WWTPs). Only recently, the novel NOB ‘Candidatus Nitrotoga arctica'' was identified in permafrost soil and a close relative was enriched from activated sludge. Still, little is known about diversity, distribution and functional importance of Nitrotoga in natural and engineered ecosystems. Here we developed Nitrotoga 16S rRNA-specific PCR primers and fluorescence in situ hybridization (FISH) probes, which were applied to screen activated sludge samples from 20 full-scale WWTPs. Nitrotoga-like bacteria were detected by PCR in 11 samples and reached abundances detectable by FISH in seven sludges. They coexisted with Nitrospira in most of these WWTPs, but constituted the only detectable NOB in two systems. Quantitative FISH revealed that Nitrotoga accounted for nearly 2% of the total bacterial community in one of these plants, a number comparable to Nitrospira abundances in other WWTPs. Spatial statistics revealed that Nitrotoga coaggregated with ammonia-oxidizing bacteria, strongly supporting a functional role in nitrite oxidation. This activity was confirmed by FISH in combination with microradiography, which revealed nitrite-dependent autotrophic carbon fixation by Nitrotoga in situ. Correlation of the presence or absence with WWTP operational parameters indicated low temperatures as a main factor supporting high Nitrotoga abundances, although in incubation experiments these NOB remained active over an unexpected range of temperatures, and also at different ambient nitrite concentrations. In conclusion, this study demonstrates that Nitrotoga can be functionally important nitrite oxidizers in WWTPs and can even represent the only known NOB in engineered systems.     

In Situ Characterization of Nitrospira-Like Nitrite-Oxidizing Bacteria Active in Wastewater Treatment Plants

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO₃⁻ or as CO₂) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.     

Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ± 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ± 0.28% of the biosludge population in the municipal WWTP and 0.37% ± 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.     

Identification and Activities In Situ of Nitrosospira and Nitrospira spp. as Dominant Populations in a Nitrifying Fluidized Bed Reactor

Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques. The microprofiles of O₂, NH₄⁺, NO₂⁻, and NO₃⁻ demonstrated the occurrence of complete nitrification in the outer 125 μm of the aggregates. The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH). No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter, respectively, could be detected by FISH. To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced. The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis. 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms. FISH analysis showed that the dominant populations of Nitrospira spp. and Nitrosospira spp. formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate. A second, smaller, morphologically and genetically different population of Nitrospira spp. was restricted to the outer nitrifying zones.     

Interactions of Nitrifying Bacteria and Heterotrophs: Identification of a Micavibrio-Like Putative Predator of Nitrospira spp.

Chemolithoautotrophic nitrifying bacteria release soluble organic compounds, which can be substrates for heterotrophic microorganisms. The identities of these heterotrophs and the specificities of their interactions with nitrifiers are largely unknown. In this study, we incubated nitrifying activated sludge with ¹³C-labeled bicarbonate and used stable isotope probing of 16S rRNA to monitor the flow of carbon from uncultured nitrifiers to heterotrophs. To facilitate the identification of heterotrophs, the abundant 16S rRNA molecules from nitrifiers were depleted by catalytic oligonucleotides containing locked nucleic acids (LNAzymes), which specifically cut the 16S rRNA of defined target organisms. Among the ¹³C-labeled heterotrophs were organisms remotely related to Micavibrio, a microbial predator of Gram-negative bacteria. Fluorescence in situ hybridization revealed a close spatial association of these organisms with microcolonies of nitrite-oxidizing sublineage I Nitrospira in sludge flocs. The high specificity of this interaction was confirmed by confocal microscopy and a novel image analysis method to quantify the localization patterns of biofilm microorganisms in three-dimensional (3-D) space. Other isotope-labeled bacteria, which were affiliated with Thermomonas, colocalized less frequently with nitrifiers and thus were commensals or saprophytes rather than specific symbionts or predators. These results suggest that Nitrospira spp. are subject to bacterial predation, which may influence the abundance and diversity of these nitrite oxidizers and the stability of nitrification in engineered and natural ecosystems. In silico screening of published next-generation sequencing data sets revealed a broad environmental distribution of the uncultured Micavibrio-like lineage.     

Enrichment and Physiological Characterization of a Novel Nitrospira-Like Bacterium Obtained from a Marine Sponge

Members of the nitrite-oxidizing genus Nitrospira are most likely responsible for the second step of nitrification, the conversion of nitrite (NO₂⁻) to nitrate (NO₃⁻), within various sponges. We succeeded in obtaining an enrichment culture of Nitrospira derived from the mesohyl of the marine sponge Aplysina aerophoba using a traditional cultivation approach. Electron microscopy gave first evidence of the shape and ultrastructure of this novel marine Nitrospira-like bacterium (culture Aa01). We characterized these bacteria physiologically with regard to optimal incubation conditions, especially the temperature and substrate range in comparison to other Nitrospira cultures. Best growth was obtained at temperatures between 28°C and 30°C in mineral medium with 70% North Sea water and a substrate concentration of 0.5 mM nitrite under microaerophilic conditions. The Nitrospira culture Aa01 is very sensitive against nitrite, because concentrations higher than 1.5 mM resulted in a complete inhibition of growth. Sequence analyses of the 16S rRNA gene revealed that the novel Nitrospira-like bacterium is separated from the sponge-specific subcluster and falls together with an environmental clone from Mediterranean sediments (98.6% similarity). The next taxonomically described species Nitrospira marina is only distantly related, with 94.6% sequence similarity, and therefore the culture Aa01 represents a novel species of nitrite-oxidizing bacteria.Numerous sponges have the capacity to accommodate large amounts of diverse microbes and represent significant sources for bioactive natural compounds (13). Many marine invertebrates excrete ammonium as a metabolic waste product (9), and the excretion of nitrite and nitrate has been taken as primary evidence that nitrifiers are active in these animals (10). By modulation of their pumping, sponges are a suitable habitat not only for aerobic microbes but also for anaerobic microbes. Accordingly, Hoffmann et al. (19) were able to detect major microbial pathways of the nitrogen cycle in the sponge Geodia barretti, including nitrification, the anammox process, and denitrification.Nitrification involves the biological oxidation of ammonia (NH₃) to nitrite (NO₂⁻) and further to nitrate (NO₃⁻) for energy purposes. It is of fundamental importance for the global nitrogen cycle in aquatic and terrestrial habitats. Nitrification is catalyzed by two phylogenetically distinct groups of microorganisms: in the first step, ammonia-oxidizing bacteria and archaea (AOB and AOA) take part in the oxidation of ammonia to nitrite, and in the second step nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate (38).Nitrite has a central position in the nitrogen cycle, connecting aerobic and anaerobic pathways. Nitrite-oxidizing bacteria play a major role in removing nitrite from the environment because it is toxic for living organisms (31). Based on morphological characteristics, NOB have been divided into five genera. This classification also reflects the phylogenetic diversity of NOB, which includes Nitrobacter and Nitrococcus (Alpha- and Gammaproteobacteria), Nitrospina (putative Deltaproteobacteria), and the candidate genus “Candidatus Nitrotoga” (Betaproteobacteria) (2). The genus Nitrospira is more distantly related to the other known NOB because it is part of its own deep-branching bacterial phylum Nitrospirae. Marine species are present in all genera of NOB except in the newly identified genus “Candidatus Nitrotoga.”As all known nitrifying prokaryotes are slow growing and hard to maintain, their enrichment and isolation from environmental samples is difficult. Most physiological studies have been performed with pure cultures of a few “model” nitrifiers, in particular AOB related to the genus Nitrosomonas and NOB of the genus Nitrobacter. For the genus Nitrospira there are only four pure cultures available: the marine species Nitrospira marina (37), Nitrospira moscoviensis (12), “Candidatus Nitrospira bockiana” (25), and Nitrospira calida (E. Lebedeva, personal communication).Sponges of the family Aplysinidae contain large amounts of bacteria embedded within the sponge tissue matrix (15). For example, the biomass of Aplysina aerophoba consists of up to 40% bacteria (36). These sponges are able to differentiate between food bacteria and their own bacterial symbionts (41). Investigations of the diversity of sponge-associated bacteria, including different genetic and also cultivation approaches, have been made with several specimens (15, 16, 39). In terms of nitrification, Hentschel et al. (17) gave first evidence for the presence of nitrite oxidizers, and it has been verified that sponges harbor AOB and AOA (8). Most of the recognized NOB in sponges are Nitrospira-like bacteria (17, 32, 35), although in the beginning, there were further hints to 16S rRNA sequences, which are most closely related to Nitrospina gracilis (17). However, as these sequences were found only once, it could be assumed that Nitrospira is the main nitrite oxidizer in this environment. Nitrospira-like bacteria are deemed to be recalcitrant and fastidious, and they are easily overgrown by other bacteria under suboptimal conditions. Despite these limitations in the laboratory, Nitrospira was determined to be the most important nitrite oxidizer during wastewater treatment (21, 33), in aquaculture biofilters (14) and in freshwater systems (20, 29).Identification of sponge-associated microorganisms has been performed largely with culture-independent methods, which are 16S rRNA gene based (denaturing gradient gel electrophoresis [DGGE], terminal restriction fragment-length polymorphism [TRFLP]) or visual (fluorescence in situ hybridization [FISH], electron microscopy) (8, 11). Nevertheless, the cultivation of microorganisms is still essential for the investigation of their physiological potential and function in the environment. Information about physiological characteristics helps us to understand the metabolism and possible nutritional interactions of nitrifiers with the host sponge (8).This is the first report about cultivation of nitrifying bacteria originating from a marine sponge. We obtained a nitrite-oxidizing enrichment culture of a Nitrospira-like bacterium derived from Aplysina aerophoba, characterized it phylogenetically, and analyzed the most important physiological features.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Identification of active denitrifiers in full‐scale nutrient removal wastewater treatment systems

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full‐scale wastewater treatment plants (WWTPs) is lacking. In this study, stable‐isotope probing (SIP) was applied in combination with microautoradiography (MAR)‐fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full‐scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully ¹³C‐labelled complex substrate was used for SIP incubations, under nitrite‐reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in ¹³C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR‐FISH represents an excellent approach for identifying and characterizing an un‐described diversity of active denitrifiers in full‐scale systems.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

Culturability and In Situ Abundance of Pelagic Bacteria from the North Sea

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.     

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10⁵ cells ml⁻¹ and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.     

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

Exploring the upper pH limits of nitrite oxidation: diversity,ecophysiology, and adaptive traits of haloalkalitolerant Nitrospira

Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo- and subsaline (1.3–12.8 g salt/l), highly alkaline (pH 8.9–10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members from  Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, “Candidatus Nitrospira alkalitolerans”, revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. “Ca. Nitrospira alkalitolerans” also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.Subject terms: Environmental microbiology, Microbial ecology     

Quantitative Molecular Analysis of the Microbial Community in Marine Arctic Sediments (Svalbard)

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ± 7.5% of total DAPI (4′,6′-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ± 1.5% hybridized to the archaeal probe ARCH915. Besides δ-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the γ-proteobacteria also constituted a significant fraction in this sediment (6.1% ± 2.5% of total cell counts, 14.4% ± 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ± 0.7% of total DAPI cell counts, 13.2% ± 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the β-proteobacteria were near the detection limit in all sediments.     

Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Identification of the Most Abundant Lactobacillus Species in the Crop of 1- and 5-Week-Old Broiler Chickens

Bacteria from crops of 1- and 5-week-old broiler chickens fed with two brands (diets A and B) of wheat-based diets were isolated on Lactobacillus-selective medium and identified (n = 300) based on partial 16S rRNA gene sequence. The most abundant Lactobacillus species were L. reuteri (33%), L. crispatus (18.7%), and L. salivarius (13.3%). Regardless of farm and feed, L. reuteri was the most abundant species (P < 0.005) in the crops of the younger chickens. However, the amount of L. reuteri was significantly reduced in the crops of the 5-week-old chickens regardless of the feed (P = 0.016). The diversity of L. reuteri isolates was studied by fatty acid analysis, and the 94 L. reuteri isolates could be arranged into several clusters. The nisin sensitivities of the L. reuteri isolates were determined because nisin is a candidate coccidiostat. Sensitive isolates were found more frequently in younger chickens (77%) than in 5-week-old chickens (23%), whereas chickens fed with commercial feed B had a higher proportion of nisin-resistant isolates (73%) than did chickens fed with feed A (45%). Nisin-resistant strains are potential candidates for adjunct cultures for maintaining L. reuteri in its natural niche in the crop and are potential targets for genetic engineering with nisin-selectable food-grade vectors. The diversity of the L. reuteri population suggested that one should consider including several strains representing different clusters and nisin resistance phenotypes in candidate probiotic feed supplements for chickens.     

Monitoring of Ralstonia eutropha KT1 in Groundwater in an Experimental Bioaugmentation Field by In Situ PCR

Ralstonia eutropha KT1, which degrades trichloroethylene, was injected into the aquifer after activation with toluene, and then the number of bacteria was monitored by in situ PCR targeting the phenol hydroxylase gene and by fluorescent in situ hybridization (FISH) targeting 16S rRNA. Before injection of the bacterial suspension, the total concentration of bacteria in the groundwater was approximately 3 × 10⁵ cells/ml and the amount of Ralstonia and bacteria carrying the phenol hydroxylase gene as a percentage of total bacterial cells was less than 0.1%. The concentration of bacteria carrying the phenol hydroxylase gene detected by in situ PCR was approximately 3 × 10⁷ cells/ml 1 h after injection, and the concentration of Ralstonia detected by FISH was similar. The number of bacteria detected by in situ PCR was similar to that detected by FISH 4 days after the start of the extraction of groundwater. On and after day 7, however, the number of bacterial cells detected by FISH was less than that detected by in situ PCR.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Nitrifying bacterial community structures and their nitrification performance under sufficient and limited inorganic carbon conditions

This study examined the hypothesis that different inorganic carbon (IC) conditions enrich different ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) populations by operating two laboratory-scale continuous-flow bioreactors fed with 15 and 100 mg IC/L, respectively. During this study, both bioreactors maintained satisfactory nitrification performance and stably oxidized 250 mg?N/L of influent ammonium without nitrite accumulation. Based on results of cloning/sequencing and terminal restriction fragment length polymorphism targeting on the ammonia monooxygenase subunit A (amoA) gene, Nitrosomonas nitrosa lineage was identified as the dominant AOB population in the high-IC bioreactor, while Nitrosomonas europaea and Nitrosomonas nitrosa lineage AOB were dominant in the low-IC bioreactor. Results of real-time polymerase chain reactions for Nitrobacter and Nitrospira 16S rRNA genes indicated that Nitrospira was the predominant NOB population in the high-IC bioreactor, while Nitrobacter was the dominant NOB in the low-IC bioreactor. Furthermore, batch experiment results suggest that N. europaea and Nitrobacter populations are proliferated in the low-IC bioreactor due to their higher rates under low IC conditions despite the fact that these two populations have been identified as weak competitors, compared with N. nitrosa and Nitrospira, under low ammonium/nitrite environments. This study revealed that in addition to ammonium/nitrite concentrations, limited IC conditions may also be important in selecting dominant AOB/NOB communities of nitrifying bioreactors.     

Molecular Detection of Novel Anammox Bacterial Clusters in the Sediments of the Shallow Freshwater Lake Taihu

Previous studies have demonstrated the wide occurrence of anaerobic ammonium oxidizing (anammox) bacteria; however, there is very limited information on the distribution of these bacteria in freshwater habitats. In this study, the anammox bacterial communities were detected by molecular analysis targeting the 16S rRNA genes in the sediments of Lake Taihu, a large and shallow eutrophic freshwater lake in China. The recovery of specific 16S rRNA sequences with two stable monophyletic clusters indicated that anammox bacteria were present in Lake Taihu. A phylogenetic analysis indicated that these two groups represent two novel lineages within the first subgroup of anammox bacteria, independent of the treeing methods. High intra-lake variability in anammox bacterial diversity and community composition was observed, in particular, based on a 1% cut-off of 16S rRNA sequence variation. The spatial variability was largely related to the substrate availability, which was denoted by the correlations between the relative abundance of the two Taihu anammox bacterial groups and the concentrations of ammonium and nitrite. This indicates that the niche differentiation of anammox bacteria is linked to the environmental heterogeneity. These findings suggest that the freshwater lakes may accommodate different anammox bacterial communities and, thus, expand our knowledge on the diversity and distribution of anammox bacteria. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.