A novel Nitrospira lineage isolated from activated sludge using elevated temperatures.

The genus Nitrospira represents the dominant nitrite-oxidizing clade in most wastewater treatment plants (WWTPs) globally, and several Nitrospira strains have been isolated from activated sludge. Using a pre-enrichment strategy with alternating nitrifying and denitrifying conditions, followed by incubation at elevated temperatures, we isolated a novel Nitrospira species, named Nitrospira tepida. This moderately thermophilic species with optimal growth between 37 and 45°C is only distantly related to other Nitrospira and forms a novel lineage VII within the genus, together with few environmental 16S rRNA gene sequences predominantly detected in thermal wastewater or oxygen-limited systems. Genomic and physiological analyses revealed remarkable differences between N. tepida and two other isolates previously obtained from the same WWTP, suggesting niche differentiation between these nitrite oxidizers. N. tepida grows in aggregates, and tolerates nitrite and nitrate concentrations of up to 20 mM and 40 mM, respectively. The Km value for nitrite of N. tepida is 77 ± 26 µM. In summary, this novel Nitrospira lineage seems to be well-adapted for wastewater treatment processes at elevated temperatures and limited aeration, conditions that potentially reduce operational costs of such systems.

Some like it cold: the cellular organization and physiological limits of cold-tolerant nitrite-oxidizing Nitrotoga.

Chemolithoautotrophic production of nitrate is accomplished by the polyphyletic functional group of nitrite-oxidizing bacteria (NOB). A widely distributed and important NOB clade in nitrogen removal processes at low temperatures is Nitrotoga, which however remains understudied due to the scarcity of cultivated representatives. Here, we present physiological, ultrastructural and genomic features of Nitrotoga strains from various habitats, including the first marine species enriched from an aquaculture system. Immunocytochemical analyses localized the nitrite-oxidizing enzyme machinery in the wide irregularly shaped periplasm, apparently without contact to the cytoplasmic membrane, confirming previous genomic data suggesting a soluble nature. Interestingly, in two strains we also observed multicellular complexes with a shared periplasmic space, which seem to form through incomplete cell division and might enhance fitness or survival. Physiological tests revealed differing tolerance limits towards dissolved inorganic nitrogen concentrations and confirmed the generally psychrotolerant nature of the genus. Moreover, comparative analysis of 15 Nitrotoga genomes showed, e.g. a unique gene repertoire of the marine strain that could be advantageous in its natural habitat and confirmed the lack of genes for assimilatory nitrite reduction in a strain found to require ammonium for growth. Overall, these novel insights largely broaden our knowledge of Nitrotoga and elucidate the metabolic variability, physiological limits and thus potential ecological roles of this group of nitrite oxidizers.

Divergent fate of coccolithophores in a warming tropical ecosystem.

Rising ocean temperatures will alter the diversity of marine phytoplankton communities, likely leading to modifications in food-web and biogeochemical dynamics. Here we focus on coccolithophores, a prominent group of calcifying phytoplankton that plays a central role in the global carbon cycle. Using both new (2017-2020) and historical (1975-1976) data from the northern Red Sea, we found that during 'mild summers', the most common coccolithophores - Emiliania huxleyi and Gephyrocapsa ericsonii - co-exist at similar densities. Both species then particularly flourish during subsequent winter periods where nutrient availability is higher due to convective mixing. However, during 'hot summers', which have become progressively the norm over the last decades with average surface temperatures exceeding 27°C for long time-periods, G. ericsonii density markedly declined. Moreover, G. ericsonii remains at low background levels even during winter mixing periods, while E. huxleyi succession and development during winter appears unchanged. Further incubation assays using native assemblages confirmed that G. ericsonii's growth over 27°C is significantly reduced relative to E. huxleyi. Additional factors likely contribute to impair G. ericsonii populations at sea, but temperature is a key factor. Our results illustrate the divergent impact of ongoing ocean warming in tropical phytoplankton species.

Relevance of Candidatus Nitrotoga for nitrite oxidation in technical nitrogen removal systems.

Many biotechnological applications deal with nitrification, one of the main steps of the global nitrogen cycle. The biological oxidation of ammonia to nitrite and further to nitrate is critical to avoid environmental damage and its functioning has to be retained even under adverse conditions. Bacteria performing the second reaction, oxidation of nitrite to nitrate, are fastidious microorganisms that are highly sensitive against disturbances. One important finding with relevance for nitrogen removal systems was the discovery of the mainly cold-adapted Cand. Nitrotoga, whose activity seems to be essential for the recovery of nitrite oxidation in wastewater treatment plants at low temperatures, e.g., during cold seasons. Several new strains of this genus have been recently described and ecophysiologically characterized including genome analyses. With increasing diversity, also mesophilic Cand. Nitrotoga representatives have been detected in activated sludge. This review summarizes the natural distribution and driving forces defining niche separation in artificial nitrification systems. Further critical aspects for the competition with Nitrospira and Nitrobacter are discussed. Knowledge about the physiological capacities and limits of Cand. Nitrotoga can help to define physico-chemical parameters for example in reactor systems that need to be run at low temperatures. KEY POINTS: • Characterization of the psychrotolerant nitrite oxidizer Cand. Nitrotoga • Comparison of the physiological features of Cand. Nitrotoga with those of other NOB • Identification of beneficial environmental/operational parameters for proliferation.

Defining Culture Conditions for the Hidden Nitrite-Oxidizing Bacterium Nitrolancea.

Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation - the second step of nitrification - is catalyzed by a phylogenetically diverse bacterial group, and only recently bacteria of the phylum Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, its distribution remains cryptic as very few closely related environmental 16S rRNA gene sequences have been retrieved so far. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a wastewater side-stream reactor operated at 39.5°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different strains of Nitrolancea, closely related to Nl. hollandica. The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C inoculated with biomass from a geothermal spring in the Copahue volcano area in Neuquen, Argentina. Here, an additional distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit (nxrA) gene sequence identities to Nl. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and Nl. hollandica of 91.9% indicates that it indeed represents a distinct species. All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35-37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea strains share 2399 out of 3387 orthologous gene clusters and encode similar key functions. Our results define general growth conditions that enable the selective enrichment of Nitrolancea from artificial and natural environments. In most natural habitats these NOB apparently are of low abundance and their proliferation depends on the balanced presence of nitrite and ammonium, with an optimal incubation temperature of 37°C.

Spatial homogeneity of bacterial and archaeal communities in the deep eastern Mediterranean Sea surface sediments.

The diversity of microorganisms inhabiting the deep sea surface sediments was investigated in 9 stations (700-1900 m depth) in the Levantine basin by 454 massive tag sequencing of the 16S rDNA V4 region using universal primers. In total, 108,811 reads (an average of 10,088 per sample) were assigned to 5014 bacterial and 966 archaeal operational taxonomic units (OTUs; at 97% cut off). The 55% of the reads were of archaea, indicating dominance of archaea over bacteria at eight of the stations. The diversity and estimated richness values were high (e.g., H´ ranged from 5.66 to 7.41 for bacteria). The compositions of the microorganisms at all stations were remarkably similar, with Bray-Curtis similarities of 0.53-0.91 and 0.74-0.99 for bacterial and archaeal orders respectively. At two stations, very high abundances of only a few genera (Marinobacterium, Bacillus, Vibrio, Photobacterium) were accountable for the dissimilarities documented compared to the other deep sea stations. Half of the bacterial reads (51%) belonged to the phylum Proteobacteria, comprising mainly Gammaproteobacteria (41-72% of the proteobacterial reads per sample), Deltaproteobacteria (12-29%), Alphaproteobacteria (7-18%) and Betaproteobacteria (3-14%). The most abundant bacterial family was Sinobacteraceae (order Xanthomonadales) with 5-10% of total bacterial reads per sample. Most abundant reads (15.4% of all microbial reads) were affiliated with Marine Group 1 archaea, putatively capable of ammonia oxidation (213 OTUs), and bacteria involved in nitrification were found in all samples. The data point to the significant role that chemolithotrophic carbon assimilation and nitrification fill in the oligotrophic deep sea Levant sediments. [Int Microbiol 19(2): 109-119 (2016)].

Distribution patterns of bacterioplankton in the oligotrophic south-eastern Mediterranean Sea.

The spatial variability in the composition of bacterioplankton communities from the Levantine basin off the Israeli coast (south-eastern Mediterranean Sea) was studied using water samples from nine stations down to 1880 m depth. An nMDS (non-metric multidimensional scaling) ordination plot of ARISA (automated rRNA intergenic spacer analyses) fingerprints performed on 39 water samples revealed three groups of communities based on depth (epi, meso and bathypelagial waters), while the stations' geographical location did not seem to have an effect on the distribution of bacterioplankton assemblages. Interestingly, communities from the two deep pelagic zones were further grouped depending on the three prevailing water masses in the Levantine basin, and depth, salinity, temperature and silicic acid were significantly related to the variations of the bacterial community compositions. Cloning of internal transcribed spacer (ITS) sequences of dominant ARISA peaks showed that abundant bacteria in all depths were ubiquitous uncultured Alpha- and Gammaproteobacteria. The results further indicate that distinct microbial community profiles in the Levantine basin are associated with prevailing water masses that have formed due to different physico-chemical parameters and thus might act as physical barriers rather than physiological discriminators, as has been suggested in studies from other ocean regions.

Characterization of a new marine nitrite oxidizing bacterium, Nitrospina watsonii sp. nov., a member of the newly proposed phylum "Nitrospinae".

Nitrite oxidizing bacteria are an integral part of the nitrogen cycle in marine waters, but the knowledge about their diversity is limited. Recently, a high abundance of Nitrospina-like 16S rRNA gene sequences has been detected in oceanic habitats with low oxygen content by molecular methods. Here, we describe a new strain of Nitrospina, which was sampled in 100m depth from the Black Sea. It coexisted with a not-yet cultivated chemoorganotrophic gammaproteobacterium and could be purified by classical isolation methods including Percoll density gradient centrifugation. The new Nitrospina-like bacterium grew lithoautotrophically at 28°C in diluted seawater supplemented with inorganic salts and nitrite. Gram-negative rods were characterized morphologically, physiologically and partly biochemically. The 16S rRNA gene of the new strain of Nitrospina is 97.9% similar to the described species N. gracilis and DNA/DNA hybridization experiments revealed a relatedness of 30.0%. The data from both Nitrospina species and environmental clones were used for an extensive 16S rRNA based phylogenetic study applying high quality filtering. Treeing analyses confirm the newly defined phylum status for "Nitrospinae" [18]. The results of phylogenetic and genotypic analyses support the proposal of a novel species Nitrospina watsonii sp. nov. (type strain 347(T), LMG 27401(T), NCIMB 14887(T)).

Relevance and diversity of Nitrospira populations in biofilters of brackish RAS.

Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with (13)C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of Nitrospira-related labeled lipid markers were found confirming that Nitrospira is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as Nitrobacter or Nitrotoga were functionally not relevant in the investigated biofilters. Nitrospira-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of Nitrospira, though most sequences clustered with the type strain of Nitrospira marina and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of Nitrospira sublineage IV organisms. The membranes included two marker lipids (16∶1 cis7 and 16∶1 cis11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine Nitrospira sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.

Relevance of Nitrospira for nitrite oxidation in a marine recirculation aquaculture system and physiological features of a Nitrospira marina-like isolate.

In biofilters of recirculation aquaculture systems (RAS), nitrification by lithoautotrophic microorganisms is essential to prevent the cultivated organisms from intoxication with ammonium and nitrite. In moving-bed biofilters nitrifying microorganisms are immobilized together with heterotrophic bacteria in dense biofilms on carrier elements like plastic beads. Analyses of fatty acid profiles of these biofilms from a marine biofilter revealed a high abundance of Nitrospira-related lipid markers (8-12% of total fatty acids). Further results of a labeling experiment with (13) C-bicarbonate in mineral salts medium with 3 mM nitrite confirmed that Nitrospira is the major autotrophic nitrite oxidizer in the biofilter system. According to 16S rRNA gene sequence analyses the nitrite-oxidizing community in the biofilter consisted of at least two different representatives of Nitrospira, one of which could be successfully isolated. The marine isolate 'Ecomares 2.1' belongs to cluster IVa and showed 98.8% 16S rRNA gene sequence similarity to Nitrospira marina, whereas the enrichment 'M1 marine' is only distantly related (94.0% 16S rRNA gene sequence similarity to N. marina). In laboratory experiments, the isolate exhibited remarkable tolerances against high substrate and product concentrations (30 mM nitrite and 80 mM nitrate) as well as ammonium (50 mM). During the isolation process a strong tendency of this strain to develop biofilms became apparent. Thus, Ecomares 2.1 seems to be well adapted to the attached lifestyle in biofilters and the nitrogenous load prevailing in the effluent waters of RAS. Both members of Nitrospira could be detected by PCR-based methods in environmental samples of marine and brackish RAS biofilters and are therefore considered to be characteristic for these engineered ecosystems.