Nitrotoga is selected over Nitrospira in newly assembled biofilm communities from a tap water source community at increased nitrite loading.

Community assembly is a central topic in microbial ecology: how do assembly processes interact and what is the relative contribution of stochasticity and determinism? Here, we exposed replicate flow-through biofilm systems, fed with nitrite-supplemented tap water, to continuous immigration from a source community, present in the tap water, to determine the extent of selection and neutral processes in newly assembled biofilm communities at both the community and the functional guild (of nitrite-oxidizing bacteria, NOB) levels. The community composition of biofilms assembled under low and high nitrite loading was described after 40 days of complete nitrite removal. The total community assembly, as well as the NOB guild assembly were largely governed by a combination of deterministic and stochastic processes. Furthermore, we observed deterministic enrichment of certain types of NOB in the biofilms. Specifically, elevated nitrite loading selected for a single Nitrotoga representative, while lower nitrite conditions selected for a number of Nitrospira. Therefore, even when focusing on ecologically coherent ensembles, assembly is the result of complex stochastic and deterministic processes that can only be interrogated by observing multiple assemblies under controlled conditions.

Relative Abundance of Nitrotoga spp. in a Biofilter of a Cold-Freshwater Aquaculture Plant Appears To Be Stimulated by Slightly Acidic pH.

The functioning of recirculation aquaculture systems (RAS) is essential to maintain water quality for fish health, and one crucial process here is nitrification. The investigated RAS was connected to a rainbow trout production system and operated at an average temperature of 13°C and pH 6.8. Community analyses of the nitrifying biofilm revealed a coexistence of Nitrospira and Nitrotoga, and it is hypothesized that a slightly acidic pH in combination with lower temperatures favors the growth of the latter. Modification of the standard cultivation approach toward lower pH values of 5.7 to 6.0 resulted in the successful enrichment (99% purity) of Nitrotoga sp. strain HW29, which had a 16S rRNA sequence similarity of 99.0% to Nitrotoga arctica. Reference cultures of Nitrospira defluvii and the novel Nitrotoga sp. HW29 were used to confirm differentiation of these nitrite oxidizers in distinct ecological niches. Nitrotoga sp. HW29 revealed pH and temperature optima of 6.8 and 22°C, respectively, whereas Nitrospira defluvii displayed the highest nitrite oxidation rate at pH 7.3 and 32°C. We report here the occurrence of Nitrotoga as one of the main nitrite-oxidizing bacteria in freshwater aquaculture systems and indicate that a slightly acidic pH, in addition to temperatures below 20°C, can be applied as a selective isolation criterion for this microorganism.

Gallionella spp. in trickling filtration of subsurface aerated and natural groundwater.

The growth of iron-oxidizing bacteria, generally regarded as obligate microaerophilic at neutral pH conditions, has been reported in a wide range of environments, including engineered systems for drinking water production. This research focused on intensively aerated trickling filters treating deep anaerobic and subsurface aerated groundwater. The two systems, each comprising groundwater abstraction and trickling filtration, were monitored over a period of 9 months. Gallionella spp. were quantified by qPCR with specifically designed 16S rRNA primers and identified directly in the environmental samples using clone libraries with the same primers. In addition, enrichments in gradient tubes were evaluated after DGGE separation with general bacterial primers. No other iron-oxidizing bacteria than Gallionella spp. were found in the gradient tubes. qPCR provided an effective method to evaluate the growth of Gallionella spp. in these filter systems. The growth of Gallionella spp. was stimulated by subsurface aeration, but these bacteria hardly grew in the trickling filter. In the uninfluenced, natural anaerobic groundwater, Gallionella spp. were only present in low numbers, but they grew extensively in the trickling filter. Identification revealed that Gallionella spp., growing in the trickling filter were phylogenetically distinct from the species found growing during subsurface aeration, indicating that the different conditions in both systems selected for niche organisms, while inhibiting other groups. The results suggest a minor direct significance for inoculation of Gallionella spp. during filtration of subsurface aerated groundwater.

Microbial Fe(II) oxidation by Sideroxydans lithotrophicus ES-1 in the presence of Schlöppnerbrunnen fen-derived humic acids.

Controlled laboratory experiments were combined with field measurements to better understand the interactions between dissolved organic matter (DOM) and reduced iron in organic-rich peatlands. Addition of peat-derived humic acid extract (HA) to Sideroxydans lithotrophicus ES-1 liquid cultures led to higher cell numbers and up to 1.4 times higher Fe(II) oxidation rates compared to chemical controls. This effect was positively correlated with increasing HA concentrations. Similar Fe(III) (oxyhydr)oxide mineralogies were formed both abiotically and biotically irrespective of HA amendment, but minerals formed in the presence of ES-1 and HA were smaller. ES-1 growth with HA promoted aggregation of Fe(III) products in agarose-stabilized gradient tubes as shown by voltammetric profiling. In situ voltammetry in an acidic, iron-rich peatland revealed a gap between oxygen penetration and iron reduction that may reflect active Fe(II)-oxidizing microorganisms. The highest abundance of Fe(II) oxidizers Sideroxydans (4.9 × 107 gene copies gww-1) and Gallionella (1.5 × 107 gene copies gww-1) in the upper peat layer coincided with small-sized minerals resembling nanoparticulate ferrihydrite or goethite. Our results suggest that microbially mediated Fe(II) oxidation dominates in the presence of DOM leading to the formation of nano-sized biogenic Fe(III) (oxyhydr)oxides that might be readily bioavailable and likely important to iron and carbon cycling.

Characterization of the First "Candidatus Nitrotoga" Isolate Reveals Metabolic Versatility and Separate Evolution of Widespread Nitrite-Oxidizing Bacteria.

Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus "Candidatus Nitrotoga" are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure "Ca Nitrotoga" culture was available. Here we obtained the first "Ca Nitrotoga" isolate from activated sludge. This organism, "Candidatus Nitrotoga fabula," prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous "Ca Nitrotoga" enrichments, which were described as cold-adapted NOB. "Ca Nitrotoga fabula" also showed an unusually high tolerance to nitrite (activity at 30 mM NO2-) and nitrate (up to 25 mM NO3-). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent Km (Km(app)) of ~89 µM nitrite and a Vmax of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of "Ca Nitrotoga fabula" were reconstructed from the closed genome. "Ca Nitrotoga fabula" possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in "Ca Nitrotoga" and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, "Ca Nitrotoga fabula" uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable "Ca Nitrotoga fabula" to survive nitrite depletion and colonize new niches.IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus "Candidatus Nitrotoga" and performed a detailed physiological and genomic characterization of this organism ("Candidatus Nitrotoga fabula"). Differences between key phenotypic properties of "Ca Nitrotoga fabula" and those of previously enriched "Ca Nitrotoga" members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that "Ca Nitrotoga" are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in "Ca Nitrotoga fabula" expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.

New cultivation medium for "Ferrovum" and Gallionella-related strains.

Since the first isolation of the well-known iron oxidizer Acidithiobacillus ferrooxidans various media and techniques have been developed to isolate new species of acidophilic iron-oxidizing bacteria. A successful strategy in many cases was the use of iFeo medium in double-layer plates with a heterotrophic strain in the underlayer. However, even with samples which had been shown by molecular techniques to be dominated by "Ferrovum myxofaciens" and Gallionella-related bacteria, these bacteria were isolated considerably less frequently than Acidithiobacillus spp. on iFeo. Therefore, a new medium was designed which corresponded largely to the chemical composition of the mine water in a treatment plant dominated by the bacterial groups mentioned and was called artificial pilot-plant water (APPW). The analyses of approximately 500 colonies obtained from mine waters of two different sampling sites by PCR with primers specific for Acidithiobacillus spp., "Ferrovum" spp., Gallionella relatives, and Acidiphilium spp. revealed higher abundances of "Ferrovum" spp. and Gallionella relatives on the newly designed APPW medium than on iFeo which favored Acidithiobacillus spp. Molecular analysis of the colonies obtained indicated the occurrence of at least two species of iron-oxidizing bacteria and/or the heterotrophic Acidiphilium spp. in most of the colonies. Furthermore, the influence on the isolation of the concentrations of iron, phosphate, and ammonium of APPW, in levels of the iFeo medium previously described was studied.

Biological iron oxidation by Gallionella spp. in drinking water production under fully aerated conditions.

Iron oxidation under neutral conditions (pH 6.5-8) may be a homo- or heterogeneous chemically- or a biologically-mediated process. The chemical oxidation is supposed to outpace the biological process under slightly alkaline conditions (pH 7-8). The iron oxidation kinetics and growth of Gallionella spp. - obligatory chemolithotrophic iron oxidizers - were assessed in natural, organic carbon-containing water, in continuous lab-scale reactors and full-scale groundwater trickling filters in the Netherlands. From Gallionella cell numbers determined by qPCR, balances were made for all systems. The homogeneous chemical iron oxidation occurred in accordance with the literature, but was retarded by a low water temperature (13 °C). The contribution of the heterogeneous chemical oxidation was, despite the presence of freshly formed iron oxyhydroxides, much lower than in previous studies in ultrapure water. This could be caused by the adsorption of natural organic matter (NOM) on the iron oxide surfaces. In the oxygen-saturated natural water with a pH ranging from 6.5 to 7.7, Gallionella spp. grew uninhibited and biological iron oxidation was an important, and probably the dominant, process. Gallionella growth was not even inhibited in a full-scale filter after plate aeration. From this we conclude that Gallionella spp. can grow under neutral pH and fully aerated conditions when the chemical iron oxidation is retarded by low water temperature and inhibition of the autocatalytic iron oxidation.

A novel lineage of proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities.

BACKGROUND: For decades it has been recognized that neutrophilic Fe-oxidizing bacteria (FeOB) are associated with hydrothermal venting of Fe(II)-rich fluids associated with seamounts in the world's oceans. The evidence was based almost entirely on the mineralogical remains of the microbes, which themselves had neither been brought into culture or been assigned to a specific phylogenetic clade. We have used both cultivation and cultivation-independent techniques to study Fe-rich microbial mats associated with hydrothermal venting at Loihi Seamount, a submarine volcano. METHODOLOGY/PRINCIPLE FINDINGS: Using gradient enrichment techniques, two iron-oxidizing bacteria, strains PV-1 and JV-1, were isolated. Chemolithotrophic growth was observed under microaerobic conditions; Fe(II) and Fe(0) were the only energy sources that supported growth. Both strains produced filamentous stalk-like structures composed of multiple nanometer sized fibrils of Fe-oxyhydroxide. These were consistent with mineralogical structures found in the iron mats. Phylogenetic analysis of the small subunit (SSU) rRNA gene demonstrated that strains PV-1 and JV-1 were identical and formed a monophyletic group deeply rooted within the Proteobacteria. The most similar sequence (85.3% similarity) from a cultivated isolate came from Methylophaga marina. Phylogenetic analysis of the RecA and GyrB protein sequences confirmed that these strains are distantly related to other members of the Proteobacteria. A cultivation-independent analysis of the SSU rRNA gene by terminal-restriction fragment (T-RF) profiling showed that this phylotype was most common in a variety of microbial mats collected at different times and locations at Loihi. CONCLUSIONS: On the basis of phylogenetic and physiological data, it is proposed that isolate PV-1(T) ( = ATCC BAA-1019: JCM 14766) represents the type strain of a novel species in a new genus, Mariprofundus ferrooxydans gen. nov., sp. nov. Furthermore, the strain is the first cultured representative of a new candidatus class of the Proteobacteria that is widely distributed in deep-sea environments, Candidatus zeta (zeta)-Proteobacteria cl. nov.