Dissimilatory nitrate reduction to ammonia (DNRA) is a common biochemical process in the nitrogen cycle in natural and man-made habitats, but its significance in wastewater treatment plants is not well understood. Several ammonifying Trichlorobacter strains (former Geobacter) were previously enriched from activated sludge in nitrate-limited chemostats with acetate as electron (e) donor, demonstrating their presence in these systems. Here, we isolated and characterized the new species Trichlorobacter ammonificans strain G1 using a combination of low redox potential and copper-depleted conditions. This allowed purification of this DNRA organism from competing denitrifiers. T. ammonificans is an extremely specialized ammonifier, actively growing only with acetate as e-donor and carbon source and nitrate as e-acceptor, but H₂ can be used as an additional e-donor. The genome of G1 does not encode the classical ammonifying modules NrfAH/NrfABCD. Instead, we identified a locus encoding a periplasmic nitrate reductase immediately followed by an octaheme cytochrome c that is conserved in many Geobacteraceae species. We purified this octaheme cytochrome c protein (TaNiR), which is a highly active dissimilatory ammonifying nitrite reductase loosely associated with the cytoplasmic membrane. It presumably interacts with two ferredoxin subunits (NapGH) that donate electrons from the menaquinol pool to the periplasmic nitrate reductase (NapAB) and TaNiR. Thus, the Nap-TaNiR complex represents a novel type of highly functional DNRA module. Our results indicate that DNRA catalyzed by octaheme nitrite reductases is a metabolic feature of many Geobacteraceae, representing important community members in various anaerobic systems, such as rice paddy soil and wastewater treatment facilities.

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Three highly alkaliphilic bacterial strains designated as A1^T, H1^T and B1^T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative and rod-shaped. Strains A1^T, H1^T and B1^T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1^T, H1^T and B1^T was 2574013, 2475906 and 2623236 bp, and the G+C content was 66.0, 66.2 and 66.1mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera Malikia (95.1–96.4%), Macromonas (93.0–93.6%) and Hydrogenophaga (93.0–96.6%) in the family Comamonadaceae. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1^T, H1^T and B1^T against the genomes of related members in this family were below 67%, which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA– DNA hybridization among the three strains were below 92.0 and 46.6% respectively, which are below the suggested thresholds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1^T=NBRC 111848^T=DSM 103917^T), Serpentinimonas barnesii sp. nov. (type strain H1^T= NBRC 111849^T=DSM 103920^T) and Serpentinimonas maccroryi sp. nov. (type strain B1^T=NBRC 111850^T=DSM 103919^T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

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Continuous culture is an “open”-culture system for the cultivation of microorganisms or cells in which fresh sterilized medium is introduced at a steady flow rate and from which the culture fluid emerges at the same rate. Many types of continuous culture methods exist of which the most common is the chemostat. Chemostats allow for steady-state concentrations of growth-limiting substrates to be maintained at a fixed level in the culture fluid, which results in highly reproducible “steady-state” growth conditions in which changes in cell density, physiological state, and medium composition of the culture are no longer detectable. Growth under nutrient limitation results in submaximal growth rates, which can usually emulate the state of most natural environments far better than what can be achieved with batch cultures. In continuous cultures, nearly all environmental parameters including pH, oxygen tension, population density, and concentration of excretion products can easily be controlled.

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When looking back and wonder how we did it, I became even more aware of how my wanderings in microbiology are all linked, from the start of my PhD with Hans Veldkamp on sulphur-oxidizing bacteria in chemostats. My interests broadened from obligate chemolithoautotrophic bacteria to facultative organisms and the question about the ecological niches of these different metabolic types. The sulphide oxidizing bacteria also may be used to produce elemental sulphur, which can easily be removed from wastewater. This fitted in a long-standing collaboration with Dimitry Sorokin on the ecophysiology and application of alkaliphilic sulphur bacteria. Then came the denitrifying sulphur-oxidizing bacteria and their application to remove sulphide from wastewater, which lead to our interest in nitrate, nitrite and ammonium removal in general. The big surprise was the serendipitous discovery of the ‘anammox’-process, whereby ammonium is anaerobically oxidized to dinitrogen gas with nitrite as electron acceptor. The early days of our anammox research are the main focus of this article, which describes the struggle of growing and identifying the most peculiar bacteria we ever came across. A specialized organelle, the anammoxosome was shown to be responsible for the key ammonium oxidation, whereby a rocket fuel, hydrazine, turned out to be an intermediate. Soon after we became aware that anammox is everywhere and in the marine environment makes up a major portion of the nitrogen cycle. The intense scientific collaboration with Mike Jetten and Mark van Loosdrecht and colleagues led to our further understanding and application of this fascinating process, which is briefly summarized in this article. My broader interest in environmental microbiology and microbial ecology has been a regularly returning theme, taking me all over the world to great collaborations lasting to this very day.

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Reduction of the greenhouse gas N₂O to N₂ is a trait among denitrifying and non-denitrifying microorganisms having an N₂O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N₂O emissions from ecosystems. To increase our understanding of the ecophysiology of N₂O reducers, we determined the thermodynamic growth efficiency of N₂O reduction and the selection of N₂O reducers under N₂O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N₂O as electron acceptor and acetate as electron donor. The biomass yields were higher during N₂O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N₂O. Denitrifiers were favored over non-denitrifying N₂O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N₂O, as defined by μ_max/K_s. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N₂O respiration under growth-limited conditions.

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N₂O is a potent greenhouse gas, but also a potent electron acceptor. In search of thermodynamically favourable – yet undescribed – metabolic pathways involving N₂O reduction, we set up a continuous microbial enrichment, inoculated with activated sludge, fed with N₂O as the sole electron acceptor and acetate as an electron donor. A nitrogen-free mineral medium was used with the intention of creating a selective pressure towards organisms that would use N₂O directly as source of nitrogen for cell synthesis. Instead, we obtained a culture dominated by microorganisms of the Rhodocyclaceae family growing by N₂O reduction to N₂ coupled to N₂ fixation. Biomass yields of this culture were 40% lower than those of a previously reported culture grown under comparable conditions but with an NH⁺ ₄ -amended medium, as expected from the extra energy expense of N₂ fixation. Interestingly, we found no significant difference in yields whether N₂O or acetate was the growth-limiting substrate in the chemostat in contrast to the study with NH⁺ ₄-amended medium, in which biomass yields were roughly 30% lower during acetate limiting conditions.

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Nitrous oxide (N₂O) reducing microorganisms may be key in the mitigation of N₂O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N₂O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (K_s) for N₂O. We compared N₂O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N₂O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N₂O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N₂O, defined by μ_max/K_s, in organisms with nosZ clade II.

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Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=∼12), highly reducing (E h <'550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.

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Denitrification and dissimilatory reduction to ammonium (DNRA) are competing nitrate-reduction processes that entail important biogeochemical consequences for nitrogen retention/removal in natural and man-made ecosystems. The nature of the available carbon source and electron donor have been suggested to play an important role on the outcome of this microbial competition. In this study, the influence of lactate as fermentable carbon source on the competition for nitrate was investigated for varying ratios of lactate and nitrate in the influent (Lac/N ratio). The study was conducted in an open chemostat culture, enriched from activated sludge, under strict anoxia. The mechanistic explanation of the conversions observed was based on integration of results from specific batch tests with biomass from the chemostat, molecular analysis of the biomass enriched, and a computational model. At high Lac/N ratio (2.97 mol/mol) both fermentative and respiratory nitrate reduction to ammonium occurred, coupled to partial oxidation of lactate to acetate, and to acetate oxidation respectively. Remaining lactate was fermented to propionate and acetate. At a decreased Lac/N ratio (1.15 mol/mol), the molar percentage of nitrate reduced to ammonium decreased to 58%, even though lactate was supplied in adequate amounts for full ammonification and nitrate remained the growth limiting compound. Data evaluation at this Lac/N ratio suggested conversions were comparable to the higher Lac/N ratio, except for lactate oxidation to acetate that was coupled to denitrification instead of ammonification. Respiratory DNRA on acetate was likely catalyzed by two Geobacter species related to G. luticola and G. lovleyi. Two Clostridiales members were likely responsible for lactate fermentation and partial lactate fermentation to acetate coupled to fermentative DNRA. An organism related to Propionivibrio militaris was identified as the organism likely responsible for denitrification. The results of this study clearly show that not only the ratio of available substrates, but also the nature of the electron donor influences the outcome of competition between DNRA and denitrification. Apparently, fermentative bacteria are competitive for the electron donor and thereby alter the ratio of available substrates for nitrate reduction.@en

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two microbial processes that compete for oxidized nitrogen compounds in the environment. The objective of this work was to determine the role of nitrite versus nitrate as terminal electron acceptor on the competition between DNRA and denitrification. Initially, a mixed culture chemostat was operated under nitrate limitation and performed DNRA. Stepwise, the influent nitrate was replaced with nitrite until nitrite was the sole electron acceptor and N-source present. Despite changing the electron acceptor from nitrate to nitrite, the dominant process remained DNRA and the same dominant organism closely related to Geobacter lovleyi was identified. Contrary to previous studies conducted with a complex substrate in marine microbial communities, the conclusion of this work is that nitrate versus nitrite as electron acceptor does not generally control the competition between DNRA and denitrification. Our results show that the effect of this ratio must be interpreted in combination with other environmental factors, such as the type and complexity of the electron donor, pH, or sulfide concentrations.@en

Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=∼12), highly reducing (E h <'550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.@en

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.@en

The spring waters trom The Cedars ultra-mafic rocks have extremely high pH (-12) and relatively high concentrations of Ca2+, H2 and CH4 due to the active serpentinization. In addition, the waters contain low concentrations of dissolved carbon and sodium, and undetectable levels of ammonium, phosphate and electron acceptors. This combination of water chemistry is supposed to be very challenging tor life; however, certain Betaproteobacteria have been trequently observed in serpentinization sites globally. Three closely-related, highly-alkaliphilic Betaprotebacterial strains were isolated trom The Cedars springs and we have proposed these as a new genus "Serpentinomonas'. All three strains grow autotrophically with H2 as the electron donor and CaCO3 as the carbon source at high pH (optimum pH 11 ), and the Serpentinomonas strains are numerically dominant in The Cedars and other terrestrial serpentinizing ecosystems. These features indicate that Serpentinomonas strains are highly adapted to the geochemistry of serpentinizing environment. Having the strains in culture has allowed both physiological and genomic studies of the metabolic capabilities of Serpentinomonas strains (Suzuki. et al., 2014}. However it remains unknown how they manage to grow in these harsh environments where low levels of protons and sodium make it extremely difficult to establish a cation motive force, and where high levels of calcium reduce the available phosphate and inorganic carbon significantly. A model alkaliphilic organism, Bacillus pseudofirmus OF4, is known to have a unique W-ATP synthase that is capable of translocating protons into the cytosol at high pH effectively (Preiss Let al., 2013}. However, the proteins in our Serpentinomonas strains lack the specific motif in the c-rings that is proposed to be critica! tor the success of the alkaliphilic Bacilli; therefore, Serpentinomonas strains must have acquired different, as yet unknown, adaptations. Genomic analyses revealed that genome size of Serpentinomonas is significantly smaller (-2.5 Mbp) than those of the most closely related genus Hydrogenophaga species (- 5Mb). To identify proteins involved in alkaliphilic properties of Serpentinomonas strains, we employed a comparative genomics approach that subtracted proteins encoded in the genomes of Serpentinomonas strains trom the shared proteins encoded in the genomes of closely-related neutrophilic Betaproteobacteria. The approach led to the identification of 380 genes that are present exclusively in Serpentinomonas strains. The identified genes mostly encoded proteins tor cation transporters and alkaline phosphatases, and those genes were further shown to be of significance by direct evidence of their transcription in The Cedars springs. Meanwhile Serpentinomonas lost 2386 genes compared with a type genus Hydrogenophaga flava1. The deleted genes include amino acids and sugar transporters, RNA polymerase sigma-70 factor, and fatty acids metabolisms. While studies of alkaliphiles trom calcium-rich highly-alkaline environment are limited, the alkaliphilic mechanisms are likely different from those of alkaliphiles from high salt environment. The genomic features of Serpentinomonas indicate that extremely low availability of proton and phosphate are the major selective forces that drive the genomic evolution of this genus in a serpentinizing ecosystem. @en