Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle.

Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.

Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

Positron-emitting radiotracers spatially resolve unexpected biogeochemical relationships linked with methane oxidation in Arctic soils.

Arctic soils are marked by cryoturbic features, which impact soil-atmosphere methane (CH4 ) dynamics vital to global climate regulation. Cryoturbic diapirism alters C/N chemistry within frost boils by introducing soluble organic carbon and nutrients, potentially influencing microbial CH4 oxidation. CH4 oxidation in soils, however, requires a spatio-temporal convergence of ecological factors to occur. Spatial delineation of microbial activity with respect to these key microbial and biogeochemical factors at relevant scales is experimentally challenging in inherently complex and heterogeneous natural soil matrices. This work aims to overcome this barrier by spatially linking microbial CH4 oxidation with C/N chemistry and metagenomic characteristics. This is achieved by using positron-emitting radiotracers to visualize millimeter-scale active CH4 uptake areas in Arctic soils with and without diapirism. X-ray absorption spectroscopic speciation of active and inactive areas shows CH4 uptake spatially associates with greater proportions of inorganic N in diapiric frost boils. Metagenomic analyses reveal Ralstonia pickettii associates with CH4 uptake across soils along with pertinent CH4 and inorganic N metabolism associated genes. This study highlights the critical relationship between CH4 and N cycles in Arctic soils, with potential implications for better understanding future climate. Furthermore, our experimental framework presents a novel, widely applicable strategy for unraveling ecological relationships underlying greenhouse gas dynamics under global change.

In vivo quantification of polyhydroxybutyrate (PHB) in the alphaproteobacterial methanotroph, Methylocystis sp. Rockwell.

Methane is a common industrial by-product that can be used as feedstock for production of the biopolymer polyhydroxybutyrate (PHB) by alphaproteobacterial methanotrophs. In vivo assessment of PHB production would shed light on the biosynthesis process and guide design of improved production strategies, but it is currently difficult to perform efficiently. In this study, the alphaproteobacterial methanotroph Methylocystis sp. Rockwell was grown on methane with three different nitrogen sources (ammonium, nitrate, and atmospheric nitrogen), and biomass samples were harvested at defined time points during lag, exponential, and stationary growth phases. PHB cell content was analyzed at these sampling points via a standard gas chromatography-flame ionization detector method, which requires hydrolysis of PHB and esterification of the resulting monomer under acidic conditions, and a novel, rapid, cost-effective approach based on fixation and staining of bacterial cells via Nile Blue A fluorescent dye enabling differential staining of cell membranes and intracellular PHB granules for single-cell analysis through fluorescence microscopy. Overall, the two PHB quantification approaches were in agreement at all stages of growth and in all three growing conditions tested. The PHB cell content was greatest with atmospheric nitrogen as a nitrogen source, followed by ammonium and nitrate. Under atmospheric nitrogen and ammonium conditions, PHB cell content decreased with growth progression, while under nitrate conditions PHB cell content remained unchanged in all growth phases. In addition to presenting a rapid, efficient method enabling in vivo quantification of PHB production, the present study highlights the impact of nitrogen source on PHB production by Methylocystis sp. Rockwell. KEY POINTS: • A novel fluorescence microscopy method to quantify PHB in single cells was developed • The microscopy method was validated by the derivation/gas chromatography method • Methylocystis sp. Rockwell synthesizes PHB granules without nutrient stress.

Transcriptomic and Metabolomic Responses to Carbon and Nitrogen Sources in Methylomicrobium album BG8.

Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled the phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated hydroxylamine dehydrogenase (haoAB) and overall central metabolic activity, whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCE Methanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism vary across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

Individual and Site-Specific Variation in a Biogeographical Profile of the Coyote Gastrointestinal Microbiota.

Most knowledge of the vertebrate gut microbiota comes from fecal samples; due to difficulties involved in sample collection, the upper intestinal microbiota is poorly understood in wild animals despite its potential to inform broad interpretations about host-gut microbe relationships under natural conditions. Here, we used 16S rRNA gene sequencing to characterize the microbiota of wild coyotes (Canis latrans) along the gastrointestinal tract, including samples from the duodenum, jejunum, ileum, caecum, ascending and descending colon, and feces. We used this intestinal profile to (1) quantify how intestinal site and individual identity interact to shape the microbiota in an uncontrolled setting, and (2) evaluate whether the fecal microbiota adequately represent other intestinal sites. Microbial communities in the large intestine were distinct from those in the small intestine, with higher diversity and a greater abundance of anaerobic taxa. Within each of the small and large intestine, individual identity explained significantly more among-sample variation than specific intestinal sites, revealing the importance of individual variation in the microbiota of free-living animals. Fecal samples were not an adequate proxy for studying upper intestinal environments, as they contained only half the amplicon sequence variants (ASVs) present in the small intestine at three- to four-fold higher abundances. Our study is a unique biogeographical investigation of the microbiota using free-living mammals rather than livestock or laboratory organisms and provides a foundational understanding of the gastrointestinal microbiota in a wild canid.

Development of an aquaponics microbial inoculum for efficient nitrification at acidic pH.

Maintaining an optimal pH that simultaneously supports plants, fish, and nitrifying microorganisms is a challenge in recirculating aquaponics systems as nitrification is optimal at a slightly alkaline pH and plant growth is optimal at a slightly acidic pH. Freshwater fish tolerate pH > 5.5. Our aim was to adapt a microbial inoculum for a recirculating aquaponics system from an operational pH of 7.6 to 5.6, compare nitrification activity and production of N2O, and describe changes in the adapted versus unadapted microbial communities. Four adaptation strategies were tested; our results indicated that a gradual reduction from pH 7.6 to 5.6, along with a gradual reduction followed by a gradual return of available ammonium, was the best strategy resulting in retention of 81% nitrification activity at pH 5.6 compared to pH 7.6. 16S rRNA gene amplicon sequencing and qPCR enumeration of nitrification-related genes showed that the composition of pH 5.6 adapted microbial communities from all four adaptation strategies was similar to one another and distinct from those operating at pH 7.6, with enrichment of comammox clade B bacteria over ammonia-oxidizing bacteria and thaumarchaeota. N2O production of the pH 5.6 adapted microbial communities was below detection in all adaptation experiments, likely due to the increased proportion of comammox bacteria. Aquaponics biofilters enriched with comammox bacteria and adapted to function at pH 5.6 can be a desirable inoculum for freshwater recirculating aquaponics systems to retain nitrification activity and improve crop yields.Key points• Microbial communities adapted from pH 7.6 to pH 5.6 retained 81% nitrification activity.• Microbial communities adapted from pH 7.6 to pH 5.6 were enriched in comammox bacteria.• Comammox-enriched microbial communities did not produce N2 O.

A Physiological and Genomic Comparison of Nitrosomonas Cluster 6a and 7 Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) within the genus Nitrosomonas perform the first step in nitrification, ammonia oxidation, and are found in diverse aquatic and terrestrial environments. Nitrosomonas AOB were grouped into six defined clusters, which correlate with physiological characteristics that contribute to adaptations to a variety of abiotic environmental factors. A fundamental physiological trait differentiating Nitrosomonas AOB is the adaptation to either low (cluster 6a) or high (cluster 7) ammonium concentrations. Here, we present physiological growth studies and genome analysis of Nitrosomonas cluster 6a and 7 AOB. Cluster 6a AOB displayed maximum growth rates at ≤ 1 mM ammonium, while cluster 7 AOB had maximum growth rates at ≥ 5 mM ammonium. In addition, cluster 7 AOB were more tolerant of high initial ammonium and nitrite concentrations than cluster 6a AOB. Cluster 6a AOB were completely inhibited by an initial nitrite concentration of 5 mM. Genomic comparisons were used to link genomic traits to observed physiological adaptations. Cluster 7 AOB encode a suite of genes related to nitrogen oxide detoxification and multiple terminal oxidases, which are absent in cluster 6a AOB. Cluster 6a AOB possess two distinct forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and select species encode genes for hydrogen or urea utilization. Several, but not all, cluster 6a AOB can utilize urea as a source of ammonium. Hence, although Nitrosomonas cluster 6a and 7 AOB have the capacity to fulfill the same functional role in microbial communities, i.e., ammonia oxidation, differentiating species-specific and cluster-conserved adaptations is crucial in understanding how AOB community succession can affect overall ecosystem function.

Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra".

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1.

Obligate methanotrophs belonging to the Phyla Proteobacteria and Verrucomicrobia require oxygen for respiration and methane oxidation; nevertheless, aerobic methanotrophs are abundant and active in low oxygen environments. While genomes of some aerobic methanotrophs encode putative nitrogen oxide reductases, it is not understood whether these metabolic modules are used for NOx detoxification, denitrification or other purposes. Here we demonstrate using microsensor measurements that a gammaproteobacterial methanotroph Methylomonas denitrificans sp. nov. strain FJG1(T) couples methane oxidation to nitrate reduction under oxygen limitation, releasing nitrous oxide as a terminal product. Illumina RNA-Seq data revealed differential expression of genes encoding a denitrification pathway previously unknown to methanotrophs as well as the pxmABC operon in M. denitrificans sp. nov. strain FJG1(T) in response to hypoxia. Physiological and transcriptome data indicate that genetic inventory encoding the denitrification pathway is upregulated only upon availability of nitrate under oxygen limitation. In addition, quantitation of ATP levels demonstrates that the denitrification pathway employs inventory such as nitrate reductase NarGH serving M. denitrificans sp. nov. strain FJG1(T) to conserve energy during oxygen limitation. This study unravelled an unexpected metabolic flexibility of aerobic methanotrophs, thereby assigning these bacteria a new role at the metabolic intersection of the carbon and nitrogen cycles.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Revision of N2O-producing pathways in the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC 19718.

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2 tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2 (-)) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2 tensions. Anoxic resting-cell assays and measurements of instantaneous NO2 (-) reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.

Agritech to Tame the Nitrogen Cycle.

While the Haber-Bosch process for N-fixation has enabled a steady food supply for half of humanity, substantial use of synthetic fertilizers has caused a radical unevenness in the global N-cycle. The resulting increases in nitrate production and greenhouse gas (GHG) emissions have contributed to eutrophication of both ground and surface waters, the growth of oxygen minimum zones in coastal regions, ozone depletion, and rising global temperatures. As stated by the Food and Agriculture Organization of the United Nations, agriculture releases ∼9.3 Gt CO2 equivalents per year, of which methane (CH4) and nitrous oxide (N2O) account for 5.3 Gt CO2 equivalents. N-pollution and slowing the runaway N-cycle requires a combined effort to replace chemical fertilizers with biological alternatives, which after a 10-yr span of usage could eliminate a minimum of 30% of ag-related GHG emissions (∼1.59 Gt), protect waterways from nitrate pollution, and protect soils from further deterioration. Agritech solutions include bringing biological fertilizers and biological nitrification inhibitors to the marketplace to reduce the microbial conversion of fertilizer nitrogen into GHGs and other toxic intermediates. Worldwide adoption of these plant-derived molecules will substantially elevate nitrogen use efficiency by crops while blocking the dominant source of N2O to the atmosphere and simultaneously protecting the biological CH4 sink. Additional agritech solutions to curtail N-pollution, soil erosion, and deterioration of freshwater supplies include soil-free aquaponics systems that utilize improved microbial inocula to enhance nitrogen use efficiency without GHG production. With adequate and timely investment and scale-up, microbe-based agritech solutions emphasizing N-cycling processes can dramatically reduce GHG emissions on short time lines.

Greenhouse gas mitigation requires caution.

Strategies to mitigate emissions must consider methane and nitrous oxide together.

Inoculum and pH effects on ammonium removal and microbial community dynamics in aquaponics systems.

Understanding the ecology of microorganisms is essential for optimizing aquaponics systems. Effects of pH and inoculum on ammonium removal and dynamics of microbial community composition from all compartments of lab-scale aquaponics systems were examined. Initial ammonium accumulation in systems with comammox-enriched inocula were 47% and 69% that of systems enriched with ammonia-oxidizing bacteria (AOB), with higher rates of ammonium removal and transient nitrite accumulation measured in the latter systems. By the end of operation, Nitrosomonas and Nitrosospira AOB were dominant nitrifiers in systems at pH 7.6-7.8, whereas comammox (Nitrospira) nitrifiers and plant growth-promoting microbes were abundant in systems operating at pH 5.8-6.0. Lower pH systems supported more robust plant growth with no significant effects on fish health. This study demonstrated functional redundancy of aquaponics microbiota, with selectivity of nitrifying taxa as a function of pH. The results suggest that inoculum and pH are important considerations for aquaponics system initiation and optimization.

Leveraging genome-scale metabolic models to understand aerobic methanotrophs.

Genome-scale metabolic models (GEMs) are valuable tools serving systems biology and metabolic engineering. However, GEMs are still an underestimated tool in informing microbial ecology. Since their first application for aerobic gammaproteobacterial methane oxidizers less than a decade ago, GEMs have substantially increased our understanding of the metabolism of methanotrophs, a microbial guild of high relevance for the natural and biotechnological mitigation of methane efflux to the atmosphere. Particularly, GEMs helped to elucidate critical metabolic and regulatory pathways of several methanotrophic strains, predicted microbial responses to environmental perturbations, and were used to model metabolic interactions in cocultures. Here, we conducted a systematic review of GEMs exploring aerobic methanotrophy, summarizing recent advances, pointing out weaknesses, and drawing out probable future uses of GEMs to improve our understanding of the ecology of methane oxidizers. We also focus on their potential to unravel causes and consequences when studying interactions of methane-oxidizing bacteria with other methanotrophs or members of microbial communities in general. This review aims to bridge the gap between applied sciences and microbial ecology research on methane oxidizers as model organisms and to provide an outlook for future studies.