Enigmatic persistence of aerobic methanotrophs in oxygen-limiting freshwater habitats.

Methanotrophic bacteria mitigate emissions of the potent greenhouse gas methane (CH4) from a variety of anthropogenic and natural sources, including freshwater lakes, which are large sources of CH4 on a global scale. Despite a dependence on dioxygen (O2) for CH4 oxidation, abundant populations of putatively aerobic methanotrophs have been detected within microoxic and anoxic waters and sediments of lakes. Experimental work has demonstrated active aerobic methanotrophs under those conditions, but how they are able to persist and oxidize CH4 under O2 deficiency remains enigmatic. In this review, we discuss possible mechanisms that underpin the persistence and activity of aerobic methanotrophs under O2-limiting conditions in freshwater habitats, particularly lakes, summarize experimental evidence for microbial oxidation of CH4 by aerobic bacteria under low or no O2, and suggest future research directions to further explore the ecology and metabolism of aerobic methanotrophs in O2-limiting environments.

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.

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.

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.

An overlooked influence of reactive oxygen species on ammonia-oxidizing microbial communities in redox-fluctuating aquifers.

Reactive oxygen species (ROS) are ubiquitous in O2-perturbed aquifers, but their role in shaping ammonia-oxidizing microbial communities is not clear. This study examined the dynamic responses of ammonia-oxidizing microorganisms (AOMs) in redox-fluctuating aquifers to ROS via field investigation and in-lab verification using transcriptomes/ metatranscriptome and RT-qPCR. Ammonia-oxidizing archaea (AOA) dominated recharge aquifers with lower ROS levels, whereas ammonia-oxidizing bacteria (AOB) and heterotrophic nitrifying aerobic bacteria (HNB) predominated in discharge areas with higher ROS levels. Similar succession in AOM enrichments was found in that the dominant AOMs changed from AOA Nitrosopumilus to AOB Nitrosomonas with increasing ROS. Ammonia oxidation and antioxidant capacity differed significantly among three AOM isolates exposed to ROS. ROS decreased the amoA gene expression of AOA strain Nitrososphaera viennensis PLX03, accompanied by inhibited ammonia oxidation capacity. By contrast, the catalase and superoxide dismutase activities of the AOB strain Nitrosomonas oligotropha PLL12 and HNB strain Pseudomonas aeruginosa PLL01 increased, and the antioxidant genes katG, sodA, ahpC, and ahpF were significantly upregulated. These results demonstrate that ROS exert an important influence on AOMs in redox-fluctuating aquifers. This study improves our understanding of the ecological niches of AOMs in surface/subsurface environments.

The influence of self-cycling fermentation long- and short-cycle schemes on Saccharomyces cerevisiae and Escherichia coli.

Self-cycling fermentation (SCF), a cyclic process in which cells, on average, divide once per cycle, has been shown to lead to whole-culture synchronization and improvements in productivity during bioconversion. Previous studies have shown that the completion of synchronized cell replication sometimes occurs simultaneously with depletion of the limiting nutrient. However, cases in which the end of cell doubling occurred before limiting nutrient exhaustion were also observed. In order to better understand the impact of these patterns on bioprocessing, we investigated the growth of Saccharomyces cerevisiae and Escherichia coli in long- and short-cycle SCF strategies. Three characteristic events were identified during SCF cycles: (1) an optimum in control parameters, (2) the time of completion of synchronized cell division, and (3) the depletion or plateau of the limiting nutrient. Results from this study and literature led to the identification of three potential trends in SCF cycles: (A) co-occurrence of the three key events, (B) cell replication ending prior to the co-occurrence of the other two events, and (C) depletion or plateau of the limiting nutrient occurring later than the co-occurrence of the other two events. Based on these observations, microbial physiological differences were analyzed and a novel definition for SCF is proposed.

Microbiome of the freshwater sponge Ephydatia muelleri shares compositional and functional similarities with those of marine sponges.

Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome.

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.

Integrative Genome-Scale Metabolic Modeling Reveals Versatile Metabolic Strategies for Methane Utilization in Methylomicrobium album BG8.

Methylomicrobium album BG8 is an aerobic methanotrophic bacterium with promising features as a microbial cell factory for the conversion of methane to value-added chemicals. However, the lack of a genome-scale metabolic model (GEM) of M. album BG8 has hindered the development of systems biology and metabolic engineering of this methanotroph. To fill this gap, a high-quality GEM was constructed to facilitate a system-level understanding of the biochemistry of M. album BG8. Flux balance analysis, constrained with time-series data derived from experiments with various levels of methane, oxygen, and biomass, was used to investigate the metabolic states that promote the production of biomass and the excretion of carbon dioxide, formate, and acetate. The experimental and modeling results indicated that M. album BG8 requires a ratio of ∼1.5:1 between the oxygen- and methane-specific uptake rates for optimal growth. Integrative modeling revealed that at ratios of >2:1 oxygen-to-methane uptake flux, carbon dioxide and formate were the preferred excreted compounds, while at ratios of <1.5:1 acetate accounted for a larger fraction of the total excreted flux. Our results showed a coupling between biomass production and the excretion of carbon dioxide that was linked to the ratio between the oxygen- and methane-specific uptake rates. In contrast, acetate excretion was experimentally detected during exponential growth only when the initial biomass concentration was increased. A relatively lower growth rate was also observed when acetate was produced in the exponential phase, suggesting a trade-off between biomass and acetate production. IMPORTANCE A genome-scale metabolic model (GEM) is an integrative platform that enables the incorporation of a wide range of experimental data. It is used to reveal system-level metabolism and, thus, clarify the link between the genotype and phenotype. The lack of a GEM for Methylomicrobium album BG8, an aerobic methane-oxidizing bacterium, has hindered its use in environmental and industrial biotechnology applications. The diverse metabolic states indicated by the GEM developed in this study demonstrate the versatility in the methane metabolic processes used by this strain. The integrative GEM presented here will aid the implementation of the design-build-test-learn paradigm in the metabolic engineering of M. album BG8. This advance will facilitate the development of a robust methane bioconversion platform and help to mitigate methane emissions from environmental systems.

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 analysis of synchrony and productivity in self-cycling fermentation of engineered yeast producing shikimic acid.

Industrial fermentation provides a wide variety of bioproducts, such as food, biofuels and pharmaceuticals. Self-cycling fermentation (SCF), an advanced automated semi-continuous fermentation approach, has shown significant advantages over batch reactors (BR); including cell synchrony and improved production. Here, Saccharomyces cerevisiae engineered to overproduce shikimic acid was grown under SCF operation. This led to four-fold increases in product yield and volumetric productivity compared to BR. Transcriptomic analyses were performed to understand the cellular mechanisms leading to these increases. Results indicate an up-regulation of a large number of genes related to the cell cycle and DNA replication in the early stages of SCF cycles, inferring substantial synchronization. Moreover, numerous genes related to gluconeogenesis, the citrate cycle and oxidative phosphorylation were significantly up-regulated in the late stages of SCF cycles, consistent with significant increases in shikimic acid yield and productivity.

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.

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.

Metabolome profiles of the alphaproteobacterial methanotroph Methylocystis sp. Rockwell in response to carbon and nitrogen source.

Methanotrophs use methane as a sole carbon source and thus play a critical role in its global consumption. Intensified interest in methanotrophs for their low-cost production of value-added products and large-scale industrialization has led to investigations of strain-to-strain variation in parameters for growth optimization and metabolic regulation. In this study, Methylocystis sp. Rockwell was grown with methane or methanol as a carbon source and ammonium or nitrate as a nitrogen source. The intracellular metabolomes and production of polyhydroxybutyrate, a bioplastic precursor, were compared among treatments to determine how the different combinations of carbon and nitrogen sources affected metabolite production. The methane-ammonium condition resulted in the highest growth, followed by the methane-nitrate, methanol-nitrate and methanol-ammonium conditions. Overall, the methane-ammonium and methane-nitrate conditions directed metabolism toward energy-conserving pathways, while methanol-ammonium and methanol-nitrate directed the metabolic response toward starvation pathways. Polyhydroxybutyrate was produced at greater abundances in methanol-grown cells, independent of the nitrogen source. Together, the results revealed how Methylocystis sp. Rockwell altered its metabolism with different combinations of carbon and nitrogen source, with implications for production of industrially relevant metabolites.

An altered microbiome in urban coyotes mediates relationships between anthropogenic diet and poor health.

Generalist species able to exploit anthropogenic food sources are becoming increasingly common in urban environments. Coyotes (Canis latrans) are one such urban generalist that now resides in cities across North America, where diseased or unhealthy coyotes are frequently reported in cases of human-wildlife conflict. Coyote health and fitness may be related to habitat use and diet via the gut microbiome, which has far-reaching effects on animal nutrition and physiology. In this study, we used stomach contents, stable isotope analysis, 16S rRNA gene amplicon sequencing, and measures of body condition to identify relationships among habitat use, diet, fecal microbiome composition, and health in urban and rural coyotes. Three distinct relationships emerged: (1) Urban coyotes consumed more anthropogenic food, which was associated with increased microbiome diversity, higher abundances of Streptococcus and Enterococcus, and poorer average body condition. (2) Conversely, rural coyotes harbored microbiomes rich in Fusobacteria, Sutterella, and Anaerobiospirillum, which were associated with protein-rich diets and improved body condition. (3) Diets rich in anthropogenic food were associated with increased abundances of Erysipelotrichiaceae, Lachnospiraceae, and Coriobacteriaceae, which correlated with larger spleens in urban coyotes. Urban coyotes also had an increased prevalence of the zoonotic parasite Echinococcus multilocularis, but there were no detectable connections between parasite infection and microbiome composition. Our results demonstrate how the consumption of carbohydrate-rich anthropogenic food by urban coyotes alters the microbiome to negatively affect body condition, with potential relationships to parasite susceptibility and conflict-prone behavior.