Grand Challenges in Terrestrial Microbiology

Understanding the functional role of different microbial populations is essential to ascertain whether environmental factors affecting their diversity, activity, and physiology will impact the functioning of terrestrial ecosystems. The soil environment represents one of the largest reservoirs of microbes in the biosphere and is the most significant in linking the activity of humans with the interaction and alteration of the major biogeochemical cycles. The global nitrogen cycle has been massively accelerated through the annual removal of over 100 Tg of atmospheric nitrogen for the production and use of fertilizers (Grube

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity

Production, isotopic composition, and atmospheric fate of biologically produced nitrous oxide

The anthropogenic production of greenhouse gases and their consequent effects on global climate have garnered international attention for years. A remaining challenge facing scientists is to unambiguously quantify both sources and sinks of targeted gases. Microbiological metabolism accounts for the largest source of nitrous oxide (N₂O), mostly due to global conversion of land for agriculture and massive usage of nitrogen-based fertilizers. A most powerful method for characterizing the sources of N₂O lies in its multi-isotope signature. This review summarizes mechanisms that lead to biological N₂O production and how discriminate placement of ¹⁵N into molecules of N₂O occurs. Through direct measurements and atmospheric modeling, we can now place a constraint on the isotopic composition of biological sources of N₂O and trace its fate in the atmosphere. This powerful interdisciplinary combination of biology and atmospheric chemistry is rapidly advancing the closure of the global N₂O budget

Control of Defined Methanotrophic Populations in Soils by Co-metabolism of Ammonium

Summary Metabolism of inorganic nitrogen (N) by soil microbial communities is heavily impacted by increasing N-loads from anthropogenic sources such as fertilizers and nitrogenous air pollutants. Nitrification, the oxidation of ammonia-N to nitrite/nitrate-N, and denitrification, the reduction of nitrite/nitrate-N to nitrogen oxides and dinitrogen, are well-characterized processes. Likewise, microbial communities performing these processes have been intensively studied. Less well characterized are methane-oxidizing bacteria (MOB), which predominantly convert methane to carbon dioxide, in their capacity to perform both nitrification and partial denitrification in terrestrial ecosystems. In this project we: 1) compared growth kinetics of four methanotrophic bacterial strains in media with ammonia versus nitrate as the N source, 2) examined the capacity of each strain to oxidize ammonia and hydroxylamine (the intermediate of ammonia oxidation) to nitrite, 3) examined the influence of ammonia and nitrite on methane oxidation potential, 4) determined differences in methane-oxidizing enzymes that could account for differences in ammonia oxidation rates, and 5) identified a hydroxylamine oxidoreductase homologue in one strain. The ultimate goals of this project were to: 1) determine the point at which ammonia (or nitrite) becomes a deterrent rather than a benefit to methane oxidation, and 2) characterize the enzymatic components in diverse MOB that oxidize ammonia to nitrite via hydroxylamine. We discovered that MOB respond very differently to ammonia; while the bacteria all grew efficiently with ammonia as an N-source, they had significantly different capacities for oxidizing ammonia to nitrite. This difference was not attributable to differences in pmoA gene sequences that encode the catalytic subunit of methane monooxygenase. While three of the four isolates could oxidize ammonia to nitrite via hydroxylamine, only one of the three was found to have a conserved gene encoding hydroxylamine oxidoreductase. This study demonstrated for the first time that not all MOB are capable of dissimilatory ammonia oxidation nor do they all have identifiable gene inventories to carry out ammonia oxidation to nitrite. The capacity for MOB to co-metabolize ammonia rather than assimilate it, especially in N-impacted soils, influences the composition and fitness of the MOB community, which in turn determines the methane oxidizing capacity of soils. Objectives Objective 1: We grew cultivated methanotrophic species in AMS (ammonium mineral salts) and NMS (nitrate mineral salts) media (30% CH 4 ) and monitored methane, carbon dioxide, nitrous oxide, and nitrite concentrations in addition to cell numbers from lag to stationary phase. Objective 2: We determined the kinetics of ammonia oxidation to nitrite by each species in the absence and presence of reductant. As co-metabolism by methane monooxygenase require

Defining Nutrient Combinations for Optimal Growth and Polyhydroxybutyrate Production by Methylosinus trichosporium OB3b Using Response Surface Methodology

Methane and methanol are common industrial by-products that can be used as feedstocks for the production of value-added products by methylotrophic bacteria. Alphaproteobacterial methanotrophs are known to produce and accumulate the biopolymer polyhydroxybutyrate (PHB) under conditions of nutrient starvation. The present study determined optimal production of biomass and PHB by Methylosinus trichosporium OB3b as a function of carbon source (methane or methanol), nitrogen source (ammonium or nitrate), and nitrogen-to-carbon ratio during growth. Statistical regression analysis with interactions was performed to assess the importance of each factor, and their respective interactions, on biomass and PHB production. Higher biomass concentrations were obtained with methane as carbon source and with ammonium as nitrogen source. The nitrogen source that favored PHB production was ammonium for methane-grown cells and nitrate for methanol-grown cells. Response surface methodology (RSM) was used to determine conditions leading to optimal biomass and PHB production. As an example, the optimal PHB concentration was predicted to occur when a mixture of 30% methane and 70% methanol (molar basis) was used as carbon source with nitrate as nitrogen source and a nitrogen-to-carbon molar ratio of 0.017. This was confirmed experimentally, with a PHB concentration of 48.7 ± 8.3 mg/L culture, corresponding to a cell content of 52.5 ± 6.3% (cell dry weight basis). Using RSM to simultaneously interrogate multiple variables toward optimized growth and production of biopolymer serves as a guide for establishing more efficient industrial conditions to convert single-carbon feedstocks into value-added products

Combined Effects of Carbon and Nitrogen Source to Optimize Growth of Proteobacterial Methanotrophs

Methane, a potent greenhouse gas, and methanol, commonly called wood alcohol, are common by-products of modern industrial processes. They can, however, be consumed as a feedstock by bacteria known as methanotrophs, which can serve as useful vectors for biotransformation and bioproduction. Successful implementation in industrial settings relies upon efficient growth and bioconversion, and the optimization of culturing conditions for these bacteria remains an ongoing effort, complicated by the wide variety of characteristics present in the methanotroph culture collection. Here, we demonstrate the variable growth outcomes of five diverse methanotrophic strains – Methylocystis sp. Rockwell, Methylocystis sp. WRRC1, Methylosinus trichosporium OB3b, Methylomicrobium album BG8, and Methylomonas denitrificans FJG1 – grown on either methane or methanol, at three different concentrations, with either ammonium or nitrate provided as nitrogen source. Maximum optical density (OD), growth rate, and biomass yield were assessed for each condition. Further metabolite and fatty acid methyl ester (FAME) analyses were completed for Methylocystis sp. Rockwell and M. album BG8. The results indicate differential response to these growth conditions, with a general preference for ammonium-based growth over nitrate, except for M. denitrificans FJG1. Methane is also preferred by most strains, with methanol resulting in unreliable or inhibited growth in all but M. album BG8. Metabolite analysis points to monitoring of excreted formic acid as a potential indicator of adverse growth conditions, while the magnitude of FAME variation between conditions may point to strains with broader substrate tolerance. These findings suggest that methanotroph strains must be carefully evaluated before use in industry, both to identify optimal conditions and to ensure the strain selected is appropriate for the process of interest. Much work remains in addressing the optimization of growth strategies for these promising microorganisms since disregarding these important steps in process development could ultimately lead to inefficient or failed bioprocesses

Phylogenomic Analysis of the Gammaproteobacterial Methanotrophs (Order Methylococcales) Calls for the Reclassification of Members at the Genus and Species Levels

The order Methylococcales constitutes the methanotrophs – bacteria that can metabolize methane, a potent greenhouse gas, as their sole source of energy. These bacteria are significant players in the global carbon cycle and can produce value-added products from methane, such as biopolymers, biofuels, and single-cell proteins for animal feed, among others. Previous studies using single-gene phylogenies have shown inconsistencies in the currently established taxonomic structure of this group. This study aimed to determine and resolve these issues by using whole-genome sequence analyses. Phylogenomic analysis and the use of similarity indexes for genomic comparisons – average amino acid identity, digital DNA–DNA hybridization (dDDH), and average nucleotide identity (ANI) – were performed on 91 Methylococcales genomes. Results suggest the reclassification of members at the genus and species levels. Firstly, to resolve polyphyly of the genus Methylomicrobium, Methylomicrobium alcaliphilum, “Methylomicrobium buryatense,” Methylomicrobium japanense, Methylomicrobium kenyense, and Methylomicrobium pelagicum are reclassified to a newly proposed genus, Methylotuvimicrobium gen. nov.; they are therefore renamed to Methylotuvimicrobium alcaliphilum comb. nov., “Methylotuvimicrobium buryatense” comb. nov., Methylotuvimicrobium japanense comb. nov., Methylotuvimicrobium kenyense comb. nov., and Methylotuvimicrobium pelagicum comb. nov., respectively. Secondly, due to the phylogenetic affinity and phenotypic similarities of Methylosarcina lacus with Methylomicrobium agile and Methylomicrobium album, the reclassification of the former species to Methylomicrobium lacus comb. nov. is proposed. Thirdly, using established same-species delineation thresholds (70% dDDH and 95% ANI), Methylobacter whittenburyi is proposed to be a later heterotypic synonym of Methylobacter marinus (89% dDDH and 99% ANI). Also, the effectively but not validly published “Methylomonas denitrificans” was identified as Methylomonas methanica (92% dDDH and 100% ANI), indicating that the former is a later heterotypic synonym of the latter. Lastly, strains MC09, R-45363, and R-45371, currently identified as M. methanica, each represent a putative novel species of the genus Methylomonas (21–35% dDDH and 74–88% ANI against M. methanica) and were reclassified as Methylomonas sp. strains. It is imperative to resolve taxonomic inconsistencies within this group, first and foremost, to avoid confusion with ecological and evolutionary interpretations in subsequent studies

Draft genome sequences of gammaproteobacterial methanotrophs isolated from marine ecosystems

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems

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

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems

The genomes of the aerobic methanotrophs “Methyloterricola oryzae” strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum