A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

How and why in microbial ecology: An appeal for scientific aims, questions, hypotheses and theories.

This article precedes a series of articles on the important questions, hypotheses and theories in microbial ecology. It considers why, as scientists, we ask questions and propose hypotheses and what makes them important, good or significant. Emphasis is placed on 'scientific' questions, the need for scientific aims and on possible reasons for, and inadequacy of aim-less studies and question free studies. Current global issues surrounding the climate crisis, pandemics and antibiotic resistance focus attention on science and scientists. They exemplify the urgent need for greater understanding of the interactions between microbes and their biological and physicochemical environments, that is, of microbial ecology. They also provide examples of reaction against science and scientists and highlight why we must be clear regarding what defines (good) science, its power and limitations, and ensure that this is communicated to stakeholders and the general public.

Chronic Environmental Perturbation Influences Microbial Community Assembly Patterns.

Acute environmental perturbations are reported to induce deterministic microbial community assembly, while it is hypothesized that chronic perturbations promote development of alternative stable states. Such acute or chronic perturbations strongly impact on the pre-adaptation capacity to the perturbation. To determine the importance of the level of microbial pre-adaptation and the community assembly processes following acute or chronic perturbations in the context of hydrocarbon contamination, a model system of pristine and polluted (hydrocarbon-contaminated) sediments was incubated in the absence or presence (discrete or repeated) of hydrocarbon amendment. The community structure of the pristine sediments changed significantly following acute perturbation, with selection of different phylotypes not initially detectable. Conversely, historically polluted sediments maintained the initial community structure, and the historical legacy effect of chronic pollution likely facilitated community stability. An alternative stable state was also reached in the pristine sediments following chronic perturbation, further demonstrating the existence of a legacy effect. Finally, ecosystem functional resilience was demonstrated through occurrence of hydrocarbon degradation by different communities in the tested sites, but the legacy effect of perturbation also strongly influenced the biotic response. This study therefore demonstrates the importance of perturbation chronicity on microbial community assembly processes and reveals ecosystem functional resilience following environmental perturbation.

Differential Ecosystem Function Stability of Ammonia-Oxidizing Archaea and Bacteria following Short-Term Environmental Perturbation.

Rapidly expanding conversion of tropical forests to oil palm plantations in Southeast Asia leads to soil acidification following intensive nitrogen fertilization. Changes in soil pH are predicted to have an impact on archaeal ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and complete (comammox) ammonia oxidizers and, consequently, on nitrification. It is therefore critical to determine whether the predicted effects of pH on ammonia oxidizers and nitrification activity apply in tropical soils subjected to various degrees of anthropogenic activity. This was investigated by experimental manipulation of pH in soil microcosms from a land-use gradient (forest, riparian, and oil palm soils). The nitrification rate was greater in forest soils with native neutral pH than in converted acidic oil palm soils. Ammonia oxidizer activity decreased following acidification of the forest soils but increased after liming of the oil palm soils, leading to a trend of a reversed net nitrification rate after pH modification. AOA and AOB nitrification activity was dependent on pH, but AOB were more sensitive to pH modification than AOA, which demonstrates a greater stability of AOA than AOB under conditions of short-term perturbation. In addition, these results predict AOB to be a good bioindicator of nitrification response following pH perturbation during land-use conversion. AOB and/or comammox species were active in all soils along the land-use gradient, even, unexpectedly, under acidic conditions, suggesting their adaptation to native acidic or acidified soils. The present study therefore provided evidence for limited stability of soil ammonia oxidizer activity following intensive anthropogenic activities, which likely aggravates the vulnerability of nitrogen cycle processes to environmental disturbance.IMPORTANCE Physiological and ecological studies have provided evidence for pH-driven niche specialization of ammonia oxidizers in terrestrial ecosystems. However, the functional stability of ammonia oxidizers following pH change has not been investigated, despite its importance in understanding the maintenance of ecosystem processes following environmental perturbation. This is particularly true after anthropogenic perturbation, such as the conversion of tropical forest to oil palm plantations. This study demonstrated a great impact of land-use conversion on nitrification, which is linked to changes in soil pH due to common agricultural practices (intensive fertilization). In addition, the different communities of ammonia oxidizers were differently affected by short-term pH perturbations, with implications for future land-use conversions but also for increased knowledge of associated global nitrous oxide emissions and current climate change concerns.

Putting science back into microbial ecology: a question of approach.

Microbial ecology, the scientific study of interactions between natural microbial communities and their environments, has been facilitated by the application of molecular and 'omics'-based techniques that overcome some of the limitations of cultivation-based studies. This has increased emphasis on community ecology and 'microbiome' studies, but the majority address technical, rather than scientific challenges. Most are descriptive, do not address scientific aims or questions and are not designed to increase understanding or test hypotheses. The term 'hypothesis' is increasingly misused and critical testing of ideas or theory is restricted to a small minority of studies. This article discusses current microbial ecology research within the context of four approaches: description, induction, inference to the best explanation and deduction. The first three of these do not follow the established scientific method and are not based on scientific ecological questions. Observations are made and sometimes compared with published data, sometimes with attempts to explain findings in the context of existing ideas or hypotheses, but all lack objectivity and are biased by the observations made. By contrast, deductive studies address ecological questions and attempt to explain currently unexplained phenomena through the construction of hypotheses, from mechanism-based assumptions, that generate predictions that are then tested experimentally. Identification of key scientific questions, research driven by meaningful hypotheses and adoption of scientific method are essential for progress in microbial ecology, rather than the current emphasis on descriptive approaches that address only technical challenges. It is, therefore, imperative that we carefully consider and define the fundamental scientific questions that drive our own research and focus on ideas, concepts and hypotheses that can increase understanding, and only then consider which techniques are required for experimental testing. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Nitrous oxide production by ammonia oxidizers: Physiological diversity, niche differentiation and potential mitigation strategies.

Oxidation of ammonia to nitrite by bacteria and archaea is responsible for global emissions of nitrous oxide directly and indirectly through provision of nitrite and, after further oxidation, nitrate to denitrifiers. Their contributions to increasing N2 O emissions are greatest in terrestrial environments, due to the dramatic and continuing increases in use of ammonia-based fertilizers, which have been driven by requirement for increased food production, but which also provide a source of energy for ammonia oxidizers (AO), leading to an imbalance in the terrestrial nitrogen cycle. Direct N2 O production by AO results from several metabolic processes, sometimes combined with abiotic reactions. Physiological characteristics, including mechanisms for N2 O production, vary within and between ammonia-oxidizing archaea (AOA) and bacteria (AOB) and comammox bacteria and N2 O yield of AOB is higher than in the other two groups. There is also strong evidence for niche differentiation between AOA and AOB with respect to environmental conditions in natural and engineered environments. In particular, AOA are favored by low soil pH and AOA and AOB are, respectively, favored by low rates of ammonium supply, equivalent to application of slow-release fertilizer, or high rates of supply, equivalent to addition of high concentrations of inorganic ammonium or urea. These differences between AOA and AOB provide the potential for better fertilization strategies that could both increase fertilizer use efficiency and reduce N2 O emissions from agricultural soils. This article reviews research on the biochemistry, physiology and ecology of AO and discusses the consequences for AO communities subjected to different agricultural practices and the ways in which this knowledge, coupled with improved methods for characterizing communities, might lead to improved fertilizer use efficiency and mitigation of N2 O emissions.

Genome Sequence of "Candidatus Nitrosocosmicus franklandus" C13, a Terrestrial Ammonia-Oxidizing Archaeon.

"Candidatus Nitrosocosmicus franklandus" C13 is an ammonia-oxidizing archaeon (AOA) isolated from soil. Its complete genome is 2.84 Mb and possesses predicted AOA metabolic pathways for energy generation and carbon dioxide fixation but no typical surface layer (S-layer) proteins, only one ammonium transporter, and divergent A-type ATP synthase genes.

The application of high-throughput sequencing technology to analysis of amoA phylogeny and environmental niche specialisation of terrestrial bacterial ammonia-oxidisers.

BACKGROUND: Characterisation of microbial communities increasingly involves use of high throughput sequencing methods (e.g. MiSeq Illumina) that amplify relatively short sequences of 16S rRNA or functional genes, the latter including ammonia monooxygenase subunit A (amoA), a key functional gene for ammonia oxidising bacteria (AOB) and archaea (AOA). The availability of these techniques, in combination with developments in phylogenetic methodology, provides the potential for better analysis of microbial niche specialisation. This study aimed to develop an approach for sequencing of bacterial and archaeal amoA genes amplified from soil using bioinformatics pipelines developed for general analysis of functional genes and employed sequence data to reassess phylogeny and niche specialisation in terrestrial bacterial ammonia oxidisers. RESULTS: amoA richness and community composition differed with bioinformatics approaches used but analysis of MiSeq sequences was reliable for both archaeal and bacterial amoA genes and was used for subsequent assessment of potential niche specialisation of soil bacteria ammonia oxidisers. Prior to ecological analysis, phylogenetic analysis of Nitrosospira, which dominates soil AOB, was revisited using a phylogenetic analysis of 16S rRNA and amoA genes in available AOB genomes. This analysis supported congruence between phylogenies of the two genes and increased previous phylogenetic resolution, providing support for additional gene clusters of potential ecological significance. Analysis of environmental sequences using these new sequencing, bioinformatics and phylogenetic approaches demonstrated, for the first time, similar niche specialisation in AOB to that in AOA, indicating pH as a key ecological factor controlling the composition of soil ammonia oxidiser communities. CONCLUSIONS: This study presents the first bioinformatics pipeline for optimal analysis of Illumina MiSeq sequencing of a functional gene and is adaptable to any amplicon size (even genes larger than 500 bp). The pipeline was used to provide an up-to-date phylogenetic analysis of terrestrial betaproteobacterial amoA genes and to demonstrate the importance of soil pH for their niche specialisation and is broadly applicable to other ecosystems and diverse microbiomes.

Soil bacterial networks are less stable under drought than fungal networks.

Soil microbial communities play a crucial role in ecosystem functioning, but it is unknown how co-occurrence networks within these communities respond to disturbances such as climate extremes. This represents an important knowledge gap because changes in microbial networks could have implications for their functioning and vulnerability to future disturbances. Here, we show in grassland mesocosms that drought promotes destabilising properties in soil bacterial, but not fungal, co-occurrence networks, and that changes in bacterial communities link more strongly to soil functioning during recovery than do changes in fungal communities. Moreover, we reveal that drought has a prolonged effect on bacterial communities and their co-occurrence networks via changes in vegetation composition and resultant reductions in soil moisture. Our results provide new insight in the mechanisms through which drought alters soil microbial communities with potential long-term consequences, including future plant community composition and the ability of aboveground and belowground communities to withstand future disturbances.

The consequences of niche and physiological differentiation of archaeal and bacterial ammonia oxidisers for nitrous oxide emissions.

High and low rates of ammonium supply are believed to favour ammonia-oxidising bacteria (AOB) and archaea (AOA), respectively. Although their contrasting affinities for ammonium are suggested to account for these differences, the influence of ammonia concentration on AOA and AOB has not been tested under environmental conditions. In addition, while both AOB and AOA contribute to nitrous oxide (N2O) emissions from soil, N2O yields (N2O-N produced per NO2--N generated from ammonia oxidation) of AOA are lower, suggesting lower emissions when AOA dominate ammonia oxidation. This study tested the hypothesis that ammonium supplied continuously at low rates is preferentially oxidised by AOA, with lower N2O yield than expected for AOB-dominated processes. Soil microcosms were supplied with water, urea or a slow release, urea-based fertiliser and 1-octyne (inhibiting only AOB) was applied to distinguish AOA and AOB activity and associated N2O production. Low ammonium supply, from mineralisation of organic matter, or of the fertiliser, led to growth, ammonia oxidation and N2O production by AOA only, with low N2O yield. High ammonium supply, from free urea within the fertiliser or after urea addition, led to growth of both groups, but AOB-dominated ammonia oxidation was associated with twofold greater N2O yield than that dominated by AOA. This study therefore demonstrates growth of both AOA and AOB at high ammonium concentration, confirms AOA dominance during low ammonium supply and suggests that slow release or organic fertilisers potentially mitigate N2O emissions through differences in niche specialisation and N2O production mechanisms in AOA and AOB.

Approaches to understanding the ecology and evolution of understudied terrestrial archaeal ammonia-oxidisers.

Ammonia-oxidising archaea (AOA) form a phylogenetic group within the phylum Thaumarchaeota and are of ecological significance due to their role in nitrification, an important biogeochemical process. Previous research has provided information on their ecosystem role and potential physiological characteristics, for example, through analyses of their environmental distribution, ecological adaptation and evolutionary history. However, most AOA diversity, assessed using several environmental marker genes, is not represented in laboratory cultures, with consequent gaps in knowledge of their physiology and evolution. The present study critically reviews existing and developing approaches for the assessment of AOA function and diversity and their potential to provide a deeper understanding of these ecologically important, but understudied microorganisms.

Ammonia-oxidising archaea living at low pH: Insights from comparative genomics.

Obligate acidophilic members of the thaumarchaeotal genus Candidatus Nitrosotalea play an important role in nitrification in acidic soils, but their evolutionary and physiological adaptations to acidic environments are still poorly understood, with only a single member of this genus (Ca. N. devanaterra) having its genome sequenced. In this study, we sequenced the genomes of two additional cultured Ca. Nitrosotalea strains, extracted an almost complete Ca. Nitrosotalea metagenome-assembled genome from an acidic fen, and performed comparative genomics of the four Ca. Nitrosotalea genomes with 19 other archaeal ammonia oxidiser genomes. Average nucleotide and amino acid identities revealed that the four Ca. Nitrosotalea strains represent separate species within the genus. The four Ca. Nitrosotalea genomes contained a core set of 103 orthologous gene families absent from all other ammonia-oxidizing archaea and, for most of these gene families, expression could be demonstrated in laboratory culture or the environment via proteomic or metatranscriptomic analyses respectively. Phylogenetic analyses indicated that four of these core gene families were acquired by the Ca. Nitrosotalea common ancestor via horizontal gene transfer from acidophilic representatives of Euryarchaeota. We hypothesize that gene exchange with these acidophiles contributed to the competitive success of the Ca. Nitrosotalea lineage in acidic environments.

Links between seawater flooding, soil ammonia oxidiser communities and their response to changes in salinity.

Coastal areas worldwide are challenged by climate change-associated increases in sea level and storm surge quantities that potentially lead to more frequent flooding of soil ecosystems. Currently, little is known of the effects of inundation events on microorganisms controlling nitrification in these ecosystems. The goal of this study was to investigate the impact of seawater flooding on the abundance, community composition and salinity tolerance of soil ammonia oxidisers. Topsoil was sampled from three islands flooded at different frequencies by the Wadden Sea. Archaeal ammonia oxidiser amoA genes were more abundant than their betaproteobacterial counterparts, and the distribution of archaeal and bacterial ammonia oxidiser amoA and 16S rRNA gene sequences significantly differed between the islands. The findings indicate selection of ammonia oxidiser phylotypes with greater tolerance to high salinity and slightly alkaline pH (e.g. Nitrosopumilus representatives) in frequently flooded soils. A cluster phylogenetically related to gammaproteobacterial ammonia oxidisers was detected in all samples analysed in this survey. Nevertheless, no gammaprotebacterial amoA genes could be amplified via PCR and only betaproteobacterial ammonia oxidisers were detected in enrichment cultures. A slurry-based experiment demonstrated the tolerance of both bacterial and archaeal ammonia oxidisers to a wide range of salinities (e.g. Wadden Sea water salinity) in soil naturally exposed to seawater at a high frequency.

Abiotic Conversion of Extracellular NH2OH Contributes to N2O Emission during Ammonia Oxidation.

Abiotic processes involving the reactive ammonia-oxidation intermediates nitric oxide (NO) or hydroxylamine (NH2OH) for N2O production have been indicated recently. The latter process would require the availability of substantial amounts of free NH2OH for chemical reactions during ammonia (NH3) oxidation, but little is known about extracellular NH2OH formation by the different clades of ammonia-oxidizing microbes. Here we determined extracellular NH2OH concentrations in culture media of several ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as one complete ammonia oxidizer (comammox) enrichment (Ca. Nitrospira inopinata) during incubation under standard cultivation conditions. NH2OH was measurable in the incubation media of Nitrosomonas europaea, Nitrosospira multiformis, Nitrososphaera gargensis, and Ca. Nitrosotenuis uzonensis, but not in media of the other tested AOB and AOA. NH2OH was also formed by the comammox enrichment during NH3 oxidation. This enrichment exhibited the largest NH2OH:final product ratio (1.92%), followed by N. multiformis (0.56%) and N. gargensis (0.46%). The maximum proportions of NH4+ converted to N2O via extracellular NH2OH during incubation, estimated on the basis of NH2OH abiotic conversion rates, were 0.12%, 0.08%, and 0.14% for AOB, AOA, and Ca. Nitrospira inopinata, respectively, and were consistent with published NH4+:N2O conversion ratios for AOB and AOA.

Ammonia oxidisers in a non-nitrifying Brazilian savanna soil.

Low nitrification rates in Brazilian savanna (Cerrado) soils have puzzled researchers for decades. Potential mechanisms include biological inhibitors, low pH, low microbial abundance and low soil moisture content, which hinders microbial activity, including ammonia oxidation. Two approaches were used to evaluate these potential mechanisms: (i) manipulation of soil moisture and pH in microcosms containing Cerrado soil and (ii) assessment of nitrification inhibition in slurries containing mixtures of Cerrado soil and an actively nitrifying agricultural soil. Despite high ammonium concentration in Cerrado soil microcosms, little NO3- accumulation was observed with increasing moisture or pH, but in some Cerrado soil slurries, ammonia-oxidising archaea (AOA) amoA transcripts were detected after 14 days. In mixed soil slurries, the final NO3- concentration reflected the initial proportions of agricultural and Cerrado soils in the mixture, providing no evidence of nitrification inhibitors in Cerrado soil. AOA community denaturing gradient gel electrophoresis profiles were similar in the mixed and nitrifying soils. These results suggest that nitrification in Cerrado soils is not constrained by water availability, ammonium availability, low pH or biological inhibitors, and alternative potential explanations for low nitrification levels are discussed.

Kinetics of NH3 -oxidation, NO-turnover, N2 O-production and electron flow during oxygen depletion in model bacterial and archaeal ammonia oxidisers.

Ammonia oxidising bacteria (AOB) are thought to emit more nitrous oxide (N2 O) than ammonia oxidising archaea (AOA), due to their higher N2 O yield under oxic conditions and denitrification in response to oxygen (O2 ) limitation. We determined the kinetics of growth and turnover of nitric oxide (NO) and N2 O at low cell densities of Nitrosomonas europaea (AOB) and Nitrosopumilus maritimus (AOA) during gradual depletion of TAN (NH3 + NH4+) and O2 . Half-saturation constants for O2 and TAN were similar to those determined by others, except for the half-saturation constant for ammonium in N. maritimus (0.2 mM), which is orders of magnitudes higher than previously reported. For both strains, cell-specific rates of NO turnover and N2 O production reached maxima near O2 half-saturation constant concentration (2-10 µM O2 ) and decreased to zero in response to complete O2 -depletion. Modelling of the electron flow in N. europaea demonstrated low electron flow to denitrification (≤1.2% of the total electron flow), even at sub-micromolar O2 concentrations. The results corroborate current understanding of the role of NO in the metabolism of AOA and suggest that denitrification is inconsequential for the energy metabolism of AOB, but possibly important as a route for dissipation of electrons at high ammonium concentration.

Chemotaxonomic characterisation of the thaumarchaeal lipidome.

Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia-oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane-spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose-phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties.