Microbial ecology provides insights into the ecological and evolutionary dynamics of microbial communities underpinning every ecosystem on Earth. Microbial communities can now be investigated in unprecedented detail, although there is still a wealth of open questions to be tackled. Here we identify 50 research questions of fundamental importance to the science or application of microbial ecology, with the intention of summarising the field and bringing focus to new research avenues. Questions are categorised into seven themes: host–microbiome interactions; health and infectious diseases; human health and food security; microbial ecology in a changing world; environmental processes; functional diversity; and evolutionary processes. Many questions recognise that microbes provide an extraordinary array of functional diversity that can be harnessed to solve real-world problems. Our limited knowledge of spatial and temporal variation in microbial diversity and function is also reflected, as is the need to integrate micro- and macro-ecological concepts, and knowledge derived from studies with humans and other diverse organisms. Although not exhaustive, the questions presented are intended to stimulate discussion and provide focus for researchers, funders and policy makers, informing the future research agenda in microbial ecology.

Background: Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils. Results: Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using ¹³C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the ¹³C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a ¹³CO₂ atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere. Conclusion: In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle.

The transition of the martian climate from the wet Noachian era to the dry Hesperian (4.1–3.0 Gya) likely resulted in saline surface waters that were rich in sulfur species. Terrestrial analogue environments that possess a similar chemistry to these proposed waters can be used to develop an understanding of the diversity of microorganisms that could have persisted on Mars under such conditions. Here, we report on the chemistry and microbial community of the highly reducing sediment of Colour Peak springs, a sulfidic and saline spring system located within the Canadian High Arctic. DNA and cDNA 16S rRNA gene profiling demonstrated that the microbial community was dominated by sulfur oxidising bacteria, suggesting that primary production in the sediment was driven by chemolithoautotrophic sulfur oxidation. It is possible that the sulfur oxidising bacteria also supported the persistence of the additional taxa. Gibbs energy values calculated for the brines, based on the chemistry of Gale crater, suggested that the oxidation of reduced sulfur species was an energetically viable metabolism for life on early Mars.

Background: Conventional methods of agricultural pest control and crop fertilisation are unsustainable. To meet growing demand, we must find ecologically responsible means to control disease and promote crop yields. The root-associated microbiome can aid plants with disease suppression, abiotic stress relief, and nutrient bioavailability. The aim of the present work was to profile the community of bacteria, fungi, and archaea associated with the wheat rhizosphere and root endosphere in different conditions. We also aimed to use ¹³CO₂ stable isotope probing (SIP) to identify microbes within the root compartments that were capable of utilising host-derived carbon. Results: Metabarcoding revealed that community composition shifted significantly for bacteria, fungi, and archaea across compartments. This shift was most pronounced for bacteria and fungi, while we observed weaker selection on the ammonia oxidising archaea-dominated archaeal community. Across multiple soil types we found that soil inoculum was a significant driver of endosphere community composition, however, several bacterial families were identified as core enriched taxa in all soil conditions. The most abundant of these were Streptomycetaceae and Burkholderiaceae. Moreover, as the plants senesce, both families were reduced in abundance, indicating that input from the living plant was required to maintain their abundance in the endosphere. Stable isotope probing showed that bacterial taxa within the Burkholderiaceae family, among other core enriched taxa such as Pseudomonadaceae, were able to use root exudates, but Streptomycetaceae were not. Conclusions: The consistent enrichment of Streptomycetaceae and Burkholderiaceae within the endosphere, and their reduced abundance after developmental senescence, indicated a significant role for these families within the wheat root microbiome. While Streptomycetaceae did not utilise root exudates in the rhizosphere, we provide evidence that Pseudomonadaceae and Burkholderiaceae family taxa are recruited to the wheat root community via root exudates. This deeper understanding crop microbiome formation will enable researchers to characterise these interactions further, and possibly contribute to ecologically responsible methods for yield improvement and biocontrol in the future.

Sulfur (S) isotopes are used to trace metabolic pathways associated with biological S-cycling in past and present environments on Earth. These pathways (sulfate reduction, sulfur disproportionation and sulfide oxidation) can produce unique S isotope signals that provide insight into biogeochemical S-cycling. The S cycle is also relevant for extraterrestrial environments and processes. On early Mars, sulfur existed in different redox states and was involved in a large range of surface processes (e.g. volcanic, atmospheric, hydrothermal and aqueous brines). Sulfur compounds have also been detected on Europa's icy moon surface, with the S cycle implicated in Europa's surface and ocean geochemistry. Given the well-established utility of S isotopes in providing a record for past life on Earth, S isotopes are a valuable tool for identifying biosignatures on Mars and Europa. Here, we review S isotopes as a biosignature, in light of two recent advances in understanding the S cycle in both Mars and Europa: (1) the measurements of δ ³⁴ S in situ at Gale Crater and quadruple S isotopes (QSI) in Martian meteorites; (2) the identification of a likely exogenous origin of sulfur on Europa's surface. We discuss important considerations for unravelling QSI biosignatures in Martian environments, considering high- and low-sulfur environments, atmospheric mass-independent fractionation of sulfur isotopes (S-MIF) signals and metabolic energy-limited niches. For Europa, we describe the potential for S isotopes to probe biogeochemistry, and identify key knowledge gaps to be addressed to unlock S isotopic tools for future life detection efforts. The resulting picture demonstrates how S isotopes will be a valuable tool for Mars sample return, and how future missions can focus on the search for environments where QSI signatures of microbial S-cycling processes have a greater chance of being preserved. For Europa, the first step will be to account for the S isotope composition of the various S pools, to recognize or rule out non-biologically mediated S isotope values, with a focus on experimental examination of potential S isotope signatures from exogenous sulfur sources. Thematic collection: This article is part of the Sulfur in the Earth system collection available at: https://www.lyellcollection.org/cc/sulfur-in-the-earth-system

Hydrothermal systems that formed as a result of impact events possess all the key requirements for life: liquid water, a supply of bio‐essential elements, and potential energy sources. Therefore, they are prime locations in the search for life on other planets. Here, we apply thermochemical modeling to determine secondary mineral formation within an impact‐generated hydrothermal system, using geochemical data returned for two soils on Mars found in regions that have previously experienced alteration. The computed mineral reaction pathways provide a basis for Gibbs energy calculations that enable both the identification of available geochemical energy, obtained from Fe‐based redox reactions, that could be utilized by potential microbial life within these environments, and an estimate of potential cell numbers. Our results suggest that water–rock interactions occurring within impact‐generated hydrothermal systems could support a range of Fe‐based redox reactions. The geochemical energy produced from these reactions would be substantial and indicates that crater environments have the potential to support microbial cell numbers similar to what has been identified in terrestrial environments.