Assessing the potential of mRNA-FISH FACS for isolation of functional soil bacterial populations for quantifying biogeochemical cycle interactions

The normal growth of all living entities depends on an adequate source of essential elements (e.g. C, N, S, P) and, in this respect, the Earth can be considered a closed system with the supply of essential elements being finite. Therefore, the recycling of these elements through the environment is fundamental to avoid exhaustion and microbes can be viewed as the 'engine room' that drive the component processes responsible for the recycling of these elements in the Earth's biogeochemical cycles. In cycling carbon, soil microbes utilise different organic and inorganic forms of carbon as energy and carbon sources resulting in the transfer carbon between environmental compartments. However, the carbon cycle does not operate on its own but it is closely metabolically linked with that of other essential elements either via the use of these as reductants and oxidants in energy transduction or via their incorporation into biomass (or release from decaying dead biomass) as part of multiple essential element- containing biomolecules (e.g proteins, DNA). Hence, the availability of carbon is a key factor in determining the transformations and cycling of other essential elements whilst the availability of other key elements control the rate at which microbes consume and respire carbon. Such biogeochemical cycle interactions can be illustrated by the soil microbial process of denitrification: the decomposition of organic carbon under low oxygen conditions through the respiration of nitrate resulting in the step-wise reduction of nitrate to dinitrogen gas (N2) with nitrous oxide (N2O) produced as an intermediate.

A central goal in microbial ecology is to link biogeochemical processes to specific microbial taxa in the environment so that the role of microbial community structure can be better represented in predictive models. A suite of methods have been developed in the last decade in order achieve this goal without the need for cultivation and characterization of isolates but none of these offer the opportunity to quantify the interactions between biogeochemical cycles in a microbially-oriented way, for example, with respect to the use of a particular carbon source as a reductant to drive denitrification. Gaining the quantitative understanding of the interactions that is required to predict essential element fluxes and feedbacks under perturbed carbon cycle and environmental change scenarios is therefore method- limited.

This project will provide proof-of-concept of a new method to quantify use of carbon by bacteria whilst transforming another essential element. The bacterial denitrification pathway will serve as a case study with a focus on the bacteria using carbon to reduce N2O to N2 (the final step in denitrification) due to the crucial role that this group play in regulating the atmospheric concentration of N2O, a potent greenhouse gas. The new method involves: (i) use of C isotopes to trace microbial C consumption; (ii) labelling actively N2O-reducing microbial cells with a fluorescent dye; (iii) sorting the fluorescent cells and quantifying the C isotope content.

The proof of concept will be in simple experimental systems involving known N2O-reducing bacteria and soil microcosms incubated under conditions known to promote denitrification. As a case study, we will test a theory concerning the carbon source preference of the N2O-reducing bacteria.The project brings together the complimentary expertise of the investigators (use of C isotopes, fluorescence-labelling and sorting of bacteria, denitrification biogeochemistry) and the project partner (fluorescence labelling of bacteria active in biogeochemical cycling). We will use state-of-the art stable isotope techniques to quantify microbial N2O reduction and exploit advances in instrumentation for cell sorting that enables the accurate detection of bacterial cells extracted from soil.

This proposal seeks to assess the potential of a novel combination of methodologies that when established can be adapted and employed widely by environmental microbiologists in curiosity-driven and applied microbial biogeochemistry research. The immediate impacts are therefore in the academic community as explained in the academic beneficiaries section and the techniques advance arising from this proposal is not directly relevant to users outside the academic community immediately and on its own.

However, it is possible to identify groups for whom this work could be relevant in the future after exploitation of the proposed technique by the academic community. The increase in quantitative knowledge and understanding of the links between microbial populations and ecosystem processes in situ (as exemplified by our nosZ case study) that will facilitate incorporation of microbial data into (agri)ecosystem process measurements and models will be of interest to: environmental managers and their advisory bodies (e.g. Farmers, ADAS), regulators (EA) and policy-driven agencies (DEFRA and DECC). As interactions in biogeochemical cycles have implications for the production and reduction of greenhouse gases with implications for climate change, the research will, via outputs in the peer-reviewed literature, feed into future IPCC reports and thus form the basis of global climate change policy in the long term.

As a result of working on the project, the Researcher Co-Investigator will develop new research skills (stable isotope techniques and mRNA-FISH) which will be available to future positions and projects within the Environmental Science employment sector. In particular, the collaboration with the Project Partner will result in transfer of expertise in bacterial mRNA-FISH to the UK, thus enhancing the UK research portfolio and contributing to UK competitiveness at the forefront of international research.

Finally, beneficiaries of the research outputs are students of Environmental Science and Management, Soil Science, and Ecology as well as the general public through promoting more sustainable management of the environment and public understanding of the science issues including climate change.