Profiling active nitrifiers in coastal sediments with single cell approaches and metagenomics

Biogeochemical Cycles

IAP2-19-135

Overview

Microorganisms within coastal sediments are the engines of these ecosystems driving essential biogeochemical cycles that underpin ecosystem functioning. Of particular importance are those that transform nitrogen and in doing so mediate nutrient loads entering coastal ecosystems. Central to this process is nitrification conducted by organisms that oxidise ammonia to nitrate and then nitrite (Fig 1). Recently there have been a number of exciting revelations that have turned current understanding of this ‘simple’ process on its head, including the discovery of comammox, complete nitrification within a single organism (Daims et al 2015, van Kessel et al 2015, Pinto et al 2015). The received wisdom was that two processes contributed to nitrification: 1) ammonia oxidation, driven by functionally similar but phylogenetically different ammonia oxidising bacteria (AOB) or archaea (AOA) and 2) nitrite oxidation driven by nitrite oxidising bacteria (NOB).
The recent discovery of comammox, complete oxidation of ammonia to nitrate by a single organism, has disproved this long-held assumption, revealing a third group of organisms whose contribution to nitrification in the environment has been overlooked. To-date the contribution of comammox to nitrification in coastal zones is unknown. However, a major hindrance to expanding our understanding of these organisms, beyond their detection, is linking activity and identify of the active nitrifiers within complex environmental samples based on DNA approaches alone. Therefore, we are proposing a new approach to identify active nitrifiers, AO, NOB or comammox, within coastal sediments. This approach is based on our state-of-the-art microfluidics platform that couples Stable Isotope Probing (SIP) to Resonance Raman and Raman Activated Cell Sorting (RR-RACS) (McIlvanna et al 2016, Song et al 2017) to label and sort active cells. Subsequently the identify of active sorted cells will be achieved by sequencing approaches. This SIP-RR-RACs approaches is detailed in the methodology section below. With this approach will address fundamental ecological questions on the distribution and activity of nitrifiers within coastal sediments.

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Image Captions

Fig. 1. Nitrification showing the key steps, organisms involved and functional marker genes for each step.

Methodology

The project will apply state-of-the art approaches to label, capture and identify active single cell nitrifiers from within coastal sediments. To achieve this, we will combine Stable Isotope Probing (SIP), Resonance Raman and Raman Activated Cell Sorting (RR-RACS) and metagenomics within time-series sediment microcosm experiments. Initially SIP will be used to label active nitrifiers via the incorporation of heavy labelled isotopes (e.g. 13CO2). Characteristic Raman shifts are generated when cells incorporate heavy isotopes and this Raman shift can be used to select labelled cells using our Raman Activated Cell Sorting (RACS) platform4,5. Active sorted cells will be identified via metagenomics. As such the experimental workflow of SIP-RR-RAC will be used to select yet-to-be cultured cells incorporating 13C within sediment microcosms and metagenomics will be used to identify and further classify the nitrifiers present. A further advantage of RR-RACS approach is that intact viable cells are recovered6 and we can use the metagenomics information to provide a targeted approach to culture the sorted cells. The project will start with a suite of nitrifier enrichments from coastal sediments to optimise methods and then move on to apply the approach to sediment microcosms. For this we will select sediments along the salinity gradients of estuaries incorporating sediments from freshwater to marine. Initial field work will select sites that offer contrasting nitrifier communities to increase our understanding of the ecology and distribution of these organisms.

Project Timeline

Year 1

An ambitious but achievable timeline and work-plan to deliver this research will include the following, building from working with in house enrichment culture, to complex environmental communities. Individual work-packages will provide the template for each PhD chapter and associated journal papers. Thesis and journal paper writing will be a continuous effort throughout.
Year 1: A literature survey and initial laboratory training will be undertaken from months 0 to 3. Months 4 to 12 will focus on development of SIP-RR-RACS approaches using suite of in-house nitrifier enrichments. Milestones: 1) Delivery of literature review and 2) first experimental thesis chapter and manuscript on SIP-RR-RACS approaches to identify growth and activity of nitrifiers within enrichment cultures.

Year 2

Year 2: Transfer and optimisation of SIP-RR-RACS approaches developed in year 1 working with enrichments to sediment microcosms (months 12 to 17), including methods for removal of cells from sediments for RR-RACS and optimisation of molecular protocols to work with low numbers of labelled cells on-chip. Milestone: 3) Optimisation of SIP-RR-RACS approaches to sediments (second experimental thesis chapter and journal manuscript).

Year 3

Year 3: Field survey of nitrifiers along estuarine salinity gradient and corresponding SIP-RR-RACS microcosm experiments to identify growth and activity of active nitrifiers (months 24 to 36). The field study will be complemented by three months of microcosm incubations and SIP-RR-RACS cell sorting (months 24-29) followed by metagenomics and bioinformatics analysis of heavy labelled cells (months 30-36). Milestone: 4) Field and experimental study applying SIP-RR-RACS and metagenomics (third experimental thesis chapter and journal manuscript).

Year 3.5

Year 4: Months 36 to 42 will be dedicated to completing thesis and journal paper write up for submission at end of month 42. Milestone 5) submission of PhD thesis.

Training
& Skills

This project offers a unique opportunity to develop expertise in the microbial ecology of the nitrogen cycle of coastal ecosystems. It will be underpinned by state-of-the-art molecular approaches to label, sort and identify diverse, active, yet-to-be cultured nitrifiers. The unique combination of internationally leading expertise required to deliver this project are provided by the supervisory team – Prof Smith (Glasgow) has extensive expertise in the microbial ecology of the nitrogen cycle in coastal ecosystems and the molecular approaches to study them (Zhange et al 2018; Cholet et al 2019); Prof. Yin (Glasgow) is internationally leading in the development and application of SIP-RR-RACS single cell approaches; Dr Ijaz (Glasgow) is driving the very latest developments in bioinformatics, informatics and ecological statistics (http://userweb.eng.gla.ac.uk/umer.ijaz/bioinf ormatics/ecological.html
); Prof. Curtis (Newcastle) is a world leading expert in experimental and theoretical microbial ecology of nitrifiers. At Newcastle University, training in FLOW-FISH (Flow cytometry and Fluorescent in situ hybridisation) will be undertaken to benchmark RAMAN-SIP methods against. The advanced facilitates required to undertake this project are in place and the student will receive up-to-date training in all of the required approaches including molecular microbiology and ecology, field and laboratory experiments, single-cell RR-RACS and bioinformatics. This highly desirable skill set will result in a uniquely trained individual with the practical and theoretical background to tackle emerging challenges in microbial ecology and ecosystem function.

References & further reading

1. Daims H et al 2015 Nature 528: 504-509. 2. van Kessel MA et al 2015 Nature 528: 555- 559.
3. Pinto A et al 2015 mSphere 1 e00054. 4. McIlvanna D et al 2016 Lab on a Chip 16: 1420-1429 (doi:10.1039/C6LC00251J) 5. Song Y et al 2017 Microbial Biotechnology 10: 125-137 (doi:10.1111/17517915.12420) 6. Yuan X et al (2018) Applied and Environmental Microbiology 8: e02508-17. (doi:10.1128/AEM.02508-17) 7. Zhang LM et al (2018) Environmental Microbiology 20: 2834-2853 (doi:10.1111/1462-2920.14238) 8: Cholet F et al (2018) Environmental Microbiology 21 (2), 827-844 (doi.org/10.1111/1462-2920.14516)

Further Information

To apply for this PhD please use the url: https://www.gla.ac.uk/study/applyonline/?CAREER=PGR&PLAN_CODES=CF18-7316

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