Anaerobic quaternary amine degradation: from single bacterium to salt marsh ecosystem.

Lead Research Organisation: Plymouth Marine Laboratory
Department Name: Plymouth Marine Lab

Abstract

Quaternary amines (QAs) such as glycine betaine (GBT) are ubiquitous in marine organisms. GBT is used by marine organisms as a compatible solute in response to changes in environmental conditions, such as increasing salinity, because GBT does not interfere with cell metabolism. QA compounds are frequently released into the ocean ecosystems due to environmental changes, such as viral attack and grazing. The degradation of these compounds, especially in intertidal coastal areas, contributes significantly to the production of climate-active trace gases. These include the potent greenhouse gas methane and volatile methylated amines, which are thought to be involved in cloud formation in the marine environment.

Coastal sediments are estimated to contribute approximately 75% of the global oceanic methane emissions (8-13 Tg per year) and much of this is likely to be derived from the degradation of QAs. Although we know that microorganisms are mainly responsible for the degradation of GBT to methane and volatile methylamines, we know little about the genes and enzymes involved in the degradation pathway. Furthermore, the identity of those microorganisms responsible for the transformation has not yet been determined. Our current knowledge of these two aspects remains speculative, at least partially due to the lack of definitive research.

This timely proposal aims to fill in this major gap in our knowledge of marine carbon cycle.

Using cultivated model microorganisms, we aim to define the key genes and the encoding enzymes in the anaerobic degradation of GBT.

Using molecular ecology techniques and the resultant data from the study of the model microorganisms, we aim to further determine the key microbial players involved in the anaerobic production of methane and methylamines from GBT in the marine environment.

The work will generate novel knowledge about our understanding of microbial GBT transformations and will therefore fill in a serious gap in our knowledge of the marine carbon cycle.

Planned Impact

This project addresses fundamental questions relating to the marine carbon cycle and greenhouse emissions from the natural environments, both of which are crucial for our understanding of global biogeochemical cycles and in the drive for a more sustainable future. The work is therefore of the utmost relevance to NERC's strategic aims, particularly the Biodiversity Science and Climate Change themes. The proposed research will have considerable impact on the following communities, the Environmental Agency, policy-makers and the wider public.

The Environmental Agency has launched schemes and activities in the last five years to protect the salt marsh ecosystems and also to create around 500 hectares of salt marsh along the Essex and Suffolk coastlines. However, whether these activities will affect local emissions of climate-active trace gases is uncertain. Our research can contribute to the decision making by the Environmental Agency and inform environmental policy making decisions.
The other beneficiaries of knowledge arising from this research will be policy-makers. They will be alerted to the positive and negative impacts of salt marsh ecosystems, which contribute not only to global warming (by releasing methane, a powerful greenhouse gas) but also potentially to global cooling (by releasing volatile small amines which are involved in aerosol formation).

Since our research has immediate relevance to climate and marine biology, it will be of interest to the wider public. People are always interested in scientific topics related to climate and marine life, and these two aspects of this type of research will always receive considerable attention in the media.

A variety of methods will be used to engage with the end-users, including workshops, a detailed website, publications in popular magazines such as Planet Earth, and visits and exhibitions at local schools. One of the things that we are keen to do is to have end user opinion at very early stages of our research. We consider that engagement of end users from the very early stages of the project will also benefit our research and help to formulate proper sampling activities during the research programme. For example, a workshop engaging the Environmental Agency will be set up in month 1 so that any concerns and questions from the Environmental Agency can then be defined and taken into account later on. On the website that we will set up from Month 1, we will have an online feedback form so that any queries from the wider public can be monitored and answered promptly.
 
Description An important analytical requirement of this project is to analyse glycine betaine (GBT), a quarternary ammonium compound in dissolved as well as particulate form. Previously, we have only successfully measured GBT in particulate samples (eg. particles such as phytoplankton cells that are retained on a filter after passing seawater through it), but we have extended this method to measure glycine betaine in dissolved samples down to a concentration of 5 nM. We hope to further improve this limit of detection. We have also shown the method can measure a second nitrogen osmolyte, trimethylamine-N-oxide (TMAO), simultaneously to GBT. TMAO is an intermediate in the aerobic microbial metabolism of GBT and choline, resulting in the production of volatile methylamines able to transfer to the atmosphere and participate in aerosol formation, and are thus climate-relevant compounds.
Exploitation Route One of the main developments in this award period, was reduction of ion suppression during analysis of glycine betaine and choline by LC/MS. This was found to be directly and severely affected by salt content in extracts, and reduction of the proportion of salt content significantly alleviated ion suppression. This has informed sampling and analysis of osmolytes from other environments, and enabled improved sensitivity of analysis, and acquisition of reliable data from salty environments. These findings have been published (Airs and Beale 2016), and have enabled new work to be undertaken both for the PEGASO Antarctic research campaign (2015), and have enabled work within the Antarctic Circumnavigation Expedition application (2016-2017). We have also published a datamining study on the abundance of genes encoding N-osmolyte breakdown in the marine environment and human gut (Jameson et al. 2016). Osmolytes are also important in other arenas in addition to environmental, for example agriculture and human medicine, so our findings may have wider impacts too, as exemplified by Jameson et al 2016.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

 
Description During LC/MS analysis of N-osmolytes in seawater, suppression of analyte ionisation by the matrix can be problematic. We have developed an alternate eluent composition for LC/MS analysis, that shifts main interfering ions away from N-osmolyte elution, thereby minimising interference from matrix effects. The impact is more successful low volume measurements of N-osmolytes from seawater media. Using a low volume is important to prevent loss of glycine betaine during sampling. The development has directly led to observations of higher than expected levels of glycine betaine in a seawater matrix, which has implication for the levels of breakdown products that may be observed. In addition, we have discovered that the proportion of salt in an extract also has a substantial effect on ion suppression, and reducing the proportion of salt is essential to analysing challenging samples. This finding has been used to inform sample analysis from other projects, and proposed work in funding applications, including those highly relevant to climate. Experimental approaches to osmolyte analysis reducing ion suppression has been published (Beale and AIrs, 2016), and we have also published a datamining study examining the presence of genes involved in osmolyte breakdown in the marine environment (Jameson et al. 2016).
First Year Of Impact 2014
Sector Chemicals,Environment
Impact Types Societal

 
Description Discovery grant
Amount £800,000 (GBP)
Funding ID NE/P012671/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2020
 
Description International Opportunities Fund - Pump Priming Plus
Amount £250,254 (GBP)
Funding ID NE/P008526/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 02/2017 
End 01/2020
 
Description NERC Discovery
Amount £800,000 (GBP)
Funding ID NE/R010382/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description NERC Discovery Science (Chen)
Amount £210,000 (GBP)
Funding ID NE/M003361/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 11/2014 
End 10/2017
 
Title Detection of TMAO 
Description Osmolyte method extended to include measurement of trimethylamine N-oxide 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Enables measurement of TMAO in seawater samples in addition to glycine betaine and choline. Analytical basis for NERC proposal (successful) 
 
Title Extraction modifications for osmolytes 
Description We have discovered that the proportion of salt in extracts is critical for reducing ion suppression during LC/MS analyses. We have determined what the limits are for the proportion of salt, and extended this to modify the extraction protocol for certain types of samples. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact One main impact is that it had enabled us to analyse samples collected on board the PEGASO cruise in the Antarctic in 2015. THese are important samples from a unique cruise where many atmospheric and waterside measurements were taken, and final datasets of great value are expected. 
 
Title Modification of method to improve ion suppression 
Description Ion suppression can be problematic during analysis of nitrogenous osmolytes. Our analytical method was modified to permit reduction in ion suppression in natural samples. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Detection of osmolytes in low volume samples which are required to determine accurate measurements. 
 
Title Analysis of Stiffkey samples 
Description Samples from Stiffkey Saltmarsh were analysed for osmolytes. None were detected. Results have informed future sampling and analysis strategies. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Results have informed future sampling and analysis strategies. 
 
Description Antarctic Circumnavigation Expedition Project SORPASSO: SImo et al. 
Organisation Middle East Technical University
Department Institute of Marine Sciences
Country Turkey 
Sector Academic/University 
PI Contribution Instigated the proposal and helped to assemble team. Key player in completing and submitting the proposal. Trained Dr Holly Pearson to take part in the expedition and collect N-osmolyte samples.
Collaborator Contribution Led the proposal.
Impact FUnding of project SORPASSO within Antarctic CIrcumnavigation Expedition,
Start Year 2016
 
Description PhD proposal with Jonathan Todd and Thomas Mock, UEA 
Organisation University of East Anglia
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint PhD proposal on glycine betaine research to EnvEast DTP
Collaborator Contribution Joint PhD proposal on glycine betaine research to EnvEast DTP
Impact Submission and acceptance of a PhD proposal to EnvEast DTP
Start Year 2014
 
Description Schafer et al, Arctic proposal 
Organisation University of Essex
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Contributed essential track record, to proposal planning, costing and writing.
Collaborator Contribution Led proposal (Schafer); contributed essential expertise (Steinke, McGenity, Hughes, Lohan, Chen)
Impact Submission of proposal to NERC Arctic call
Start Year 2015
 
Description Schafer et al, Arctic proposal 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Contributed essential track record, to proposal planning, costing and writing.
Collaborator Contribution Led proposal (Schafer); contributed essential expertise (Steinke, McGenity, Hughes, Lohan, Chen)
Impact Submission of proposal to NERC Arctic call
Start Year 2015
 
Description Schafer et al, Arctic proposal 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Contributed essential track record, to proposal planning, costing and writing.
Collaborator Contribution Led proposal (Schafer); contributed essential expertise (Steinke, McGenity, Hughes, Lohan, Chen)
Impact Submission of proposal to NERC Arctic call
Start Year 2015
 
Description Schafer et al, Arctic proposal 
Organisation University of York
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Contributed essential track record, to proposal planning, costing and writing.
Collaborator Contribution Led proposal (Schafer); contributed essential expertise (Steinke, McGenity, Hughes, Lohan, Chen)
Impact Submission of proposal to NERC Arctic call
Start Year 2015
 
Description Todd et al Discovery grant: A multidisciplinary study of DMSP production and lysis - from enzymes to organisms to process modelling. 
Organisation University of East Anglia
Department School of Environmental Sciences UEA
Country United Kingdom 
Sector Academic/University 
PI Contribution Preparation of proposal; track record for proposal; reponse to reviewers comments.
Collaborator Contribution Leader of proposal.
Impact Proposal submitted to NERC discovery, scored 8 for science excellence, ansfunded: A multidisciplinary study of DMSP production and lysis - from enzymes to organisms to process modelling. Collaboration is multidisciplinary: Analytical chemistry, molecular biology, environmental chemistry, biochemistry; crystallography
Start Year 2015
 
Description UK participation in PEGASO cruise 
Organisation Spanish National Research Council (CSIC)
Country European Union (EU) 
Sector Public 
PI Contribution Participation in PEGASO (Plankton-derived Emissions of trace Gases and Aerosols in the Southern Ocean) research cruise to measure methyl amines and glycine betaine in seawater off South Georgia and in the Weddel Sea. Provision of labware.
Collaborator Contribution Provision of a science berth for the PEGASO cruise. Provision of labware. Funding from higher education innovation fund. Provision of instrumentation for methylamine measurement.
Impact Preparation for research cruise. Funding secured from higher education innovation fund. Funding application submitted to International Opportunities Fund.
Start Year 2014
 
Description UK participation in PEGASO cruise 
Organisation University of Plymouth
Country United Kingdom 
Sector Academic/University 
PI Contribution Participation in PEGASO (Plankton-derived Emissions of trace Gases and Aerosols in the Southern Ocean) research cruise to measure methyl amines and glycine betaine in seawater off South Georgia and in the Weddel Sea. Provision of labware.
Collaborator Contribution Provision of a science berth for the PEGASO cruise. Provision of labware. Funding from higher education innovation fund. Provision of instrumentation for methylamine measurement.
Impact Preparation for research cruise. Funding secured from higher education innovation fund. Funding application submitted to International Opportunities Fund.
Start Year 2014
 
Description C1 GRC USA 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on "Anaerobic GBT and TMA metabolism in saltmarsh sediment: A stable-isotope probing approach" delivered at the Gordon Research COnference in a session entitled "Molecular Basis of Microbial one-carbon metabolism".
Year(s) Of Engagement Activity 2014
 
Description Communication of science at Challenger Conference 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Talk on glycine betaine researched delivered in well attended biogeochemistry session

Elected at a member of Challenger Council
Year(s) Of Engagement Activity 2014
 
Description Dissolved Organic Nitrogen workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Ten researchers from different disciplines met to share their science relating to dissolved organic nitrogen and discuss future research.

New collaboration with Jonathan Todd (UEA) resulting in submission of PhD proposal to the EnvEast DTP.
New collaboration with PML colleagues resulting in submission of a research proposal.
Year(s) Of Engagement Activity 2014
 
Description Engagement with children in science activity 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 12 school and preschool children engaged in a science activity using everyday materials found in the home.

Imagination of young minds captured by science activity
Year(s) Of Engagement Activity 2014
 
Description GRC Applied and Environmental Microbiology: Saltmarsh methanogenesis from choline. When bacteria meet archaea. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research presented from stable isotope probing used to determine which microbes were responsible for methanogenesis from choline in saltmarsh sediment, at GRC on Applied and Environmental Microbiology.
Year(s) Of Engagement Activity 2017
 
Description International Symposium on DMSP and related compounds, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Science presentations on glycine betaine and methylamine research sparked much interest and scientific discussion. Several new collaborations forged.

Invitation by Rafel Simo (conference organiser) to participate in PEGASO cruise to measure glycine betaine and methyl amines.
Year(s) Of Engagement Activity 2014
 
Description Invited Seimar University of Warwick 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Gave an invited seminar on glycine betaine research at the University of Warwick

Further collaboration with research group including NERC research proposal (successful)
Year(s) Of Engagement Activity 2014
 
Description MMEG Bangor 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation on data mining metabolic pathways delivered to molecular microbial ecologists.
Year(s) Of Engagement Activity 2014
 
Description Metagenomic datamining reveals links to cardiovascular disease, enabling engineering of a healthier human gut microbiome 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on Metagenomic Datamining reveals links to cardiovascular disease, enabling engineering of a healthier human gut microbiome, given at Rowett-INRA 2018, Gur microbiology: No longer the forgotten organ, 11-14 June, 2018.
Year(s) Of Engagement Activity 2018
 
Description PML/Plymouth Uni Science Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Science presented at local science day

Awareness of science increased locally
Year(s) Of Engagement Activity 2014