Stable Isotope Probing with Resonance Raman Cell Sorting to profile influence of ocean acidification on microbial carbon fixation

Lead Research Organisation: University of Glasgow
Department Name: School of Engineering

Abstract

When we think of photosynthesis we normally think of trees and green plants converting carbon dioxide into life giving oxygen. However, there is a panoply of other organisms that also perform this function equally efficiently, each having adapted to their own individual environment. More than 70% of Earth's surface is covered by ocean where photosynthetic micro-organisms are the major primary producers and play a critical role in CO2-fixation. A technique to determine the influence of environmental change on these organisms at the base of the food chain will be a wide-ranging asset in the environmental science toolkit. However, a major hurdle in identifying the influence of environmental factors is that the vast majority of microbes in natural environments cannot be cultivated. The enormous diversity of photosynthetic microbes in nature remains unknown.

In a timely approach that overcomes the need to cultivate the marine micro-organisms, we will combine emerging single-cell sorting and metagenomic techniques to develop an enabling technology to investigate the influence of environmental factors on microbial communities by linking physiological activities of individual micro-organisms with community diversity and function.

As a proof of concept, within this project, we will focus on the effect of ocean acidification (OA) on marine photosynthetic micro-organisms because of concerns over the global impact of OA. Studies thus far do not report a consistent OA effect on photosynthetic micro-organisms with different responses from different taxonomic groups.

Since nearly all photosynthetic micro-organisms contain carotenoids, carotenoids could be used to as an internal biomarker to identify photosynthetic micro-organisms. By providing micro-organisms with stable isotope labelled 13CO2 as the carbon source, using Raman spectroscopy, we can identify individual cells that are actively fixing CO2. Here we will combine this technique with Raman activated single cell sorting to screen and isolate community members based on a quantitative measure of CO2 fixation activity of individual cells. Metagenomic analyses of these functionally sorted cells will further identify the species in each category.

By developing this technology, we will address the following specific questions:
1. To what extend will OA affect the CO2 fixation activities of photosynthetic microorganisms?
2. Which microorganisms and their CO2 fixation activities are affected by OA?
3. What changes to the community will come about as a consequence of question 1&2?
4. Which enzymes are implicated in optimal CO2 fixation activities of photosynthetic microorganisms?

While this research will specifically answers questions on the influence of OA that underpin major issues such as food security and aquaculture, the approach can serve as a generic technique to be applied widely in environmental sciences to determine the influence of any agent of environmental change.

Planned Impact

1. Industry:
The project is relevant to industries that are closely associated with marine primary productivity such as aquaculture, since ocean acidification negatively affects many organisms that produce a calcium carbonate shell or skeleton, such as shellfish. Therefore, understanding ocean acidification will help develop mitigation strategies that could have significant effect on the UK economy.
Action:
The Scottish Aquaculture Innovation Centre (SAIC) is ideally placed as a link between academia and the industry. We would like to demonstrate our technique at the SAIC conference so that those in academic and industrial aspects of the sector are aware of any technological innovations developed through this project. In addition, we have long established links with the aquaculture sector and the food industry across the UK. We will actively engage with them during the course of this project. They will be invited at the annual Industry Day of the University of Glasgow where we will present our research findings. We will also actively engage in Knowledge Exchange activities which are often organised by the University and will utilise Glasgow Sustainable Development Network to communicate with public and industry. We will interact with the NERC Innovation and Communications team where appropriate.

2. Outreach:
It is essential that the public are aware of ocean acidification being an important environmental issue. Additionally there is no UK or international legislation that currently addresses the problem of ocean acidification, therefore educating the public as well as informing the policy makers about its profound environmental impacts on the marine systems is essential.
Action:
This project is an ideal vehicle with which to inform the public of environmental issues such as ocean acidification and to explain one way in which scientists strive to understand the impacts of environmental change. We will present this work at the Glasgow Science Festival that attracts more than 50,000 visitors each year. We intend to receive the feedback in the form of a questionnaire that will quantify the impact of the demonstration of the participants' understanding of ocean acidification.
A blog site detailing our progress will be launched at the beginning of the project. We will ensure that our publications and workshop proceedings are covered by the media and issue press releases throughout the project accordingly. By adopting an open source approach in releasing the software associated with metagenomics on the blog site, we will become a part of a community of users and developers who will have an interest in working together to support each other. Any software bugs will tend to be more visible and more rapidly corrected to improve the software. It will also establish reputation and bring intangible benefits of goodwill.

3. Training:
The innovation and high reward of interdisciplinary research is well recognized. To maintain the leading status of the UK's science on the international stage, it is essential to train more young researchers with necessary skills for multidisciplinary research. This is important for environmental microbiology, where new tools and technologies are urgently needed.
Action:
we will develop a number of short-term projects for BEng undergraduate or masters students. The focus will be on additional derivative experiments of this proposed study that will train students in multidisciplinary approaches that combine engineering, microbiology, and mathematics. For instance, the proposed technology can be used in several projects for the discovery of new enzymes where enzyme expression in "artificial cells" will be monitored and optimised for a specific function.

Publications

10 25 50
 
Description Raman spectroscopy provides a direct method to characterise biological cells without the need of sample preparation, and therefore has huge potential in a wide range of fields. Since Raman spectroscopy uses a laser, we have evaluated its influence on cell growth in novel single-cell microfluidic devices. This led to the discovery of different tolerance levels among various classes of microorganisms. This finding provides valuable guidance for characterisation of microorganisms using laser irradiation. We have also exploited machine-learning approaches and developed advanced sorting software with trained classifiers that significantly enhanced the accuracy of target identification. These key developments allows efficient and non-invasive sorting of complex microbial communities, as is often found in natural environments.
Exploitation Route The discoveries of different responses from Gram-negative and Gram-positive bacterial to laser irradiation can have profound impacts on clinical safety regimes. The machine learning methodology developed can broadly benefit those working in analytical sciences.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The project has provided a fundamental platform for an industry-led new project with Nissan Chemical Ltd in the field of industrial biotechnology.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Standard research grant
Amount £577,256 (GBP)
Funding ID NE/P011063/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 05/2017 
End 04/2020
 
Description The IB Accelerator Programme
Amount £410,026 (GBP)
Funding ID Engineering microbial cell factories for industrial carotenoids biosynthesis 
Organisation IBioIC 
Sector Academic/University
Country Unknown
Start 05/2017 
End 11/2018
 
Description Collaboration with Nissan Chem Ltd. 
Organisation Nissan Chemical Industries Ltd
Country Japan 
Sector Private 
PI Contribution We have developed a microfluidic system that will be used in hospital diagnosis.
Collaborator Contribution Directly funded research and visiting scientists.
Impact PCT application Publications in preparation
Start Year 2013
 
Description Collabration with the University of Oxford 
Organisation University of Oxford
Department Department of Paediatrics
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided our partner with new methodology and platforms to investigate fundamental microbiology questions. New phenomena have been found.
Collaborator Contribution My partner provided us with complementary expertise in molecular biology and microbiology. They guided the design of microbiological experiments and trained the postdoctoral research in their labs.
Impact This collaboration has facilitated an award of an industry led project, entitled "Engineering microbial cell factories for industrial carotenoids biosynthesis" http://www.ibioic.com/funded_projects.htm.
Start Year 2016