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.
 
Description Raman spectroscopy provides a direct method to characterize biological cells without the need for 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 the characterization 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 allow efficient and non-invasive sorting of complex microbial communities, as is often found in natural environments. With this capability, we have examined sea waters from different regions and discovered the effect of temperature on photosynthetic microorganism communities.

In addition, the platform can be used for microbiology studies in general. It provided a powerful tool for rapid antibiotic susceptibility assays and identifying antibiotic resistant cells. An advanced automated Raman based cell sorting with 3D microfluidics has been further developed, which has significantly enhanced the throughput and performance of the platform.
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. The platform and methods have now been used in rapid diagnosis of antibiotic resistance.
Sectors Agriculture

Food and Drink

Chemicals

Education

Environment

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Since the start of the project, we have developed cutting-edge single cell microfluidic technologies, including Raman activated cell sorting. This has led to several new collaborations and nine high-quality journal publications, which demonstrated the potential of these technologies to address several economic and societal challenges. We show the technology can provide an innovative route to screen novel bacterial strains for cost-effective microbial production. The foundation of this platform technology also led to several methods for rapid diagnostic of antibiotic resistance (ABR). The implementation of these tools to study the spread of antibiotic resistance has been demonstrated in the publications. With our industrial collaborators, we are implementing these technologies to screen herb medicines to search for an effective route to reduce antibiotic resistance.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Economic

 
Description An integrated microfluidic - single cell Raman technology for rapid diagnosis of pathogens and their antibiotic resistance
Amount £748,508 (GBP)
Funding ID 104984 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description EPSRC IAA project ""A gut microbiota chip for functional analysis of herbal medicine" co-funders: TianjinTCM and Horiba UK
Amount £108,739 (GBP)
Funding ID 312019/0 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 12/2020 
End 10/2021
 
Description LinkPI: Linking Phenotype function with Identity: a novel integrated single-cell technology and metagenomics approach
Amount £83,395 (GBP)
Funding ID NE/S008721/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 12/2018 
End 11/2021
 
Description NSFDEB-NERC The blueprint for marine biomineralization in a changing climate
Amount £566,464 (GBP)
Funding ID NE/W005115/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 07/2022 
End 01/2026
 
Description Nanotechnology for clean water: High throughput, In-situ analysis of biofouling in membrane separation processes
Amount £100,000 (GBP)
Funding ID NA170113 
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom
Start 11/2017 
End 10/2020
 
Description Standard research grant
Amount £577,256 (GBP)
Funding ID NE/P011063/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 04/2017 
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 United Kingdom
Start 04/2017 
End 11/2018
 
Title Automated Raman based cell sorting system and method 
Description Raman activated cell sorting has emerged as a label-free technology that can link phenotypic function with genotypic properties of cells. We have developed a three-dimensional hydrodynamic focusing microfluidic system for a fully automated, continuous Raman activated cell sorting. It has the ability to sort a range of cell sizes (from 1 µm bacteria to 10's µm mammalian cells) with stable operation over >8 hours and high throughput. 
Type Of Material Improvements to research infrastructure 
Year Produced 2021 
Provided To Others? Yes  
Impact The system provides a versatile tool for function-based flow cytometry and sorting applications in the fields of microbiology, biotechnology, life science and diagnostics. 
 
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 University of Warwick 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided a platform technology for studying microorganisms with single-cell resolution.
Collaborator Contribution Provided expertise in experimental microbiology and bioinformatics. The combination of these technologies provides us a powerful tool to understand and track the rapid spread of antibiotic resistance in the environment.
Impact This is multi-disciplinary research, including engineering, microbiology, and bioinformatics.
Start Year 2017
 
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
 
Description Prof Cha 
Organisation Chung-Ang University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and the development of "field-use" devices
Collaborator Contribution Professor Cha will bring the expertise in antimicrobial resistance gene detection from environmental samples using "SmartChip" technology for AMR monitoring and will test the "field-use" devices.
Impact The collaboration is multi-disciplinary, involves engineering, enviromental microbiology and water treatment.
Start Year 2018
 
Description Professor Cheng 
Organisation Boston University
Country United States 
Sector Academic/University 
PI Contribution Microfluidic platforms and Raman activated cell sorting technology
Collaborator Contribution Professor Cheng has been constantly at the most forefront of chemical imaging. He is world-leading in Stimulated Raman Spectroscopy technology. The combination of SRS with our microfluidic sorting platform will enable us to develop the next generation of high throughput cell sorting technology.
Impact The collaboration is multi-disciplinary, involves engineering, microbiology and optical physics.
Start Year 2019
 
Description Professor Ines Mandic Mulec 
Organisation University of Ljubljana
Country Slovenia 
Sector Academic/University 
PI Contribution Provide single-cell technology.
Collaborator Contribution Professor Mandic Mulec is a leading scientist in environmental ecology and has discovered a number of new species. The collaboration between us will significantly enhance our ability to understand the behaviour of these new species as well as their ecological roles in the community.
Impact The collaboration is multi-disciplinary, involves engineering and environmental ecology.
Start Year 2019
 
Description Professor Liu 
Organisation Chinese Academy of Sciences
Department Institute of Microbiology
Country China 
Sector Learned Society 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and single-cell microfluidic devices.
Collaborator Contribution Bring expterise in cultivation of microbes from various environments and molecular microbiology techniques.
Impact The collaboration is multi-disciplinary, involves engineering, enviromental microbiology and industrial biotechnology.
Start Year 2018
 
Description TCM 
Organisation Tianjin Modern Innovative Traditional Chinese Medicine Technology Co. Ltd
Country China 
Sector Private 
PI Contribution Provided single-cell microfluidic technology for studying antibiotic resistance of bacteria with single-cell resolution.
Collaborator Contribution The company will bring their expertise in traditional Chinese Medicine (TCM) to the collaboration. Together, we aim to explore the potential of using TCM to reduce the occurrence of antibiotic resistance.
Impact The collaboration is multi-disciplinary, involves engineering, clinical microbiology and pharmaceutical industry.
Start Year 2019
 
Description Univ. Ghent 
Organisation University of Ghent
Department Department of Biochemical and Microbial Technology
Country Belgium 
Sector Academic/University 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and the development of "field-use" devices.
Collaborator Contribution Our partner will bring flow cytometry expertise, in particular, a flow cytometric fingerprinting toolbox that can detect phenotypical and phylogenetic changes in microbial communities.
Impact The collaboration is multi-disciplinary, involves engineering, industrial biotechnology and water managment and treatment.
Start Year 2018
 
Description Public interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Dr Huabing Yin was invited as a panel member in a public event organised by Research Councils UK (RCUK) China, titled " "Beat the Bugs" - UK-China working together to tackle Antimicrobial Resistance". The event was held on Thursday 23 November, 2017, in the Naked Hub, Shanghai, (www.nakedhub.com).

More than 100 audience attended the event. They were students, postgraduates, the press, policy-related professionals, doctors and general public. The event was live streamed to reach an event broader audience.

Audience in the venue were very active, raising many questions and expressing their opinions and personal experience. It was a great success.
Year(s) Of Engagement Activity 2017