Molecular basis of algal-bacterial interactions and its implications for industrial cultivation of microalgae

Lead Research Organisation: University of Cambridge
Department Name: Plant Sciences

Abstract

The World is faced with the considerable challenge of supplementing, and ultimately replacing, its fossil fuel-based economy with one based on clean energy technologies such as biofuels. Currently, commercially available biofuels (e.g. bioethanol and biodiesel) are derived from crop plants such as maize and soybean. However, there are major concerns regarding both the use of valuable agricultural land for production of biofuel crops, and the sustainability and energy balance of such technologies. A potential alternative source of biofuels is microalgae - aquatic photosynthetic organisms that do not require fertile land for cultivation; grow considerably faster than plants, and which can accumulate significant quantities of high-energy compounds such as oils. Furthermore, such aquatic cultivation could be coupled to waste streams such as CO2 output from industry and nutrient-rich effluent, thereby using this waste to promote algal growth. However, industrial-scale cultivation of microalgae for biofuels faces considerable challenges, not just in terms of technical feasibility, but also in terms of economics and achieving a net positive energy balance. In particular, although the best rates of productivity of suitable strains are achieved in enclosed tubular systems, called photobioreactors, the energy requirement for building and operating these facilities is much greater than that in the fuel that is extracted. In contrast, growth in open raceway ponds generally results in energy savings compared to fossil-derived diesel. On the other hand, open ponds are at great risk from contamination by bacteria, viruses or competing algae. Crop protection is therefore a key issue that must be addressed to allow effective and productive commercialisation of algae. We have discovered an interaction between microalgae and bacteria that might provide a means to assist in this crop protection. Over half of all species of microalgae require vitamin B12 for growth - and they can obtain it from bacteria, in return for sugars made from photosynthesis. We have identified a possible explanation for why so many algae need this vitamin - it appears that loss of a particular gene, called METE, changes an alga from being effectively a 'hunter-gatherer', using B12 if it is available, to a 'subsistence farmer', needing to cultivate bacteria to ensure a proper supply of this vitamin. This suggests that there must be ways in which the two organisms signal to one another, and also that there is some advantage to this lifestyle, since it is so prevalent. In this project we will test our hypothesis, and determine if the growth of algae and bacteria together in cocultures affect the productivity of fuel molecules in the algal cells, and if it prevents contamination by invasive species. We will also use several molecular approaches to identify genes and proteins that might be involved in this interaction, in particular in the uptake of B12 by the algal cells.

Technical Summary

We propose a three-year project that will address a key bottleneck in the cultivation of microalgae on an industrial scale, namely the need to devise strategies to deal with contamination of cultures. We will build on our discovery of mutualistic interactions between microalgae and bacteria, in which the bacteria supply vitamin B12 to the algae in return for fixed carbon. Over half of all microalgal species have an absolute requirement for the vitamin for growth, indicating that they are dependent on this interaction. We have evidence from algal genome sequence data that whether or not an alga is a B12-auxotroph is determined by the absence or presence, respectively, of the gene for METE (a B12-independent form of methionine synthase). We have established a model system to study the interaction using Lobomonas rostrata, a close relative of the model green alga, Chlamydomonas reinhardtii, and the soil bacterium Mesorhizobium loti. We have embarked on sequencing the Lobomonas transcriptome; M. loti MAFF3030099 genome is already known. Thus the model system is tractable at the molecular level. We will use molecular, biochemical and physiological approaches to build on the preliminary work we have done to: (i) begin identification of genes involved in establishing and maintaining the interaction between algae and B12-producing bacteria, and to test the hypothesis that loss of the METE gene converts 'hunter-gatherers' (ie algae such as Chlamydomonas that use B12 if it is available) to 'subsistence farmers' (ie algal B12-auxotrophs such as Lobomonas that must cultivate interactions with B12-producing bacteria); (ii) investigate components of the B12 uptake and recognition in Lobomonas; and (iii) test whether cocultures confer advantages in terms of productivity of the fuel molecules (ie triacylglycerides), and resistance to invasive species.

Planned Impact

The topic of research in this application is relevant to a number of the major research challenges (so-called grand challenges) we face today: CO2 emissions and resulting climate change; dwindling reserves of fossil fuels, particularly those for liquid transport fuels, but also as feedstock for bulk and high-value chemical production; diminishing areas of arable land suitable for food crop production; and water management - both supplies of fresh water and waste-water treatment. Microalgae offer an enormous, as yet essentially untapped resource, which if exploited appropriately could lead to novel solutions to address ALL of the above. Many species have very fast rates of growth, and can accumulate high amounts of lipids, which can be used as fuel molecules. They can capture CO2 from flue-gas and scrub nutrients from effluent, and they do not require fertile land for cultivation. This has been recognized around the World by both governments and industry, leading to considerable investment in both research and development for algal biofuel production. Nevertheless, successful implentation of microalgal biotechnology will require much greater understanding of these organisms than we currently possess. In particular, to have both economic and sustainable algal cultivation at industrial scale will most likely involve the use of open ponds or raceways, which will be at considerable risk of contamination by adventitious organisms - predators, competing algae, or microbes. It is essential therefore that we increase our understanding of algal community biology, particularly in dense cultures that will be the norm in industrial operations. Our project will do just that, building as it does on our discovery of algal-bacterial symbiosis. We believe that - as well as enhancing our understanding of this important fundamental question in biology - the knowledge we gain will provide the means to devise strategies for algal crop protection. For example, cocultures are likely to prove more resistant to invasion by bacteria, since that niche will already be occupied. Moreover, if the cultivated alga is B12-dependent (there is a 50% chance it will be) then coculture with a B12-synthesising bacteria will obviate the need to supply this very expensive micronutrient. In the longer term, identification at the molecular level of components involved in symbiosis may provide opportunities to manipulate organisms to allow development of appropriate consortia of algae and bacteria for example to make novel products, or to maximise light capture across the spectrum by growing two or more organisms with different complements of light-harvesting pigments.

Publications


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Abalde-Cela S (2015) High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform. in Journal of the Royal Society, Interface
Helliwell KE (2013) Widespread decay of vitamin-related pathways: coincidence or consequence? in Trends in genetics : TIG
Helliwell KE (2014) Unraveling vitamin B12-responsive gene regulation in algae. in Plant physiology
Helliwell KE (2013) Widespread decay of vitamin-related pathways: coincidence or consequence? in Trends in genetics : TIG
Helliwell KE (2011) Insights into the evolution of vitamin B12 auxotrophy from sequenced algal genomes. in Molecular biology and evolution
 
Description Over half of all species of microalgae (simple aquatic plants) require a supply of vitamin B12 from their environment to grow. This compound is only made by bacteria and we have previously shown that algae and bacteria can live together in symbiosis, with the bacteria providing B12 in exchange for sugars from algal photosynthesis. In this project we wanted to explore how this common trait may have arisen in different algae, how B12 affects algal growth, and the implications for growth of microalgae for industrial purposes, for example for biofuel production.

We have shown that it is relatively straightforward for algae to become B12-dependent, by generating a strain of the green alga Chlamydomonas reinhardtii that now requires B12 simply by growing it for about 6 months in medium that contains the vitamin. The trait resulted from changes in a single gene, METE, which becomes non-functional. Interestingly, if B12 is removed the changes were reversible in some cells, so that they could now grow again without the vitamin.

We characterised the METE gene in more detail in Chlamydomonas, and found a region that could be used to regulate gene expression in response to B12. This is now being used to help generate strains of the alga that can make high value chemicals for biotechnological purposes.

We investigated whether growing algae with bacteria under outdoor conditions would help in production of compounds that could be used for biofuels, and instead found that the levels were lower. Although this was unexpected, it nonetheless provided clues about the interaction between the two organisms, in particular the way in which nitrogen was used in the cultures.
Exploitation Route METE promoter region that is B12 responsive and can be used to regulate transgenes in Chlamydomonas
Knowledge of how algal-bacterial consortia behave under natural conditions
Sectors Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology
 
Description Interactions with algal biotechnology companies has led to increased awareness of algal-bacterial consortia, how they might affect productivity and how they might be used to reduce contamination.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Chemicals,Energy,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description BBSRC IBBE Strategy Panel
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The Strategic panel provided advice and guidance for the setting up of the Networks in Industrial Biotechnology & Bioenergy (NIBBs), which aim to foster increased awareness and interaction between academics and industrialists in the bioeconomy. It was also important for the thinking behind the IB Catalysts, joint with Innovate UK & EPSRC. Many of the grants and proof-of-concept awards are aimed at increased sustainability through the use of IB solutions
URL http://www.bbsrc.ac.uk/funding/filter/networks-in-industrial-biotechnology/
 
Description FP7 Collaborative project (DEMA)
Amount € 4,900,000 (EUR)
Funding ID n° 309806 
Organisation Community Research and Development Information Service (CORDIS) 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 10/2012 
End 02/2017
 
Description Marie Curie ITN
Amount € 3,900,000 (EUR)
Organisation Community Research and Development Information Service (CORDIS) 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 11/2012 
End 10/2016
 
Description Marie Curie ITN on Quantitative Ecosystems 
Organisation Pierre and Marie Curie University - Paris 6
Country France, French Republic 
Sector Academic/University 
PI Contribution We are a partner in a consortium aiming to apply for an ITN to link statistical physics approaches and ecological/biodiversity research in marine systems. Our interests are in algal community interactions, so we provide a biological perspective
Collaborator Contribution Several of the groups are physicists and can provide a quantitative approach to tackle the questions we are interested in
Impact Biochemistry & Molecular Biology Ecology Physics Mathematics
Start Year 2014
 
Description Marie Curie ITN on Quantitative Ecosystems 
Organisation Technical University of Denmark
Country Denmark, Kingdom of 
Sector Academic/University 
PI Contribution We are a partner in a consortium aiming to apply for an ITN to link statistical physics approaches and ecological/biodiversity research in marine systems. Our interests are in algal community interactions, so we provide a biological perspective
Collaborator Contribution Several of the groups are physicists and can provide a quantitative approach to tackle the questions we are interested in
Impact Biochemistry & Molecular Biology Ecology Physics Mathematics
Start Year 2014
 
Description Marie Curie ITN on Quantitative Ecosystems 
Organisation University of Edinburgh
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We are a partner in a consortium aiming to apply for an ITN to link statistical physics approaches and ecological/biodiversity research in marine systems. Our interests are in algal community interactions, so we provide a biological perspective
Collaborator Contribution Several of the groups are physicists and can provide a quantitative approach to tackle the questions we are interested in
Impact Biochemistry & Molecular Biology Ecology Physics Mathematics
Start Year 2014
 
Description Marie Curie ITN on Quantitative Ecosystems 
Organisation University of Naples
Country Italy, Italian Republic 
Sector Academic/University 
PI Contribution We are a partner in a consortium aiming to apply for an ITN to link statistical physics approaches and ecological/biodiversity research in marine systems. Our interests are in algal community interactions, so we provide a biological perspective
Collaborator Contribution Several of the groups are physicists and can provide a quantitative approach to tackle the questions we are interested in
Impact Biochemistry & Molecular Biology Ecology Physics Mathematics
Start Year 2014
 
Description Marie Curie ITN on Quantitative Ecosystems 
Organisation University of Padova
Country Italy, Italian Republic 
Sector Academic/University 
PI Contribution We are a partner in a consortium aiming to apply for an ITN to link statistical physics approaches and ecological/biodiversity research in marine systems. Our interests are in algal community interactions, so we provide a biological perspective
Collaborator Contribution Several of the groups are physicists and can provide a quantitative approach to tackle the questions we are interested in
Impact Biochemistry & Molecular Biology Ecology Physics Mathematics
Start Year 2014
 
Description SIMS 
Organisation University of Stockholm
Country Sweden, Kingdom of 
Sector Academic/University 
PI Contribution We recently started a collaboration with researchers at the University of Stockholm and the Natural History Museum in Stockholm the metabolic exchanges between mutualistic microbes using Secondary Ion Mass Spectroscopy (SIMS).
Collaborator Contribution Our partners are experts in applying SIMS to microbial systems
Impact Molecular level understanding of microbial mutualistic metabolic interactions
Start Year 2015
 
Description Authentic Biology Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The Authentic Biology programme, funded by the Wellcome Trust, enables post 16 students to carry out real research in their schools. They then get to present their work at the symposium in London. I was invited to speak to them about my work on algal bioenergy, and then to engage with them about studying science at university. Several of the students (the majority from state schools) were encouraged to make applications to Oxbridge as a result.
Year(s) Of Engagement Activity 2015
URL http://www.authentic-biology.org/
 
Description BBC Radio 4 Frontiers - What ever happened to biofuels? 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was one of 4 invited participants in this programme, broadcast on BBC Radio 4 and available as a podcast. I had many emails and contacts from the wider public, including at the Festival of Plants exhibit, generally indicating that this had increased people's awareness of the possibility to use solar energy with algae, not just for biofuels, but also for other 'green tech'. Their attitude to biofuels per se was also more positive.
Year(s) Of Engagement Activity 2013
URL http://www.bbc.co.uk/radio/player/b02ykygv
 
Description Festival of Plants, Botanical Garden, Cambridge 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Exhibit at the Festival of Plants (was called Fascination of Plants in 2012) in Cambridge University Botanic Garden. It provided basic information about algae, showcased our scientific work, and then explained how we were using this in collaboration with companies to carry out remeidation of waste water. There were activities such as viewing microalgae with microscopes and observing algae phototaxis. Children could handcraft their own algae using pipe cleaners or take part in an interactive algae quiz to win prices. Several members of the public asked for additional information.

Interest from several visitors about how to incorporate algal growth in their activities, eg anaerobic digestion or green energy generally
Year(s) Of Engagement Activity 2012,2013,2014,2015
URL http://www.botanic.cam.ac.uk/Botanic/Event.aspx?p=27&ix=351&pid=2718&prcid=0&ppid=2718
 
Description IntoBiology resource 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact A final year undergraduate student in Plant Sciences produced a webpage describing work on algae in Plant Sciences and Biochemistry. The webpage is part of the programme established by IntoBiology, aimed at encouraging school children to consider studying plant biology.
Year(s) Of Engagement Activity 2015
URL http://intobiology.org.uk/unlocking-the-potential-of-algae-how-the-green-stuff-in-your-pond-might-go...
 
Description Pint of Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I presented a talk on the potential uses of algae for bioenergy production as part of the Pint of Science festival in Cambridge, where scientists engage with people having a drink in a local pub. This takes place in London, Cambridge, Bristol and other university towns each year. As result of my talk and questions afterwards, although most people were not aware initially what algae were, nor how they could be used to generate energy, their opinions became more positive and optimistic towards biofuels generally. Pint of Science was awarded "Points of Light" by the Prime Minister David Cameron in November 2015 and the founders were interviewed about it on London Live TV.
Year(s) Of Engagement Activity 2014,2015
URL https://en.wikipedia.org/wiki/Pint_of_Science
 
Description Science on Saturday, part of SET week in Cambridge 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We had a stand in the science tent outside Dept of Plant Sciences. We demonstrated what algae are, what we use for now and how they might be used in the future for energy production. There were activities such as viewing microalgae with microscopes, extracting different light-harvesting pigments, and observing algae phototaxis. Children could handcraft their own algae using pipe cleaners or take part in an interactive algae quiz to win prices.

School asked for visit to talk to years 10-13.

Established the material to mount a Royal Society Summer Science Exhibit
Year(s) Of Engagement Activity 2006,2007,2008,2015,2016
 
Description Science on Sunday 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact An exhibit showcasing the biology, diversity and uses of algae, with hands on activities for children and adults. Taking advantage of a 3-D printed microscope it was possible to engage people in (a) microscopic world (b) characteristics of algae and (c) use of 3-D printing. Children often could relate the hands-on activities as related to their Science Week in school the previous week. Adults reported feeling more informed about subjects that they had heard of (eg aquatic food chains, bioenergy) but did not have good grasp of the details.
Year(s) Of Engagement Activity 2016
URL http://www.botanic.cam.ac.uk/Botanic/NewsItem.aspx?p=27&ix=196
 
Description Science on Sundays 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Short talk and Q&A on the nature of algae and how their characteristics could be used for sustainable energy, water remediation, or other green tech solutions. Held in the Cambridge Botanic Garden, the audience were interested in plants, and so this was an opportunity to provide information on what algae are, and how they might be used in novel ways. The audience fed back that they felt much better informed.
Year(s) Of Engagement Activity 2015
URL http://www.botanic.cam.ac.uk/Botanic/NewsItem.aspx?p=27&ix=196
 
Description Science with Impact at SEB annual meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to present overview of using algae to capture CO2 and generate energy in "Science with Impact" session at Society for Experimental Biology (SEB) annual conference in Prague. Media interest on this session at the time, and session report published in SEB magazine (Autumn 2015, pp 50-51), prompting requests from schools and other science engagement organisations to speak.
Year(s) Of Engagement Activity 2015
URL http://www.sebiology.org/meetings/Past_Meetings/Prague2015/education.html
 
Description The Life Scientific on BBC Radio 4 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was interviewed by Jim Al-Khalili about my scientific career and interest in algae and vitamins. The programme "The Life Scientific" was broadcast on BBC Radio 4 and was also available on BBC iPlayer and as a podcast. As a result I have had numerous contacts about possible scientific and industrial collaborations, as well as enquiries from the general public about the various topics.
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/programmes/b08bzl8y