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

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.

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.

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.