Regulation of polyphosphate metabolism in Chlamydomonas and potential for exploitation as P phosphorus sink in nutrient recovery systems

Phosphorus is an essential element for life. It is a component of our genetic information (DNA and RNA). It plays critical roles in many aspects of our metabolism including the major energy currency of our cells ATP. It is a component of the membranes which surround our cells. It cannot be substituted for any other element. We obtain our phosphorus from our diet in the form of phosphate (phosphorus bonded to four oxygen atoms). Phosphorus enters the food chain through the acquisition of phosphate from the soil by plants. Plants, like all forms of life require this element and as any gardener knows it is one of the important macronutrients for plant growth- the P in your NPK fertiliser. Although it is quite an abundant element, most of the phosphorus in the environment is not in a form that plants can readily make use of (i.e., as soluble phosphate). Much of the phosphorus is locked in organic matter in the soil or bound to the surface of soil particles. This is why to get a good crop yield gardeners and farmers apply phosphate containing fertiliser. However there is a two-fold problem. To meet current needs rock phosphate is processed into phosphate fertiliser, but this non-renewable resource is being used and not replaced. At current rates of usage we will run out in between 50 and 200 years, so business as usual is not an option if we want to feed our children's children. The other problem is a more immediate one; much of the phosphorus we do use is wasted. Some of it enters water courses as agricultural run-off and can cause algal blooms in rivers and lakes. Much of it ends up at sewage works in domestic waste water from urine, faeces, organic waste and detergents. Because of strict environmental regulations on water quality, removing the unwanted phosphorus is necessary and expensive, contributing to high water bills. Use of chemicals to precipitate the phosphorus is a common method but limits reuse as a fertiliser. Biological methods are also used, and this is an active area of current research, but so far none have truly solved the problem of recovering phosphorus in an economic and re-usable way. This proposal is to investigate the fundamental mechanisms by which a species of alga called chlamydomonas, a microscopic unicellular photosynthetic organism, take up and store phosphate. Chlamydomonas is naturally occurring in the UK and grows well on waste water using either organic compounds in the waste, or light, to provide energy. It can accumulate phosphate to potentially high levels but the mechanisms by which it does so are not fully understood. To be able to use chlamydomonas or other naturally occurring algae to clear up waste water efficiently and recycle the reclaimed phosphate as fertiliser we need to understand what these mechanisms are and how and when they operate. The aims of our project are 1) to look at the effect of changing expression of genes that are thought to be involved in phosphate accumulation and storage to understand their role and importance in this process; 2) to test an hypothesis about the function of a gene of currently unknown function whose mutant phenotype implicates it in phosphate metabolism and 3) to find out which genes are important by looking for mutants which hyper accumulate phosphate. This fundamental knowledge will help engineers to design low-cost, low-carbon and environmentally friendly systems for phosphate recovery at sewage treatment works for sustainable reuse in agriculture.

Under certain conditions microalgae can take up in phosphate in excess of metabolic need and store it as polyphosphate (poly P). While phosphate uptake and poly P metabolism has been relatively well studied in bacteria and yeast (S. cerevisiae) there is a paucity of information about this process in algae which is a clear research gap that needs to be filled if the ability of algae to grow on nutrient rich wastewaters is to be exploited as a means to recovering and recycling phosphate into fertiliser for sustainable agriculture. The availability of high quality annotated genome sequence, transcriptomic data and molecular tools make chlamydomonas the organism of choice for investigations into mechanisms of phosphate uptake and poly P synthesis and turnover. Some mechanisms may be conserved with yeast but regulation of phosphate deficiency responses is known to be quite distinct. In objective 1 we will investigate the role of the key transcription factor PSR1 and plasma membrane and vacuolar phosphate transporters in poly phosphate accumulation by genetically altering their level of expression in chlamydomonas and testing the consequences for phosphate uptake and poly P levels. In objective 2 we will investigate a candidate for putative poly P phosphatase. A mutant is available which has a phenotype that is consistent with an inability to degrade poly P however the function of the protein is currently unknown. Using degradation of poly P by wild type and mutant cell lysates and measurements of poly P accumulation in mutant and wild type we will test if the corresponding gene encodes a poly P phosphatase. This would be confirmed by enzymatic measurements with the recombinant protein. In objective 3 we will implement genetic screens to identify and characterise poly P hyper accumulating strains. Strains which show increased poly P accumulation will be tested at 2L photobioreactor scale to assess suitability for larger scale process use.

This project will cover fundamental research on the mechanisms controlling phosphorous uptake by microalgae, but with clear potential to apply the expected outcomes in the delivery of sustainable processes for nutrient recovery from waste and their reuse in agriculture. Microalgae have been proven to be effective at controlling nutrients (nitrogen and phosphorus) in small wastewater treatment works (i.e., waste stabilization pond systems), particularly in the tropics and under summer conditions in temperate climate countries, when weather conditions are favourable for stimulating photosynthetic activity. Therefore, there is potential for developing engineered algal bioprocesses to retrofit existing energy-intensive wastewater treatment works and contribute to the recovery of phosphorus from wastewater. This project will use Chlamydomonas reinhardtii as a model organism, which commonly appear in sewage algal ponds in the UK, because of the availability of excellent molecular tools to successfully achieve our research objectives. It is clear that chlamydomonas can under some conditions accumulate high levels of poly phosphate but the key steps regulating this process are still unknown. This project will therefore deliver short-term impact through (a) generation of new knowledge of the mechanisms controlling phosphorus (P) transformations by microalgae in aquatic environments; (b) development of new strains of Chlamydomonas to support further research in this area; (c) development of methodologies that may allow selection of high P accumulating isolates of other algae; and (d) through the interdisciplinary training of researchers who can contribute to the solving of the problems that currently prevent commercial applications of this promising approach. In addition, our expected research outcomes will deliver medium-/long-term impact by supporting the development of an algal-based alternative to current bacterial nutrient control processes in Sewage Treatment Works (STW), which will help to deliver: (e) sustainable routes for P control, recovery and recycling from sewage, as an alternative to current energy intensive bacterial processes; and (f) alternatives for access to renewable sources of P fertiliser contributing to reduce reliance on imports and to achieve food security targets in the UK.

New mutant strains and lines will be donated to the chlamydomonas collection at the University of Minnesota or CCAP in Oban as appropriate. By acquiring this knowledge it may be possible to develop robust systems that can be monitored and controlled to reliably accumulate polyphosphate in chlamydomonas or other microalgae, which are less genetically amenable than chlamydomonas but which may be more suitable for waste water treatment. This is a clear interest of water companies, as they are currently facing the potential challenge of more stringent phosphorous discharge consents (<0.1 mg P/L) and a ban on the spread of sewage sludge on land. An algal-based technology for nutrient control in sewage treatment works will contribute to reducing operation costs as they could eliminate the use of energy intensive bacterial processes for nitrogen and phosphorus control (i.e., nitrification/denitrification, bacterial biological uptake and chemical precipitation) and stimulate sustainable reuse of nutrients in agriculture via the production of high-quality bio-fertilisers based on algal biomass cultivation. The UK has the potential to become a world leader by setting the agenda for resource recovery from waste, in line with a circular economy approach, leading to the achievement of sustainable economic growth with clear benefits to the environment, public health, industrial sector and general public. As part of our impact activities, we will engage with key stakeholders interested in achieving such goals by actively supporting meetings of existing networks including the UK Nutrient Platform, water@leeds and the Wastewater Network.