Discovering novel microbial tools to mitigate the global phosphorus crisis: Identification of unique phosphatases in abundant rhizobacteria

The demand to provide sufficient food for a rising global population in the face of several emerging global issues, such as climate change and depletion of natural resources, has resulted in an urgent need to explore novel ways of generating sustainable agricultural practices. For example, currently, agriculture massively relies on the intensive application of rock phosphate fertiliser to maintain sufficient crop yields. Unfortunately, predictions suggest that we could run out of rock phosphate, which is only produced through geological processes, within the next 50-200 years. In addition, only 10-30% of rock phosphate, applied as fertiliser, is taken up by the plant for growth. This inefficiency leads to problems such as eutrophication, which has negative effects on our environment and wildlife. Most soils are actually saturated with numerous forms of inorganic and organic phosphorus (oP) that are unfortunately unavailable to plants without prior transformation to an available form, orthophosphate.
Plants have a limited ability to mobilise phosphorus locked up in these various forms. Therefore, plants heavily rely on their associations with various microorganisms to assist them in acquiring bioavailable orthophosphate from various complex inorganic and organic forms. One of the major microbial mechanisms involved in transforming various soil oP complexes into orthophosphate is the production of extracellular phosphatases. Despite advances in understanding the role of microorganisms in mobilising soil oP, a complete and holistic understanding of this process remains unknown. Therefore, our ability to use microorganisms as a means of alleviating our reliance on rock phosphate is inadequate.
I recently discovered that a group of abundant bacteria related to the genus Flavobacterium, that inhabit the rhizosphere of Oil Seed Rape, the UK's 3rd most economically important crop, exhibited both strong and constitutive phosphatase activity. This phenotype appears to be both unique to Flavobacterium and potentially beneficial for plants. However, the specific enzymes responsible for this interesting and unique phenotype remain unknown.
The phylum Bacteroidetes (predominantly Flavobacteria), can constitute up to 65% of the plant microbiome and are major components of the human and marine microbiomes. Therefore, they likely have a major role in regulating environmental phosphorus dynamics. The major aim of this project is to therefore identify and characterise the novel unidentified phosphatases produced by these under-studied but interesting Flavobacteria. The two major outcomes of this project are 1) the discovery of novel microbial enzymes with potential agritech application 2) the discovery of new components in the soil oP cycle. Both of these outcomes will improve our knowledge regarding the global phosphorus cycle and sustainable agricultural practices, through increasing the efficiency by which plants utilise natural phosphorus resources locked up in soils.
I will undertake this Fellowship in the Department of Animal and Plant Sciences at the University of Sheffield using the lab of Prof. Tim Daniell as a host. The Daniell lab is based in the state-of-the-art Arthur Willis Environmental Centre, home of the Plant Protection and Production (P3) Centre, which unifies a diverse set of plant and soil scientists. The University of Sheffield was placed in the top 10% of research excellence in the UK (2014 Research Excellence Framework) and 99% of research at Sheffield that is internationally recognised with 48% of research in Biological Sciences classed as 4* world leading. Therefore, undertaking this prestigious Fellowship at Sheffield will provide me with the best opportunities to embark on a career in academia and tackle the emerging issues related to food security.

One solution to reducing our reliance on the non-renewable resource rock phosphate is to utilise the enormous potential for microorganisms to mobilise phosphate from various organic phosphorus (oP) complexes residing in soils. I have recently discovered that an abundant group of rhizobacteria related to Flavobacteria, elicit strong phosphomonoesterase and phosphodiesterase activity. However, the genes and enzymes responsible for this activity remain unknown. I aim to identify these uncharacterised phosphatases, using Flavobacterium johnsoniae as the model. To achieve this, I will perform reverse genetics, combining protein fractionation via size-exclusion chromatography, enzyme assays, proteomic analysis and subsequent bacterial genetics. This gained knowledge will allow me to generate a null mutant for phosphatase activity. This mutant will serve as a strain to systematically express the newly identified phosphatases and their variants found in closely related strains. Phosphatases showing strong activity will be subject to heterologous expression in E. coli BL21(DE3) as well as a Pseudomonas putida phosphatase null mutant I previously generated. This will both confirm their function and allow purification to homogeneity allowing subsequent determination of their enzyme kinetics and pH optima. Finally, I will determine the potential for Flavobacteria to enhance plant P uptake through the mineralisation of phosphate from oP complexes. To do this, I will grow plants at differing scales (agar plate, rhizobox and pot), quantify plant biomass, total phosphatase activity and determine soil dynamics using state-of-the-art analytical techniques, such as 31P-nuclear magnetic resonance spectroscopy. This project will greatly advance the fields of microbiology, microbial ecology, organic phosphorus cycling and plant microbiome research. The findings will also identify bacterial strains and individual enzymes for potential commercial exploitation.

The global demand for food is increasing. However, numerous factors, including climate change and rising costs of finite commodities such as rock phosphate, means that agriculture has to become much more efficient and sustainable to ensure food security for all. The major aim of this project is to identify the novel genes and enzymes expressed by abundant rhizosphere-dwelling bacteria, related to Flavobacteria, that mobilise the standing stocks of organic phosphorus in soils.
The discovery of novel enzymes in abundant rhizosphere-dwelling bacteria will be of significant interest to several organic farming and bioagronomic companies, such as ICL Innovations, Biotechnica and Tricet and to a lesser extent Syngenta, who all sell commercial products related to enhancing crop production. In addition, understanding how Flavobacteria mineralise organic phosphorus will determine their suitability as commercial bioinoculant products and will help form collaborations with these companies. Specifically, from the outcomes of this research, there will be potential for further trialling of these Flavobacteria isolates to determine if they can enhance current bioinoculants or enzymatic cocktails in order to increase biofertiliisation of phosphates in cropping systems.
Due to the rising cost associated with finite commodities such as rock phosphate and oil (transportation, fuel), as well as growing socioeconomic and political issues associated with the mining process itself, decreasing our reliance on intensive farming will also result in a positive economic gain for the UK. Therefore, this research project will open up longer term research avenues that could impact on the nation's wealth through two mechanisms 1) reducing our cost of crop production and 2) potential commercial exploitation and export of products to other countries.
At present, plant uptake of phosphate fertilisers is highly inefficient compared to the amount of nutrients added, which results in a proportion of rock phosphate fertilisers leaching into rivers, driving eutrophication. Therefore, the potential application of functionally beneficial bacteria would also help reduce our impact on the environment through a reduction in use of nutrient fertilisers. This would therefore have an impact on human and animal wellbeing in many areas interacting with water bodies, such as rivers and coastal areas.
The research proposal will also feed into a growing body of information that will increase public awareness in global food security issues, such as increasing food production to meet global population rise whilst mitigating the detrimental side effects of agriculture (i.e. eutrophication). Through public engagement activities the research project will aim to affect food consumers' behaviour and make them think about ways to mitigate their own food consumption footprints i.e. buying more organically sourced foods and being less wasteful with the foods they buy. Indeed, by disseminating the issues surrounding intensive farming, food consumers can also improve their health by switching to more sustainable organic foods which contain less harmful insecticides and fungicides, which have recently been linked to increases in cancer-related illnesses. Therefore, during the timeline of this project, the proposed research and its activities will be able to make very short-term impacts of public health and wellbeing as well as developing longer term strategies to help mitigate issues surrounding costs and environmental issues through reducing our use of inorganic phosphate fertilisers.