Ecological significance of surface bound enzyme activities in lichens

Lichens are symbiotic associations between fungi and photosynthetic microorganisms which can be either algae or cyanobacteria. Lichens are slow growing hardy "organisms" that typically grow on rocks, tree bark and unproductive soils. Common features of these habitats are aridity and scarcity of key nutrients, especially nitrogen and phosphorus. As is often the case, there are exceptions. Some lichens grow in almost permanently wet habitats such as below low water line in rivers and lakes, and on the rocky sea shore, while others grow at sites enriched with animal faeces where nitrogen and phosphorus are plentiful such as on bird perching stones or close to large animal colonies. However, lichens do not grow in habitats that are both permanently wet and rich in nutrients; such habitats are likely to be occupied by plants and/or algae. This new project is investigating how different species of lichen have adapted to habitats with contrasting availabilities of nitrogen and phosphorus. It is developing a new area of research in lichen biology with potential environmental and industrial applications.
Recent research at Nottingham University has shown that lichens produce a range of enzymes on their surfaces which are hypothesised to maximise their supply of available phosphorus. These enzymes, called phosphatases, catalyze the release of useful phosphate from organic compounds which otherwise cannot be used directly as a nutrient source. It turns out that species of very infertile habitats have high phosphatase activities - in fact rates of activity in such lichens are amongst the highest recorded among plants and microorganisms. The exact rates vary with background nitrogen availability (for example in rainfall) making some phosphatase activities useful indicators of nitrogen pollution. In contrast, lichens species that grow in nutrient enriched habitats have low phosphatase activities that vary little from site to site and that have functional properties that differ from those of lichen phosphatases in nutrient poor habitats. Lichens with cyanobacteria fix nitrogen from the atmosphere and therefore have a plentiful internal supply of this important nutrient but they have a high requirement for phosphorus. Preliminary results show that nitrogen fixing lichens that grow on the ground in woodlands have phosphatases that catalyze the release of phosphate from organic compounds found in leaf litter and soil. One class of such enzymes, called phytases, and shown to be active in the dog lichen (Peltigera species), is used widely in the agrochemical industry as an additive to animal feeds. Therefore novel phytases in lichens could prove to be of commercial value. Yet another enzyme, urease, might catalyze the release of useful ammonia from urea present in bird guano.
This project is examining habitat conditions that promote the production of these different surface-bound enzymes in lichens. These include the degree of nutrient enrichment, time of year and seasonal availability of leaf litter, and capacity for nitrogen fixation. The project will compare the properties of phosphatases and urease among a wide selection of lichen species from different habitats and also in single species occurring in a range of habitat conditions. It is known that phosphatases are produced by the fungal symbiont. In order to better understand their function we will find which parts of lichens are most active in producing surface enzymes, and their precise location in fungal cells. In addition, we will test whether lichen fungi produce the same enzymes when grown separately from their photosynthetic partners in the laboratory. The project will help us understand some of the physiological characteristics that adapt lichens to different habitats. It is hoped that information on phytase activities in lichens will lead to industrial collaborations and that phosphatase activity will provide a tool in nitrogen pollution monitoring.

1. Biotechnology companies, users of their products and society at large.
The research will identify the extent and properties of phytase activity in lichens and assess the capacity for enzyme production in pure cultures of lichen-forming fungi. Phytases are of considerable commercial interest: the market size for industrial enzymes is c. £2bn p.a. and c.10% of that is for phytases used in animal feeds. Phytases increase available phosphate to support animal growth and reduce phosphate loads in farm waste that cause eutrophication of water courses. Thus novel phytases will benefit manufacturers, farmers and the environment. New phytases continue to be brought to market and, while some are engineered products (e.g to improve thermostability), others are sourced from novel organisms. Colleagues in the fungal group at Nottingham already collaborate with two multinational enzyme producers (one a UK employer, the other a European employer). The PI has previously collaborated with several biotechnology companies on the exploitation of lichen-forming fungi for novel products. Hence, the Nottingham group is well placed to develop industrial collaborations on phytases. It has also initiated the first lichen fungus genome sequence programme; sequencing of Xanthoria parietina is now underway at the Joint Genome Institute in California (http://www.jgi.doe.gov/sequencing/allinoneseqplans.php). This genome will provide a major resource of genes to underpin novel enzyme discovery. The timescale for such benefits to be realized is 3-6 years.
2. Environmental agencies.
SEPA, SNIFFER, SNH and The Environment Agency have all commissioned research, or are in the process of doing so, to produce a lichen indicator scale for nitrogen/ammonia pollution that will use species investigated in the present proposal. UNECE relied heavily on lichen responses in recommending a new ammonia critical level for Europe (Ammonia Expert Workshop, Edinburgh, December 2006) which was accepted in April 2007; the PI contributed to the UNECE workshop. The proposed research will provide a physiological basis for lichen response to nitrogen and provide scientific underpinning and robustness to lichen scales under development. It will also provide data pertinent to development of new physiology-based biomarkers that have a faster response time to changes in nitrogen availability. All the above agencies could exploit such research in future field surveys. Future indirect beneficiaries will be those using the lichen scales thus developed and those who benefit from future decisions made on the basis of lichen indicators or from new air quality standards recommended by UNECE.
A prototype lichen scale for nitrogen pollution is being used c. 45000 individuals and groups in schools and community groups in the OPAL project (http://www.opalexplorenature.org/?q=LichenGuide). The use of lichen scales to indicate sulphur dioxide is now in GCSE and A-level syllabuses. This is likely to change to nitrogen pollution indicators as research results work their way from primary publications to reviews and text books to the attention of syllabus designers.
Indicators of nitrogen enrichment are important because nitrogen pollution is the principal cause of biodiversity loss in north temperate regions. Information from research projects will flow into agency programmes via contracts and communications at scientific meetings ahead of publication schedules. Therefore, benefits could be realised within 3-5 years from project launch.
3. Transferable skills
The PDRA will develop skills in lichen identification, isolation and culture of lichen-forming fungi and the measurement of phosphatases including phytase activity in addition to project management. Any company embarking on a programme to exploit lichen forming fungi for phytases would need some or all of these skills. Phosphatase assays could be applied in nitrogen pollution monitoring undertaken on behalf of agencies by, e.g., CEH