The Blue Mountains in Australia and Blue Ridge Mountains of Virginia, are so-named because of the blue haze that results from atmospheric reactions with isoprene, a gas produced in abundance by plants and especially many tree species. Trees that are starting to be grown widely as a source of bioenergy, namely willow and poplar, are among the highest isoprene emitters. Isoprene protects plants against heat and light-induced damage, and can also serve as a signalling molecule. Isoprene is so reactive with other chemicals in the lower atmosphere that it limits their capacity to react with methane and also generates ozone. Both ozone and methane are potent greenhouse gases, and ozone impairs plant growth. On the more positive side, isoprene can indirectly stimulate cloud formation which provides a cooling effect. Hundreds of studies have investigated isoprene production, primarily from trees, and examined the effect of a changing environment on its flux from tree to atmosphere.
In stark contrast, only a handful of studies have shown that microbes in the soil consume isoprene, and a few of those microbes have been grown in the laboratory. Microbes are abundant (several billion per teaspoon of soil and more than a million per square cm of leaf) and the most important catalysts for cycling chemicals in the environment. We know from studying the cycles of other climatically important gases, like methane, that microbial consumption is an extremely important process that is greatly influenced by climate change. From hundreds of methane-consuming bacteria in culture, we have extensive knowledge of their metabolic pathways, which allows the development of investigative tools to help inform land-use management decisions. For isoprene, which is produced in similar abundance to methane, we lack this knowledge and tools.
Therefore, in addition to those bacteria that we already have in culture, we propose to culture isoprene-degrading microbes, focusing on soil and leaf inhabitants. Using powerful genomic-based techniques, we will determine the DNA sequences of the genes involved in isoprene degradation. Additionally, we will use tools developed by the PI to identify and investigate those isoprene degraders that are not easy to grow. Most bacteria look alike and so we frequently use DNA sequences to study their roles in nature. Selected unique DNA sequences will be used to identify, view and count key species of isoprene-degrading bacteria in natural samples. This will enable us to determine precisely where they live, e.g. we envisage that they will be especially abundant around stomata (pores in the leaf) from where most isoprene escapes; and the use of state-of-the art imaging techniques (developed by our project partner) will allow us to identify which individual microbes are actively degrading isoprene in the soil or on the leaf surface.
Complementing this study, a PhD student will measure isoprene consumption in forest soils, and for the first time, on leaves from various tree species, comparing isoprene emitters with non-emitters as well as sun and shade leaves. We will test whether adding permutations of isoprene-degrading microbes to leaf surfaces enhances consumption, and by measuring the microbes’ ability to survive or grow on the leaves, we will obtain insights into whether this is a potential strategy for reducing isoprene flux. All of the data emanating from this project will be valuable for management of natural woodlands and bioenergy crops, in relation to greenhouse gas emissions.
Summary of results
We have isolated several isoprene degrading bacteria from the terrestrial environment and shown that isoprene monooxygenase is the key enzyme responsible for isoprene degradation in these strains of Rhodococcus. This has an inducible enzyme system which is required for growth of bacteria on isoprene. RNA sequencing has shown that epoxyisoprene is an inducer of isoprene metabolism.
We have sequenced the genomes of several ne isoprene degrading isolates. This has given us valuable information on the genomic context of the isoprene monooxygenase gene clusters in these isoprene degraders. The gene isoA, encoding the large subunit of the hydroxylase component of isoprene monooxygenase is highly conserved in all isolates and thus is an ideal functional gene marker for the presence of these microbes in the environment.
Genome analysis has shown that all of the genes required for isoprene metabolism are carried on a large plasmid in Rhodococcus strain AD45. This has profound implications for the horizontal gene transfer of isoprene degrading ability in microbes.
We have developed PCR primer sets specific for isoA, a key gene involved in isoprene metabolism. This has allowed us to retrieve isoA genes from enrichment cultures and from environmental samples in order to develop cultivation-independent techniques for these important trace gas degrading bacteria.
We have carried out proof of principle experiments with 13C labelled isoprene and DNA stable isotope probing and identified the active isoprene degrading bacteria in soils and marine sediments. Isoprene degraders also appear to be present on the leaves of plants.
We have started to carry out measurements of isoprene production and consumption in environmental samples, including different tree species and soil surrounding these trees.
The isoprene monooxygenase might yield valuable reactions that might be used in a biotechnological context. We are currently investigating the activity and substrate specificity of this enzyme.
Potential impact of these studies
The project will provide data on isoprene consumption in woodland environments, molecular tools to investigate isoprene degradation, an understanding of the abundance and distribution of isoprene-degrading microbes as well as their potential to mitigate isoprene emissions. All of this will impact on the management of natural woodland and plantations of bioenergy crops (that may supply 10% of the UK’s energy), especially high isoprene emitters such as willow and poplar. Our research will have maximum impact because of the involvement of Dr J. Morison (Forest Research) as project partner, who has close collaborations with the Energy Technology Institute (ETI) and advises government departments. It complements Forest Research’s core program on the carbon and greenhouse gas balances of UK forests, which underpins both policy and practice in support of major initiatives for enhanced woodland creation as part of UK emissions abatement policy and the new Natural Environment White Paper. A key outcome, with input from University of Essex Interdisciplinary Centre for Environment and Society (iCES) and FR, will be a briefing document aimed at stakeholders (e.g. Defra, DECC, World Meteorological Organisation, Met Office, UNEP, IPCC, and IOC), and we will participate in end-user led meetings during the course of the project. In addition to having FR as a key link, we will continue to engage with key individuals in the marine (e.g. SOLAS) and terrestrial VOC community (e.g. via ESF’s VOCBAS initiative), who will be interested in applying our data to isoprene flux models.
Future industrial potential for this work might include the discovery of novel oxygenases for production of chiral synthons as precursors to pharmaceuticals / agrichemicals. Our research may also benefit the bioremediation industry, e.g. 1) by degradation of isoprene spills (800,000 tonnes pa of isoprene are produced by the petrochemical industry, and is set to increase with the advent of BioIsoprene synthesis); 2) because many isoprene degraders also degrade other volatile hydrocarbons like BTEX; and 3) isoprene degraders co-metabolise chlorinated solvents like TCE. In other words isoprene degraders may already play a major role in remediating some of the most abundant volatile pollutants, especially on the leaf surface. Again, this will impact on woodland management (our route to impact will be Forest Research), as well as planning green spaces in cities (our route to impact will be iCES who played a major role in the Government paper “UK National Ecosystems Assessment: Understanding Nature’s Role in Society”).
Research Activity Images
Most species of oak produce a lot of isoprene. Our studies include isolation and characterisation of isoprene-degrading bacteria from leaves and comparisons of soils around isoprene-producing and non-producing trees. For a comprehensive list of a plant species’ capacity to produce isoprene, see http://www.es.lancs.ac.uk/cnhgroup/download.html.
With funding from the Konstanz-Essex Development Fund we quantified isoprene and DMS concentrations in Lake Constance in the summer of 2014. Day-trips aboard the RV Robert Lauterborn from the University of Konstanz facilitated the collection of trace gas data in depth profiles of the large oligotrophic lake.