Who is the report targeted at?
The report is for researchers and industry investors interested in the scope for new chemical products from eucalypt oil, that may have broadscale commercial or industrial application.

Background
It is estimated that around 6 million hectares are affected by dryland salinity and this will increase to 17 million hectares in the coming decades. As a result, Eucalyptus trees are being planted at a large scale to address this issue. This will lead to an abundance of eucalypt oil available for world markets and hence presents an opportunity for Australia to discover and improve on eucalypt oil applications.

Incorporating added value products derived from biocatalysis of key eucalypt oil constituents with schemes such as the integrated tree crop system of Western Australia, would assist in the exploitation of these potential opportunities.

Novel applications for the eucalypt oil constituents in the chemical, solvent, perfumery, pharmaceutical, agricultural and paint industries are possible using biocatalysis. Environmentally, microbial or enzymatic transformations are preferable to traditional chemical synthetic approaches since they are less polluting and often require less energy consumption. Microbial transformations are also more specific in their catalysis and can often carry out a complex multi-step synthesis in a single transformation. This biological approach to chemistry has already been championed recently by chemical companies such as DuPont and Cargill-Dow. The more applications found for eucalypt oil the greater the positive impact on the environment through providing an incentive for farmers to plant trees to try to address the dryland salinity issue, and improve carbon sequestration.

The long term outcome for this project (i.e. beyond this proposal) is to derive new chemical entities with new applications using biocatalysis (such as microbial biotransformations or enzymatic transformations) from Eucalyptus leaf oil constituents, with a view to addressing the large surplus forecast for eucalypt oil. As the first phase of such a project, the establishment of a microbial library is required and is the subject of this project.

Aims/Objectives
The aims for this specific project were:

Establish a microbial library that can utilize 1,8-cineole as a sole carbon source either in a purified form or from crude leaf oil from E. polybractea.

Preliminary identification of chemical products derived from microbial transformations of 1,8-cineole using GC-mass spectrometry analysis.

Methods used
Methods were:

Enrichment of microbes using 1,8-cineole as the sole carbon source. Enrichment took place using a proprietary enrichment device developed at CSIRO.

GC Mass spectrometry analysis of enriched supernatant

Obtaining pure cultures by monoculturing onto plates of defined media, with the 1,8-cineole as the carbon source.

Purified cultures (scaled up on 1,8-cineole as the sole carbon source), metabolic byproducts from supernatants, and metabolic intermediates from cell extracts analyzed on the GC Mass spectrometer.

Pure cultures which produced potentially useful products from 1,8-cineole as the sole carbon source, were adapted to grow on crude eucalypt oil from E. polybractea.

Finally microbes were catalogued accordingly and stored appropriately depending on their identification.

Results/Key findings
The findings of this project were as follows:

A) Enrichment of microbes using 1,8-cineole as the sole carbon source: A proprietary device, the Evolver© invented at CSIRO was used to enrich for microbes that can grow on 1,8-cineole as the sole carbon source. By enriching for 1,8-cineole consumers, it was anticipated that 1,8-cineole derivatives would form either as terminal by-products or as intermediates of microbial metabolic function.

Using the Evolver©, a consortia of microbes were enriched to use 1,8-cineole as the sole carbon source. Unlike previous literature which reports 1,8-cineole microbial metabolism, the Evolver© allowed for the enrichment of microbes at a constantly controlled concentration of 1,8-cineole. This constant concentration of 1,8-cineole was 0.6 to 0.8g l-1 and such conditions have not previously been reported. Such conditions are unique in that we have now enriched for microbes that can survive and indeed grow in the presence of relatively high and constant concentrations of 1,8-cineole and thus produce 1,8-cineole derivatives in a manufacturing setting.

 B) Obtaining pure cultures by monoculturing onto plates of defined media with the 1,8-cineole as the carbon source: Enriched microbes that could grow on 1,8-cineole as the sole carbon source (obtained from the Evolver©), needed to be purified to monocultures, to allow for further analysis. Fourteen (14) strains of bacteria were isolated from samples obtained from the Evolver©, nine of which may be independently different strains. Although the strains isolated in this study have not been formally identified, preliminary characterization of the isolates suggests that additional species, not previously reported to grow on 1,8-cineole, may have been isolated.

Two isolates could grow on 2 g l-1 1,8-cineole, a level higher than previously attained in other studies. This is a significant achievement in a short time frame and using the Evolver© to further acclimate microbes to even higher concentrations would be strongly recommended.

One purified bacterial isolate (B2) was also grown on eucalypt oil (90% pure 1,8-cineole) to evaluate its ability to consume 1,8-cineole in the presence of other carbon sources. In this instance it was observed that B2 could grow readily in eucalypt oil.

All microbes isolated after 1,8-cineole growth have been catalogued.

C) GC-Mass Spectrometry analysis of 1,8-cineole derivatives: Purified microbes isolated during the monoculturing phase of the project were scaled up in the presence of 1,8-cineole as the sole carbon source. The supernatants and microbial lysates from the purified microbes, as well as from the Evolver©, were analyzed on a GC mass spectrometer to identify any 1,8-cineole derivatives that may have resulted during growth of the microbes on 1,8- cineole. Four compounds were consistently observed. Three of these are considered to be intermediates as they are present whilst the bacteria were growing, while the other one was considered as product because it appeared at the final stages of bacterial growth.

Mass spectral library matching using the National Institute of Standards Technology (NIST) database did not indicate any firm matches with the 4 cineole derivatives discovered, and our initial proposed definition would have indicated entirely new chiral compounds. However, on the basis of published literature, the three ‘intermediates’ are postulated to be two isomers of 2-hydroxy-1,8-cineole and 2- oxo-1,8-cineole. The ‘product’ has not been formally identified. In one case, there is also evidence consistent with the formation of a di-hydroxy-1,8-cineole. Several other compounds were observed when eucalypt oil (crude cineole) was used as substrate, but their concentrations were too low to allow mass spectra to be obtained in the time available.

This project has thus delivered on the outcomes stated in the initial application by:

Establishing a microbial library that can utilize 1,8-cineole as a sole carbon source in a purified form and from crude leaf oil from E. polybractea.

Identifying chemical products derived from microbial transformations of 1,8-cineole using GC-mass spectrometry analysis.

Implications for relevant stakeholders:
This project represents a preliminary investigation of the scope to derive new chemical entities, and will require further investment to determine its commercial prospects.

Recommendations
Further extension of this work is now necessary if the microbes and chemicals discovered are to be applied for commercial advantage. The following recommendations are made:

A) Identify the isolated microbes to species level in order to assess for any new species discovered.
B) Given the rapid manner by which microbes can be enriched using the Evolver©, the microbial library should be expanded. This is recommended since only a single pH and temperature was used as enrichment parameters in the Evolver©. Expanding the library through use of a range of pH and temperature conditions, would also allow for isolates such as B2 to acclimate to even higher 1,8-cineole concentrations.
C) Make sufficient quantities of 1,8-cineole derivatives to isolate and confirm the structures of 1,8-cineole derivatives using Nuclear Magnetic Resonance.
D) Screen 1,8-cineole derivatives for potential anti-cancer, anti-microbial and herbicide activities. Esterification with carboxylic acids may also lead to perfumery products.
E) Evaluate the chemical modification of 1,8-cineole derivatives for use as surfactants and solvents.
F) Isolate, characterise, clone and express the enzyme(s) responsible for the production of the 1,8-cineole derivatives in 1,8-cineole tolerant microbes. Using directed evolution, improve the enzyme activity to productivity levels acceptable for manufacturing.