1. Destruction of Industrial Biofilms Grant Burgess School of Marine science and Technology, Newcastle University

2. Overview What are biofilms and why are they a problem Structure of biofilms A 3.5 billion year old problem Discovery and characterisation of a biofilm dispersing compound Applications Conclusions

3. What are biofilms ? Biofilms are sticky layers of slime that build up on surfaces exposed to non sterile liquids

5. Why are they a problem Ship fouling Increased drag and fuel costs Corrosion Microbially influenced corrosion Food processing plants Costs of cleaning Breweries Wastewater treatment Laundry cleaning

6. A 3.5 billion year old problem Bacteria evolved about 3.5 billion years ago They have been competing with each other all this time Biofilms have evolved to provide a protective layer, for example against antibiotics Also To enable greater control of their environment, (pH, nutrients) To allow the development of complex communities which can repel outsiders

7. Breaking down biofilms

8. A marine discovery Studied seaweed surfaces in Scotland Many bacteria live on these surfaces Many strains of bacteria can attack and dissolve the biofilms of their competitors We isolated a compound that could dissolve biofilms Working with Mike Hall in the School of Chemistry we identified the compound as a DNA degrading nuclease, NucB from the bacterium Bacillus licheniformis

9. NUCB Purification NUCB was purified by a combination of ammonium sulphate precipitation and chromatography on a Q Sepharose column. The purity of the protein was analysed by SDS PAGE using a 20% acrylamide separating gel (see below) using increasing loadings (3 to 12  l) of the final pool of protein. The identity of the protein was confirmed by mass spectrometry (MALDI PMF). Approximately 12.5 mg of NUCB at greater than 95% purity was recovered from 800mls of starting culture supernatant. 3 6 9 12  l

10. Solution Mr determination of NUCB by size exclusion chromatography. Buffer: 50mM KPO4 pH 7.2, 1mM DTT, 150mM NaCl. Samples and concentration: NUCB 0.25mg ml -1. 200  L of NUCB chromatographed on a Superdex 200 HR10/30 FPLC column at a flow rate of 0.5m min -1, collecting 0.5ml fractions. The column was calibrated with Mr markers consisting of blue dextran, apoferritin, alcohol dehydrogenase, BSA, carbonic anhydrase and cytochrome C. NUCB (12-kda) eluted at the same position as cytochrome C (12.4kda). NUCB is monomeric.

11. Analysis of the far-UV spectrum of NucB using the Dicroweb server. The CD spectrum of NUCB was analysed using Dicroweb. This database contains the secondary structure content of thousands of proteins known from their crystal structures and also the CD spectra of these proteins. The programme looks for the best fit between the far UV CD spectrum of the protein under investigation and those in the database NUCB has a mixed secondary structure content that is highly similar to a protein that has been analysed by X-ray crystallography

12. NucB is a robust small protein

13. Effective removal of biofilm from steel surfaces

14. Towards commercial application UK patent granted 2011 PCT filed 2011 Currently working with several multinational companies that have expressed interest in this anti- biofilm technology In addition to industrial biofilm, many examples of biofilms causing medical problems Toxicity data also confirm safety Established protocols for scale up and production

15. Conclusions Understanding of science behind the problems can lead to new biotechnology products Multi-disciplinary team essential Requires good industry academia dialogue and mutual understanding Work with your local Universities to address, understand and solve your problems Develop a culture of Industry – University team work, not just one off projects

16. Acknowledgements Dr Nick Jakubovics Lecturer in Oral Microbiology Dr Reindert Nijland Utrecht Medical Centre Dr Mike Hall Lecturer in Organic Chemistry Dr Sam Neill Commercialisation Team Protein purification and characterisation: Alastair Hawkins, Heather Lamb and Paul Thompson Ms Nithya Rajarajan PhD student Chemical Engineering

17. Thank you Anti-biofilm active agent Enzyme activity