Destruction of Industrial Biofilms

Grant Burgess

School of Marine science and Technology, Newcastle University

Overview

What are biofilms and why are they a problem

Structure of biofilmsA 3.5 billion year old problemDiscovery and characterisation of a

biofilm dispersing compoundApplicationsConclusions

What are biofilms ?

Biofilms are sticky layers of slime that build up on surfaces exposed to non sterile liquids

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

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

Breaking down biofilms

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

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 12l) 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 12l

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.

200L 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.

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

NucB is a robust small protein

Effective removal of biofilm from steel surfaces

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

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

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 RajarajanPhD studentChemical Engineering

Thank you

Anti-biofilm active agent

Enzyme activity