Transcript Grant Burgess

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 biofilms
 A 3.5 billion year old problem
 Discovery and characterisation of a
biofilm dispersing compound
 Applications
 Conclusions
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What are biofilms ?
Biofilms are sticky layers of slime that build up
on surfaces exposed to non sterile liquids
Why are they a problem
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Ship fouling
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Corrosion
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Microbially influenced corrosion
Food processing plants
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Increased drag and fuel costs
Costs of cleaning
Breweries
 Wastewater treatment
 Laundry cleaning
A 3.5 billion year old problem
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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
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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
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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
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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 12ml) 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
12ml
Solution Mr determination of NUCB by
size exclusion chromatography.
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Buffer: 50mM KPO4 pH 7.2,
1mM DTT, 150mM NaCl.
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Samples and concentration:
NUCB 0.25mg ml-1.
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200mL of NUCB
chromatographed on a
Superdex 200 HR10/30
FPLC column at a flow rate of
0.5m min-1, collecting 0.5ml
fractions.
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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.
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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 antibiofilm 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
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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 Mike Hall
Dr Reindert Nijland
Lecturer in Organic
Chemistry
Utrecht Medical
Centre
Dr Nick Jakubovics
Lecturer in Oral
Microbiology
Ms Nithya Rajarajan
PhD student
Chemical
Engineering
Dr Sam Neill
Commercialisation
Team
Protein purification and characterisation:
Alastair Hawkins, Heather Lamb and
Paul Thompson
Thank you
Anti-biofilm
active agent
Enzyme activity