Biofouling: unexpected,

underestimated,
undertreated
Biofouling happens while you do other
research or production…

Hans-Curt Flemming

Biofilm Centre

IWW Water Centre, Mülheim an der Ruhr

- Consulting - Research - Service -
 What is Fouling?
Definition adopted from heat exchanger technology:
Fouling is the undesired deposition of
material on surfaces (Epstein, 1981)

• Inorganic fouling (precipitation of inorganic crystals),

„Scaling“

• Organic fouling (deposition of fat, oil, protein etc.)

• Particle fouling (deposition of silt, clay, humic particles

etc.)

• Biofouling (deposition and growth of microorganisms on

surfaces) – particles which can multiply on the expense
of nutrients
Biofouling has a bright future
An example: Washing machines
- Slime and mold behind washing soap intake box
- Slime formation in tubes
- Clothes are smelly if not dried fast enough
- Insufficient disinfection, infections spreaded

Cause: Biofouling – due to protection of environment and clothes

h Biologically readily degradable washing soaps
h Water saving & increasing washing soap concentrations
h Liquid washing soaps stick stronger to walls – higher local
concentrations at interfaces
h Low temperatures while washing
h No more peroxides or other bleaching agents in washing soap
h Water with washing soap remaining in tubes for extended periods
of time because it is not completely drained after washing
Domestic biofouling:
Toilet rinsing water reservoir, red and
dark brown colour: bacterial and fungal
biofilms in calciferous incrustations
Potential problem: dispersion of
aerosols when flushing
Mold formation and a
head-space detector
Inner tubes of showers:
massive biofilm growth,
habitat for hygienically
relevant organisms which
can be distributed as
aerosols
(U. Szewzyk, TU Berlin)
 Medically relevant biofilms

Keratitis due to non-compatible biofilm

on contact lense
Biofouling on teeth:
cause for bad breath
and caries
 Biofouling in dialysis
unit

Microbial contamination of
an injection syringe
 Contamination pathways in
catheter application
www.apic.org/resc/ gdivfig1.gif

Intravenous
catheter with
biofilm
 Hole due to slime

Paper mills: An ideal biofouling environment: free access

of bacteria from air, water and paper components
Fouling chances increased by
- chlorine-free process
- neutral pH
- higher concentrations of nutrients
- temperatures between 30 and 45 °C
- Use of contaminated additives
Pea production
Left: coupon colonization after 3 months of operation
Right: surface of ventilation system (courtesy of Holah et al.)
Biofouling: a
serious problem for
cooling cycles in
energy industry
h Heat transfer †
h Drag resistance y
h Biocorrosion
How good intentions cause inexpected problems: Massive biofilm
development in a heat exchanger due to biodegradable corrosion
inhibitor
Biofilms in water systems affect:
! Water quality (contamination by released
microorganisms)
! Health (release of pathogens in water and
pathogen-containing aerosols)
! Hydrodynamic parameters (clogging,
friction & hydraulic resistance)
! Material (covering of surfaces, change of
surface properties, microbially
influenced corrosion [MIC])
 Health hazards by biofilms:

Legionellosis
• Every case is proof of contact with an improperly
managed water system

• Every case could have been prevented by

applying practical scientifically-proven risk
management principles

• Legionella are typical biofilm-related pathogens

as they survive in protoza which graze on biofilms
 Biofilm

Fouling layer: Secondary

membrane which participates in
the separation process
h Drag resistance to crossflow
h Hydraulic resistance (Δp) Membrane
h Improvement of separation
characteristics?
 SEM images: Dr. Susan Bradford, OCWD

Microbial degradation of a
cellulose acetate reverse
osmosis membrane
 Interfaces in drinking water systems
Drinking water reclamation: inner walls of Treatment: surfaces of filter media (sand,
wells, well plumging, pump plumbing, etc. activated carbon), membranes, etc.

Drinking water distribution: inner walls of Drinking water reservoirs: walls,

pipes, made of mineral, metallic and floors, ceilings, microlayers of
polymer surfaces, hoses etc. calciumcarbonate and bacteria
Massive biofilm development due to
release of biodegradable substances from
rubber coating.
Supporting growth of coliform bacteria

Rubber-coated
valve after
exposure for 3
months
Biofilm
thickness: 0,5 - Protozoa
3 mm

g SEM Nematodes
Establishment of pathogens in drinking water
biofilms (cultivation, FISH, immunology)
Organism Persistence
Coliforms max. 3 weeks
E. coli 1-3 days
Yersinia up to 2 weeks
Campylobacter up to 4 weeks
Helicobacter pylori 2-3 weeks
Aeromonads could not be established
Atypical mycobacteria persistent
Pseudomonas aeruginosa > 4 weeks
Giardia cysts 1-3 days
Cryptosporidium oocysts 1-3 days
Enteroviruses 7 days
Legionella persistent
If nutrients were added in low concentrations and if the
contamination was extended to 24 h, most pathogens could
be established in the biofilms
 What to do against biofouling?
The Quest for the Silver Bullet
• Get rid of biofouling once and forever!
• The ultimate anti-fouling coating: Tributyltin
paints – a Silver Bullet too strong

Normal oister shell

Oister shell,
exposed to TBT
Time scale for fouling protection
Kevin Marshall: „The organism always wins“ (Question: how fast!)

Anti-Fouling requirements in different time scales:

Hours to days
- Some Catheters
- Surfaces in contact with food

Days to weeks
- Environmental sensors
- Dialysis units

Weeks to months
- Heat exchanger systems
- Membranes and filters for water treatment

Months to years
- Ship hulls, oil rigs
- Drinking and process water pipes and reservoirs
What is Biofouling?
„Undesired deposition and growth of
microorganisms at interfaces“
! Biofouling is a biofilm problem
! Dissolved substances are converted
into locally accumulated biomass
! Operational definition: Biofilm growth
exceeding „threshold of interference“
! Biofouling: A biofilm reactor in wrong
place & time
! Understanding biofilms is the key for
sustainable anti fouling strategies
 Biofilms: The social life of
microorganisms
Aggregates of microbial cells at interfaces
- solid-liquid
- liquid-liquid
- liquid gaseous
Biofilms are kept together and adhered to surfaces by a matrix of
extracellular polymeric substances (EPS), associated with working
materials, corrosion products, debris, soil, particles etc.)
The definition includes films on surfaces, flocs („floating biofilms“),
mats and sludges – all kinds of cells in matrices
Some characteristics:
- Long retention time of cells next to each other, gene exchange
- Formation of microconsortia, synergistic use of enzymes
- Hetererogeneity in space and time
- Physiological differences between planctonic and biofilm cells
Significance of Biofilms
" Environment:
- Carbon-Cycle: binding of CO2, degradation of Corg („Self-
purification“ of soils, sediments and surface waters,
degradation of particles)
- Biogeochemical cycle of N, O, S, P and metals
- Water retention in soils

" Technological use of biofilms:

- Biological waste water treatment
- Biological waste treatment
- Biological waste air purification

" Detrimental biofilms (biofilms in wrong place & time):

- Biofouling
- Biocorrosion of metallic, mineral and synthetic materials
- Acid mining drainage
EPS = Extracellular Polymeric
Substances: The house of the cells
" Polysaccharides, proteins, nucleic acids, lipids, forming
hydrogels (> 95 % water) of microbial origin
" Attachment of cells and of entire biofilm to surfaces
" Cohesion in microbial aggregates
" Fill and form the space between the cells, shape the
three-dimensional biofilm structure: Micro-environment!
" Immobilize cells, allow long-term synergistic interactions

EPS provide functional architecture of

microbial aggregates
Separate visualisation of EPS and cells
red: alginate-bound fluorescent ConA
green: P. aeruginosa cells, Syto 9

Spatial distribution of EPS components

" Volume element: 1 µm2, 1 nm thick
" 10 molecules of Alginate (1 Mio D, 1-3 µm, black)
" 300 protein molecules (30 kD, globular, red dots)
Biofilms: the oldest form of life on earth recorded:
3,5 billion years
Sandstone cliffs South
Bondi, Sydney
Bacteria in pore,
buried in EPS layer
preventing desiccation
More Sydney sandstone biofilms,
involved in weathering
Steps in biofilm formation
a) Formation of conditioning film
b) Primary adhesion, reversible
and irreversible
c) Formation of microcolonies,
surrounded by EPS
d) Development of a continuous
biofilm
e) Sloughing off of biofilm parts
f) Transport of biofilm particles
(flocs) throughout the system,
initiation of further biofilm
formation
 AVertical CLSM view

Artistic model derived

from that view

„Mushroom-model“
of biofilms
(Center for Biofilm
Engineering, Bozeman)
Oxygen gradient profile in a biofilm
(de Beer et al., 1994)
Biofouling: What to do?
Steps in fouling cases
1. Detection
2. Sanitization
3. Prevention
Biofilm sampling:
On surfaces!
Sometimes, remote surfaces have to
be sampled

Methods currently available:

! Scratching, scratching, scratching (razor blades,
rubber scrapers, cotton swabs etc.)
! Removal of parts of support material with biofilms
⇒ laboratory
! Expose test surfaces („coupons“), remove after
given time ⇒ laboratory
Verification of biofouling:
h Field tests:
- Visual observation: slime
- Smell
- Texture (slimy)
- Smolder test: heat sample, go for smell of burnt hair

hLaboratory:
- Microscopical observation, cells/cm²
- Cultural methods (cfu/cm²)
- Chemical analysis (water content, org./inorg.)
- Spectroscopical analysis (e.g., FITR)
The medicine-based strategy
• Biofouling is considered a „technical disease“
• Microorganisms cause this disease
• Kill microorganisms and the problem is solved
• Adoption of medical term: „Disinfection“ to cure the system

Drawback of this strategy:

- In many cases ineffective because biomass, not activity is the
problem, dead or alive
- Killing does not necessarily remove biomass: dead biomass can still
cause problems (heat exchanger, membranes, process waters etc.)
- „Disinfection“ means only „Inactivation of pathogenic organisms“
- Frequent failure of this strategy creates jobs and contributes to the
gross national product but not to sustainable solution of problems
 Killing vs. removal
colony count biocide treatment total cell count biocide treatment
colony count control total cell count control
10
10 1. biocide- 2. biocide- 3. biocide- 4. biozide-
colony count and total cell count [cm ]
-2

10
9 treatment treatment treatment treatment

8
Cells/cm²
10
7
10
6
10
5
10

10
4 cfu
3
10
2
10
1
10
0
10
7 8 9 10 11
time [d]

Total cell count with DAPI

Colony counts on R2A agar, incubation 7 days at 20 °C

Repeated H2O2 treatment for 15 min

(Courtesy of Simone Schulte)
Still the best:
mechanical cleaning

Cleaning: you
must overcome
adhesion and
cohesion forces,
key parameter:
Biofilm stability
Compression
experiments
(Körstgens et al., 2000,
2001, 2002)
 Five fundamental problems in
conventional anti-fouling strategies
1. Monitoring by process performance or product quality
Æ No early warning systems

2. Sampling of water phase and not on surfaces

Æ No information on biofilm site / extent

3. „Disinfection“
Æ No cleaning: dead biomass on surface, good
regrowth conditions

4. No nutrient limitation
Æ Nutrients = potential biomass; not reduced by
biocides, maybe even increased (O3 and humics)

5. Efficacy control by process/product quality

Æ No optimization of countermeasures
Alternative: Strategies based on
better understanding of biofouling
Biofouling: „Undesired deposition and
growth of microorganisms at interfaces“
! Biofouling is a biofilm problem
! Dissolved substances are converted into
locally accumulated biomass
! Operational definition: Biofilm growth
exceeding „threshold of interference“
! Biofouling: A biofilm reactor in wrong
place & time
Elements of an integrated
Anti-Fouling Strategy:
Holistic approach, based on understanding
the principles of biofilm development
! Apply risk assessment to your system
! Surface design and modification
! Cleaning friendly design
! Biofilm management
! Biofilm analysis
! Monitoring of deposit development
The most obvious target: Surfaces
Surface modification to influence primary adhesion

- Material selection (however, on a long term, all materials can be

colonized)
- Surface properties (hydrophobic, hydrophilic; hydrogels – selection for
specialized organisms)
- Surface topography (roughness, nanostructures, lotus effect - needs 3
phases, beware of surfactants!)
- Antiadhesive coatings (Can be fouled by abiotic matter)
- Surface bound biocides (Mechanism obscure; sometimes even effect
on water phase claimed; fate of dead cells; Fouling)
- Biocide releasing surfaces (Water contamination)
- Self polishing surfaces (Water contamination)
- Functional surfaces (H2O2+Catalyst; UV-light) (Fouling)
- Conductive coatings, application of electrical current (Fouling)
- Reversible change of surface properties (Fouling)

0 How long does the effect last?

0 Toxicity
Most promising: Low adhesion surfaces, easy to clean
 Biofilm Management

Threshold of
interference
Biofouling Potential:
1. Ubiquitous, always hungry biofilms
2. Nutrients from medium and support,
representing potential biomass
3. Surfaces inviting for colonization
4. Undisturbed growth conditions, e.g.,
infrequent cleaning
Strategy: Put the
biofilm into the
right place
Example:
membrane system
for treatment of
heat exchanger
water
Biofilter ahead of
the membrane
Biofilm

Biofilm

Membrane
Membrane Porous
Porous support
support

Before biofilter After biofilter

Thin cut of biofilm on membrane

Biofilm parameters before/after filter
Parameter unit before filter after filter

Cell number [#/cm2] 1,0 x 108 5,5 x 106

CFU [#/cm2] 1,0 x 107 1,2 x 106
Protein [µg/ cm2] 78 4
Carbohydrates [µg/ cm2] 26 3
Uronic acids [µg/ cm2] 11 2
Humic subst. [µg/ cm2] 41 12

Biofilm thickness [µm] 27 3

Flux decline [%] 35 <2

Natural Anti-Fouling Strategies
h Biochemical defense by surface-bound or excreted
metabolites
* Toxins (some are very toxic)
* Pheromones (AHL´s)
h Mechanical defense
* Mucus, cilia, skinning, scraping
h Extrinsic defense
* Selected epibionts („personal“ protective flora)
* Specialized epibiont predators (grazers)

In general: more than one mechanism is

employed – strategies with defense lines
Monitoring - the key to successful
anti-fouling strategies
! Early warning saves remedy
efforts
! Efficacy control of cleaning
procedures
! Optimization of cleaning
! Control of anti-fouling strategies
Biofilm monitoring:
Observation of biofilm development over time

What do we want to know?

! Site/location
! Quantity
! Thickness
! Distribution
! Nature of deposit
- organic/inorganic
- biological/abiological
- chemical composition
! Stability (how easy to clean?)
! Kinetics of formation and removal
The optimal monitoring method:
! in situ
! in real time
! on line
! non destructive
! fast and accurate
! Integrates over large surface areas
! easy to handle, stable
) Has predictive power
) Early warning capacity
) Saves value
An example: Fibre Optical Device (FOS)
illuminated area active area

reading area

illuminating
fiber
reading fiber

Optical fiber with illuminating and reading areas;

Overlapping: active area

Onvida GmbH, Duisburg

 Biofilm growth in a brewery water pipeline
from November 2000 to February 2003

4 5
1 2 3
Sensor signal

2,5
(arb. Units)

1,5

1
0,5

1.1.01 1.7.01 1.1.02 1.7.02 1.1.03

Time
-2 -2
#/cm (DAPI) cfu cm (R2A)
_ _
1 Biofilm growth during holidays (4 days)
6 6
2 Cleaning of Sensor and direct cell enumeration 13.10 1,7.10
_ _
3 Cleaning of Pipeline
4 Cleaning of Sensor and direct cell enumeration, 6 6
53.10 0,42.10
Signal amplification 100 fold decreased
6 6
5 Cleaning of Sensor and direct cell enumeration 6200.10 260.10
 Differential turbidity
measurement device
(DTM)

Comparison of DTM, slime

board and drag resistance
device in a paper mill
 Conclusions: Five principles of
alternative anti-fouling strategies
1. Learn to live with biofilms and keep them under
control
2. Monitoring: crucial for timely detection of biofilms
and countermeasure optimization
3. Cleaning is more important than killing biofilm
organisms
4. Biofilm management: Nutrient limitation
(nutrients are potential biomass)
5. Apply risk assessment, e.g., on terms of HACCP
) It is possible to live with biofilms, when you
understand them!
Apply HACCP concept creatively
Example: Risk assessment in production pathway

Production path Critical Control Point: protection bath of the car body

Bacterial aggregates
between metal and
paint

Paint comes off in dots

Acknowledgements
Research group „Physical chemistry of biofilms“:
Sascha Broekman
Werner Borchard
Thomas Griebe
Karl-Erich Jäger
Hubert Kuhn
Volker Körstgens
Christian Mayer
Harry Ridgway
Alex Rode
Natascha Schürks
Martin Strathmann
Petra Tielen
Wiebren Veeman
Jost Wingender

" Deutsche Forschungsgemeinschaft (DFG)

" Ministery for Research (BMBF)
" Deutsche Bundesstiftung Umwelt (DBU)