2. What is Biofouling?
• Biofouling* is the unwanted adhesion of
bacteria or other organisms onto surfaces of
solution-handling systems
• Biofouling is not necessarily uniform in
space & time
• Biofouling may contain significant amounts
of inorganic materials held together by the
polymeric matrix
*(Charackis & Marshall, Biofilms, 1990)
Biofouling can be extraordinarily difficult to detect
2
and control
3. What Problems Does Biofouling
Cause?
• Off taste in food & beverage products
• Product spoilage
• Extended downtimes to clean the process
• More aggressive process cleaning
methods
• Random microbiology problems
3
4. Current Biofilm Detection Methods
• Product sampling:
– Taste (for food & beverage products)
– Microbiological plating
• Process sampling:
– Microbiological plating of water rinse effluent
– Microbiological plating of process swab
samples
– ATP or PCR analysis of swab samples
All of these methods require organisms to grow in
4
culture in the microbiology lab
5. Problems with Current Biofilm Test
Methods
• Biofilms can be remarkably difficult to find and sample in large-scale
manufacturing processes (i.e., pipes and tanks)
• Even if recovered, biofilms tend not to grow in culture in the
microbiology lab
• Culturing techniques, if they work, only indicate whether an
organism is dead, not if the organism is dormant or even if the dead
organism has been removed from the process
• Dead organisms on a process surface serve as a nutrient source for
the wave of microorganisms
• Biofilms will sacrifice the outermost layer of organisms to cleaning
chemicals but protect the hidden, innermost layer of organisms from
CIP methods
• High-tech methods of ATP and PCR analysis require a minimum
number of cells in order to generate a signal; very few cells are
necessary to generate an abundance of biofilm exopolymer (“glue”)
and biofilms can evade detection by ATP and PCR methods
5
6. Biofilm Locations
• Biofilms can be found in:
– Water systems
– Food & beverage plant product lines
– Dairy processing plants
– Pharmaceutical manufacturing processes
– Cosmetics and nutraceuticals plants
– Raw materials suppliers’ processes
– Cleaning chemicals
– Steam lines
– Fine & specialty chemicals plants
– Pulp & paper mills
– Heat exchangers
6
8. Biofouling Rate
Physical
quality of
product
fouling mass
physical degrades
Chemical
chemical quality of
secondary fouling
product
degrades
induction period
time
The goal of cleaning is to return the system to the
induction period level of fouling
8
9. Fouling Cell Techology
• Does not depend on microbial culturing
techniques to detect biofilms
• Biofilms are not removed from their surface but
instead analyzed while still in place on the
colonized surface
• Fouling cell analysis by reflection infrared
spectroscopy detects primarily the biofilm
exopolymer, not the organisms, and as a result
detects the very earliest stages of biofilm
formation
9
10. Fouling Cell: Sanitary Cross with
Polished End Caps
Insoluble material
deposits on pipe wall
and mirror-polished end
Mirror-polished
cap during product flow
end caps
Product Flow
Biofouling that adsorbs on pipe wall also adsorbs on mirror-polished end
caps (fouling cell discs)
10
11. Measuring Wall Fouling
Fourier
transform
infrared beam
Spectrum
from
reflected
infrared
beam
Fouled end cap (fouling cell disc)
11
14. Fouling Cell Analysis Tracks Impact of
Improved Mechanical Cleaning on Biofilm
0.0080
0.0075
0.0070
0.0065
0.0060
Old water flush
0.0055
0.0050
0.0045
Absorbance
0.0040
0.0035
Improved water
0.0030
0.0025
flush
0.0020
0.0015
0.0010
0.0005
0.0000
-0.0005
-0.0010
3500 3000 2500 2000 1500 1000
Wavenumbers (cm-1)
Peak height data correlate to effectiveness of cleaning: the smaller
the peak, the more effective the cleaning
14
15. Biofilm Resistance to Cleaning
• Standard CIP methods may not remove biofilm
• Biofilms able to grow after 8 months desiccation
• Biofilms withstood 80C or higher water
temperatures
• Biofilms withstood 20, 50 and 200 ppm chlorine,
25 ppm iodine
∗ Food Protection Report, 7(5):8 (1991)
15
16. Bacteria Populations in a Pipe
TRADITIONAL
SAMPLING: 1% of total
bacteria population
inside of pipe is
planktonic (free
swimming organisms
from bulk solution)
FOULING CELL SAMPLING:
99% of total bacteria
population inside of pipe is
sessile (attached biofilm on
the wall of the pipe)
Sessile organisms (biofilms) can
be very resistant to cleaning
16
18. Biofilm Resistance to Cleaning:
Bleach Treatment
Biofilm
remaining after
bleach treatment
18
19. Fouling Cell Analysis Directly
Measures Impact of Chemical
Cleaning Parameters
Impact of temperature
100%
90%
cleaning efficiency
80%
70%
60%
50%
40%
30%
20% Impact of concentration
10%
0% 100%
25 C 45 C 65 C 90%
cleaning efficiency
5% NaOH 80%
70%
60%
The higher the bars, 50%
40%
the more efficient the 30%
20%
cleaning 10%
0%
0.2% 1.0% 5.0%
NaOH wt% @ 60 C
19
20. Case Study: Mapping Process CIP
Efficacy in a Brewery
FTIR spectra of
fouling cells placed in
5 locations of a
brewery process
(stages A through E)
for 8 weeks
FTIR & epifluorescence of fouling cells can provide cleaning efficacy data from
end to end of a process
20
21. Process Mapping in a Brewery: FTIR
Peak Heights by Location
0.05
0.045
0.04
0.035
0.03
absorbance units
0.025
0.02
0.015
0.01
0.005
0
A B C D E
Process Start Packaging
21
22. Brewery Wort Line
2 weeks, 100x objective 8 weeks, 100x objective
0.05
0.045
0.04
0.035
8-week fouling
0.03
cell shows the
absorbance units
0.025
0.02 beginning of
0.015
0.01
biofilm
0.005
0
exopolymer 22
A B C D E
23. Brewery Aging Line
2 weeks, 100x objective 8 weeks, 100x objective
0.05
0.045
0.04
0.035
Fouling cells
0.03
show aging line
absorbance units
0.025
0.02
cleaning
0.015
0.01 requires more
water velocity
0.005
0
A B C D E
23
24. Brewery Filler Inlet Line
2 weeks, 100x objective 8 weeks, 100x objective
0.05
0.045
0.04
0.035
Fouling cells
0.03
determine onset of
absorbance units
0.025
0.02 biofouling in bottling
0.015
0.01
line
0.005
0
A B C D E
24
26. Case Study: Winery Bottling Line 1
After CIP
1-week exposure, 100x 4-week exposure, 100x
Bottling line 1 appears very clean
26
27. Winery Bottling Line 2 After CIP
1-week exposure, 100x 4-week exposure, 100x
Bottling line 2 appears to have some particle contamination
27
28. Winery Bottling Line 2 Before & After CIP
Removed by CIP
Not Removed
by CIP
After water flush After CIP
Fouling cell technology detects that the winery CIP removes one fouling
component but not the others from the stainless steel process surface
28
29. Case Study: Biotech Company
Fermentation
2-day exposure
before CIP
2-day exposure after
CIP
Fouling cell
technology
reveals CIP- 4-week exposure
resistant biofilm after CIP
at 4 weeks
29
30. Biotech Company Recovery
2-day exposure
before CIP
2-day exposure after
CIP
Fouling cell
technology
reveals CIP- 4-week exposure
resistant biofilm after CIP
at 4 weeks
30
31. Conclusions
• In-line fouling cells can provide:
– An early warning for issues of process cleanliness
and health
– Information on chemistry and rate of biofouling within
system
– Objective data on CIP efficacy
– Ability to determine efficacy of proposed cleaning
changes in the lab, not in production
– Ability to screen new products for fouling propensity
• These methods are complimentary to existing
process health measures
31
