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Direct Detection of Biofilms

Mark Fornalik

Ethox International

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• 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)

What is Biofouling?

Biofouling can be extraordinarily difficult to detect and control

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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

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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 culture in the microbiology lab

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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

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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

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Biofilm-Related Contaminants

• Cells (possibly pathogenic)

• Anions (acetate, formate, nitrate, etc.)• Proteins, glycoproteins, carbohydrates, fatty

acids• Enzymes• Surfactants

• Organic and inorganic particles• Substrate degradation (metal & plastics

corrosion)

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The goal of cleaning is to return the system to the induction period level of fouling

Biofouling Rate

time

fou

ling

mas

s

physical

chemical

induction period

secondary fouling

Physical quality of product degrades

Chemical quality of product degrades

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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

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Fouling Cell: Sanitary Cross with Polished End Caps

Product Flow

Biofouling that adsorbs on pipe wall also adsorbs on mirror-polished end caps (fouling cell discs)

Insoluble material deposits on pipe wall and mirror-polished end cap during product flow

Mirror-polished end caps

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Measuring Wall Fouling

Fouled end cap (fouling cell disc)

Fourier transform infrared beam

Spectrum from reflected infrared beam

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FTIR provides a “chemical fingerprint” of the biofilm, as well as an indication of biofilm amount

Fouling Identification

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Fouling Cell Analysis Tracks Biofilm Chemistry Changes Over Time

*Subtraction Result:ir1848, 610 NRX disc #26, 3-month exposure, no clean*Subtraction Result:ir1896, 610 NRX, 14 batches (4 days), disc #7 (1/30 - 2/2/98)*Subtraction Result:ir2288, 610, NRX, #10, 24 hours, 5 batches, 2/26 - 2/27/98*Subtraction Result:ir1974, disc 10, 610 NRX, 1 batch, 4 hrs, without santoprene gasket

-0.008

-0.007

-0.006

-0.005

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

0.012

0.013

0.014

0.015

0.016

0.017

0.018

0.019

0.020

Abs

orba

nce

600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000

Wavenumbers (cm-1)

Biofilm chemistry changes to cleaning-resistant exopolymer upon aging

2 hrs

8 hrs

24 hrs

6 mo

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Fouling Cell Analysis Tracks Impact of Improved Mechanical Cleaning on Biofilm

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

0.0055

0.0060

0.0065

0.0070

0.0075

0.0080

Abs

orba

nce

1000 1500 2000 2500 3000 3500

Wavenumbers (cm-1)

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

0.0055

0.0060

0.0065

0.0070

0.0075

0.0080

Abs

orba

nce

1000 1500 2000 2500 3000 3500

Wavenumbers (cm-1)

Peak height data correlate to effectiveness of cleaning: the smaller the peak, the more effective the cleaning

Old water flush

Improved water flush

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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)

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Bacteria Populations in a Pipe

TRADITIONAL SAMPLING: 1% of total bacteria population inside of pipe is planktonic (free swimming organisms from bulk solution)

Sessile organisms (biofilms) can be very resistant to cleaning

FOULING CELL SAMPLING: 99% of total bacteria population inside of pipe is sessile (attached biofilm on the wall of the pipe)

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1 day 2 days

9 days4 days

45°C Ultrapure Water Biofouling

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Biofilm Resistance to Cleaning: Bleach Treatment

Biofilm remaining after bleach treatment

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Fouling Cell Analysis Directly Measures Impact of Chemical

Cleaning Parameters

0%10%20%30%40%50%60%70%80%90%

100%

25 C 45 C 65 C5% NaOH

clea

nin

g e

ffic

ien

cy

Impact of temperature

0%10%20%30%40%50%60%70%80%90%

100%

0.2% 1.0% 5.0%NaOH wt% @ 60 C

clea

nin

g e

ffic

ien

cy

Impact of concentration

The higher the bars, the more efficient the cleaning

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Case Study: Mapping Process CIP Efficacy in a Brewery

FTIR & epifluorescence of fouling cells can provide cleaning efficacy data from end to end of a process

FTIR spectra of fouling cells placed in 5 locations of a brewery process (stages A through E) for 8 weeks

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Process Mapping in a Brewery: FTIR Peak Heights by Location

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

A B C D E

abso

rban

ce u

nit

s

PackagingProcess Start

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Brewery Wort Line

2 weeks, 100x objective 8 weeks, 100x objective

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

A B C D E

abso

rban

ce u

nit

s

8-week fouling cell shows the beginning of biofilm exopolymer

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Brewery Aging Line

2 weeks, 100x objective 8 weeks, 100x objective

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

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0.045

0.05

A B C D E

abso

rban

ce u

nit

s

Fouling cells show aging line cleaning requires more water velocity

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Brewery Filler Inlet Line

2 weeks, 100x objective 8 weeks, 100x objective

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

A B C D E

ab

sorb

an

ce u

nit

s

Fouling cells determine onset of biofouling in bottling line

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Brewery Filler Inlet Line

8 weeks, 100x objective 8 weeks, 100x objective

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Case Study: Winery Bottling Line 1 After CIP

1-week exposure, 100x 4-week exposure, 100x

Bottling line 1 appears very clean

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1-week exposure, 100x 4-week exposure, 100x

Winery Bottling Line 2 After CIP

Bottling line 2 appears to have some particle contamination

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After water flush After CIP

Removed by CIP

Not Removed by CIP

Winery Bottling Line 2 Before & After CIP

Fouling cell technology detects that the winery CIP removes one fouling component but not the others from the stainless steel process surface

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Case Study: Biotech Company Fermentation

2-day exposure before CIP

2-day exposure after CIP

4-week exposure after CIP

Fouling cell technology reveals CIP-resistant biofilm at 4 weeks

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Biotech Company Recovery

2-day exposure before CIP

2-day exposure after CIP

4-week exposure after CIP

Fouling cell technology reveals CIP-resistant biofilm at 4 weeks

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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