Early Biofilm Detection

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Early Biofilm Detection

  1. 1. Direct Detection of Biofilms Mark Fornalik Ethox International 1
  2. 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. 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. 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. 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. 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
  7. 7. 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) 7
  8. 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. 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. 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. 11. Measuring Wall Fouling Fourier transform infrared beam Spectrum from reflected infrared beam Fouled end cap (fouling cell disc) 11
  12. 12. Fouling Identification FTIR provides a “chemical fingerprint” of the biofilm, as well as an indication of biofilm amount 12
  13. 13. Fouling Cell Analysis Tracks Biofilm Chemistry Changes Over Time 0.020 *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) 0.019 *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 0.018 0.017 0.016 0.015 0.014 0.013 0.012 0.011 0.010 0.009 0.008 0.007 Absorbance 0.006 0.005 0.004 0.003 6 mo 0.002 0.001 0.000 -0.001 24 hrs -0.002 -0.003 -0.004 8 hrs -0.005 -0.006 -0.007 2 hrs -0.008 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 Wavenumbers (cm-1) Biofilm chemistry changes to cleaning-resistant exopolymer upon aging 13
  14. 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. 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. 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
  17. 17. 45° Ultrapure Water Biofouling C 1 day 2 days 17 4 days 9 days
  18. 18. Biofilm Resistance to Cleaning: Bleach Treatment Biofilm remaining after bleach treatment 18
  19. 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. 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. 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. 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. 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. 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
  25. 25. Brewery Filler Inlet Line 8 weeks, 100x objective 8 weeks, 100x objective 25
  26. 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. 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. 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. 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. 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. 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

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