Non-Formaldehyde Releasing Biofilm Control Options Putting you in control.

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1 May 20, 2014 Non-Formaldehyde Releasing Biofilm Control Options Putting you in control. Christine E. McInnis PhD, Jennifer Liboon, Ella Massie-Schuh Dow.com

2 Outline Biofouling Problems Biofilm Characteristics and Formation Laboratory Studies on Biocide Efficacy Biofilm Control Strategies 2 2

Microbial Problems with Biofilms in Metalworking Fluid Systems Surface fouling = increased microbial load Source of reinoculation (splash zones, swarf, dead spots) Blockage

3 Microbial Problems with Biofilms in Metalworking Fluid Systems Surface fouling = increased microbial load Source of reinoculation (splash zones, swarf, dead spots) Blockage of filters, weirs, and screens Microbially influenced corrosion Potential health risks (Mycobacterium, endotoxins) Visual appearance (slime) Reduced biocide performance 3 3

4 Stages of Biofilm Formation Cells + Exopolymer 4

5 Biofilm Growth on Surfaces Early - Uniform Mature - Patchy 5

6 Biofouling Activities in a Flowing Pipe System 6

7 Why Are Biofilms Harder to Kill? Diffusion / reactivity barrier biocides must penetrate through biofilm EPS biocides may react with biofilm components / dead cells metabolic byproducts deactivate biocides (sulfide) Quorum Sensing (cell-cell signaling) increased communication via homoserine lactones (HSL) exoenzymes, secondary metabolites, toxins, biofilms Expression of biofilm /stress gene products altered EPS and outer membrane components SOS repair systems activated Lower metabolic activity = slower growth resulting from nutrient gradients Persister cells survive in biofilms less susceptible to biocides personal/intro-biofilms-s4.htm 7

8 Microbial Control Testing Results 8

9 Biocides Tested Chemical Name 5-Chloro-2-methyl-4-isothiazolin-3-one 2-Methyl-4-isothiazolin-3-one 2-n-Octyl-4-isothiazolin-2-one 2,2-Dibromo-3-nitrilopropionamide 2-Bromo-3-nitropropane-1,3-diol 3-Iodopropynylbutylcarbamate 1,2-Benzisothiazolin-3-one Sodium o-phenylphenate Tris-hydroxyethyl-hexahydrotriazine 2-Methyl-4-isothiazolin-3-one 1-(3-Chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride Poly [oxyethylene(dimethylminino) ethylene (dimethyliminio)ethylene dichloride] Abbreviation CMIT/MIT OIT DBNPA Bronopol IPBC BIT NaOPP Triazine MIT CTAC PolyQuat 9

Eradication Testing Method MWF samples were challenged with either: Mixed Bacterial Culture Alcaligenes faecalis (#8750) Enterobacter cloacae (#13047) Proteus vulgaris (#29905) Pseudomonas aeruginosa

10 Eradication Testing Method MWF samples were challenged with either: Mixed Bacterial Culture Alcaligenes faecalis (#8750) Enterobacter cloacae (#13047) Proteus vulgaris (#29905) Pseudomonas aeruginosa (#9027) Pseudomonas fluorescens (#17397) Mixed Fungal Culture Aspergillus niger (# 6275) Penicillium ochro-chloron (#9112) Fusarium (oxysporon) Candida albicans (# 10231) Rhodotorula rubra (#9449) After contact for 24 hours, samples were analyzed for viable microbes using the Most Probable Number (MPN) method. 10

11 log reduction Eradication Testing 1E+06 1E+05 Soluble Oil MWF Bacteria Fungi 1E+04 1E+03 1E+02 1E+01 1E+00 CMIT/MIT 5 ppm CMIT/MIT + Bronopol ppm Biocide ppm active DBNPA 15 ppm The controls had greater than 10 4 CFU/mL. 11

12 log reduction Eradication Testing 1E+05 1E+04 Synthetic MWF Bacteria Fungi 1E+03 1E+02 1E+01 1E+00 CMIT/MIT 5 ppm CMIT/MIT + Bronopol ppm Biocide ppm active DBNPA 15 ppm The controls had greater than 10 4 CFU/mL. 12

13 High Throughput Planktonic Efficacy Method 96 well plates containing biocides diluted in synthetic MWF were inoculated with A. faecalis, E. aerogenes, P. aeruginosa, P. fluorescens and S. aureus The plates were incubated overnight at 37 o C The OD 630 value was taken postincubation 13

14 OD 630 Control of Planktonic Mixed Bacteria 0.25 Synthetic MWF- planktonic mixed bacterial culture Biocide ppm active 14

15 Synergy Index The synergy index (SI) a measure of the synergy between two compounds. Synergy Index (SI) = Ca/CA + Cb/CB CA = concentration of CMIT/MIT in ppm, acting alone, which produced an end point (MIC of CMIT/MIT). Ca = concentration of CMIT/MIT in ppm, in the mixture, which produced an end point. CB = concentration of Bronopol in ppm, acting alone, which produced an end point (MIC of Bronopol). Cb = concentration of Bronopol in ppm, in the mixture, which produced an end point. 15

16 Synergy The synergy index (SI) a measure of the synergy between two compounds. Synergy Index ("SI") = Ca/CA + Cb/CB CA CB Ca Cb = ppm CMIT/MIT = 9.65 ppm Bronopol = 0.85 ppm CMIT/MIT = 5.79 ppm Bronopol SI Result >1 Antagonism 1 Additivity <1 Agonism SI = 0.72 Agonistic synergy! 16

17 Bacterial Biofilm Recovery Plate Method Bacterial biofilm was grown on a 96 well plate pegged lid. After biofilm formation, the pegged lid was used to easily transfer biofilm into dosed MWF. Biofilms were incubated in MWF for 24 hours at 37 o C with shaking. 17

OD Recovery Plate Method The pegged lids were transferred to 96 well plates containing Tryptic Soy broth recovery medium, and incubated with shaking for 10 minutes.

18 OD Recovery Plate Method The pegged lids were transferred to 96 well plates containing Tryptic Soy broth recovery medium, and incubated with shaking for 10 minutes. The pegged lids were removed, and the recovery plates were incubated overnight at 37 o C. The optical density at 630 nm (OD630) was taken post-incubation. Higher optical densities are indicative of more growth. 18

19 OD 630 Biofilm Efficacy Recovery Plate Method Control CMIT/MIT 5 ppm CMIT/MIT + Bronopol ppm Biocide ppm active DBNPA 15 ppm Soluble Oil MWF Synthetic MWF 19

20 OD 630 Bacterial Biofilm Control Soluble Oil Biocide ppm active 20

21 OD 630 Bacterial Biofilm Control Synthetic Fluid Biocide ppm active 21

22 Bacterial Biofilm Conclusions In general, it is easier to control bacterial biofilm in synthetic fluid than in a soluble oil. CMIT/MIT, Bronopol, and a combination of the two were effective in controlling bacterial biofilm in a soluble oil and synthetic fluid. NaOPP and CTAC were less effective in the soluble oil than in the synthetic fluid. Polyquat was not able to control bacterial biofilm in a soluble oil, but was effective in a synthetic fluid. BIT was moderately effective in a soluble oil and not effective in a synthetic fluid. 22

23 log reduction Planktonic Fungal Control Biocide ppm active 23

24 Fungal Biofilm Biofilm was grown on glass cover slips in 6-well plates at 30 ºC for 24 hours. The glass cover slips were removed from the growth media, placed in MWF dosed with biocide, and allowed to incubate for 24 hours. 24

Fungal Biofilm Control Test After incubating in the MWF dosed with biocide, the glass cover slips were placed in phosphate buffer and gently agitated to remove and

25 Fungal Biofilm Control Test After incubating in the MWF dosed with biocide, the glass cover slips were placed in phosphate buffer and gently agitated to remove and disperse the biofilm. The Most Probable Number (MPN) method was used to enumerate the living organisms in the biofilm. Each biocide and dose level was replicated 6 times. 25

26 Log Reduction of Fungal Growth on Surface of Cover Slip Fungal Biofilm Control in Soluble Oil MWF Biocide ppm active 26

27 Fungal Biofilm Conclusions NaOPP is able to control fungal biofilm at both the high and low dose. CMIT/MIT and Bronopol, BIT, and Triazine are able to control fungal biofilm at the high dose. CMIT/MIT and Bronopol together offer a higher level of control than CMIT/MIT or Bronopol alone. OIT was effective at controlling planktonic fungus, but was not effective against fungal biofilm. The low dose of the polyquat increased fungal biofilm growth. 27

28 Approaches to Control Biofouling Monitor biofilm growth periodically on surfaces, screens, or coupons to track microbial buildup. Clean out or add biocide to dead spots frequently to reduce reinoculation. Routinely apply biocides to maintain low levels of planktonic microorganisms in the bulk water (to reduce the overall level of contamination). Shock dose with biocides when biofilm accumulation appears heavy. Biodispersants, enzymes or surfactants may be added to assist biocides in reducing biofilm deposits which accumulate (use low foaming products). 28

29 Things to Consider for Biofouling Control Low microbial counts in the bulk fluid are good; but counts in biofilms may still be high. Microbial counts in the bulk fluid may increase after biocide shock dosing as cells are released. Biofilms grow back quickly; be sure to keep the system adequately controlled with biocide. The microbial composition of the biofilm may differ compared to the bulk fluid, so both must be sampled for organisms of concern. 29

30 Conclusions Biofilm is a source of many microbially influenced problems in MWF systems. Monitoring for biofilms and biofouling is important. Biocides added to the MWF concentrate can remove or prevent biofilm in addition to biocides added tankside. The type of MWF influences the efficacy of the biocide. 30

31 Questions? 31

32 Thank You The Dow Chemical Company makes no representations or warranties as to the completeness or accuracy of Information. Information is supplied upon the condition that the persons receiving same will make their own determination as to its suitability for their purposes prior to use. In no event will The Dow Chemical Company be responsible for damages of any nature whatsoever resulting from the use of or reliance upon Information or the products to which Information refers. NO REPRESENTATIONS OR WARRANTIES, EITHER EXPRESS OR IMPLIED, OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE OR OF ANY OTHER NATURE ARE MADE HEREUNDER WITH RESPECT TO INFORMATION OR THE PRODUCT TO WHICH INFORMATION REFERS. Trademark of The Dow Chemical Company ( Dow ) or an affiliated company of Dow

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