WO1998013305A1 - Compositions and methods for reducing deposit formation on surfaces - Google Patents

Abstract

A method for inhibiting microbial colonization of a surface in contact with an aqueous system is disclosed, the method including adding to the system an amount of at least one compound including repeating ethylene oxide units. Also disclosed is a multi-component composition comprising a polyoxypropylene-polyoxyethylene block copolymer and a biocide, and a method including applying the composition to a surface in contact with an aqueous system in order to reduce the formation of deposits on the surface.

Description

COMPOSITIONS AND METHODS FOR REDUCING DEPOSIT FORMATION ON STmFACES

TECHNICAL FIELD AND INDUSTRIAL _Ppτ TΓARΠ.TTY OF THE INVENTION The present invention relates, in part, to methods for inhibiting the formation of deposits on surfaces in contact with aqueous systems. More particularly, the present invention, in part, relates to methods of using compounds including repeating ethylene oxide units to reduce the adherence of microorganisms and the resultant formation of bilofilms on surfaces in contact with aqueous systems. The present invention also particularly relates to multi-component compositions and to methods of using those compositions to reduce the occurrence of deposits of water-bome matter, for example, silt and microorganisms, on surfaces in contact with aqueous systems. The compositions and methods of the present invention may be used in any system in which surfaces are subjected to static or flowing aqueous media. Examples of such systems include industrial water system applications, such as, for example, process or cooling water systems and paper mills and paper processing systems BACKGROUND OF THE INVENTION

Biofilms are considered indigenous to industrial water systems and may result in a number of serious problems, including fouling of heat exchangers and cooling tower fill materials, microbially-influenced corrosion, reseeding of the water system with biofilm organisms, plugging of orifices or piping, and final product or process stream complications.

It has been determined that biofilms are not merely comprised of monoiayers of bacterial cells embedded in a polysaccharide matrix, but rather are heterogeneous assemblages of cells, extracellular polymeric substances (EPS), and abiotic panicles

(clay, diatom frustules, corrosion and mineral deposits). £e_e W.G. Characklis, "Microbial Fouling," in W.G. Characklis and K.C. Marshall (eds.), Rinfilm.s (John Wiley & Sons, N.Y., 1990), pp. 523-584; RM. Donlan. "Correlation Between Sulfate Reducing Bacterial Colonization and Metabolic Activity on Selected Metals iΛa Recirculating Cooling Water System," National Association of Corrosion Engineers,

Technical Paper No. 83, Nashville. Tennessee. Further research has demonstrated that microbially-produced polymers responsible for initial bacterial adherence to surfaces may not be the same as those involved in cell-to- cell interactions within the biofilm (the biofilm matrix polymers). M. Fletcher, et al., "Bacterial Surface Adhesives and Biofilm Matrix Polymers of Marine and Freshwater Bacteria," Biofouling. 4: 129- 140 ( 1991).

Over the past approximately 20 years, high efficiency fill material in the form of thin sheets of PVC (polyvinyl chloride) has been used as cooling tower fiU media. See

J.S. Gill et al., "Fouling of Film Forming Cooling Tower Fills - A Mechanistic Approach", Cooling Tower Institute Annual Meeting (Houston, Texas, 1994). Because of its greater heat transfer efficiency and lower weight it has been received favorably by the industry. A problem with this fill material is that it has a tendency to foul rapidly with water borne materials and develops significant deposits commonly containing microorganisms and silt. Stud es have shown that the microorganisms provide a matrix or "glue" for further deposition of silt, primarily clay, especially when the makeup is from a fresh water surface supply. U It is common for biofilms in industrial water systems to collect or capture abiotic particles including clay particles. Ss≤ E. J. Bower, "Theoretical Investigation of Particle Deposition in Biofilm Systems",

Water Research. 21: 1489- 1498 ( 1987); W.J. Drur et al, "Interactions of 1 μm Latex Particles in Pseudomonas aeruginosa Biofilms", Water Research. 27: 1119- 1126 (1993); W.J. Drury et al., 'Transport of 1 μm Latex Particles in Pseudomonas aeruginosa Biofilms", Biotechnol. Bioeng .. 42:111-117 (1993); W.G. Characklis, "Microbial Fouling", in W.G. Characklis and K.C. Marshall (eds ), Biπfiims pp. 523-

584 (John Wiley, New York, 1990). In the case of clay crystals, Marshall, in Interfaces in Microbial Ecology. (Harvard Univ. Press, Cambridge, Mass., 1976), presented electron microscopic evidence of clay-bacterial associations. He presented results indicating that the clay crystals associated in an edge-to-edge manner to carboxyl-type bacterial surfaces, with the positively-charged edges of the clay crystal attracted to the negatively charged bacterial surface. These observations were supported by J.S. Gill et al., suβia, in which a complex scanning electron microscopic procedure was used to view bacterial-clay interactions on the PVC fill surface.

Different treatments have been proposed to control fouling of PVC fill material by microorganisms and other matter in recirculating cooling water. Pearson, et al.,

"Cleaning and Maintenance of Film Fill at Florida Power Corporation", Cooling Tower Institute Annual Meeting, 1992, Technical Paper No. TP92-09, utilized a 60% acrylic

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T1TUTE SH acid, 40% 2-acrylamido-2-methylpropylsulfonic acid (AA/AMPS copolymer) to control the fouling onto PVC fill material in a seawater fed system. Mortensen and Conley, "Film Fill Fouling in Counterflow Cooling Towers: Research Results", National Association of Corrosion Engineers Annual Meeting, 1994, Paper No. 457, recommended microbiological control with the use of microbiocides and with possible pretreatment of the makeup water using some type of clarification.

Others have documented that nonionic surfactants may affect the adhesion of bacteria to surfaces. L.R. Robertson, "Prevention of Microbial Adhesion", Biological Sciences Symposium, TAPPI Proceedings, Minneapohs. MN, October 3-6, 1994, pp. 225-232; C.L. Wiatr, "Development of Biofilms", Biological Sciences Symposium,

TAPPI Proceedings, Minneapolis, MN, October 3-6, 1994, pp. 225-232; B.L. Blainey and K.C. Marshall, "The Use of Block Copolymers to Inhibit Bacterial Adhesion and Biofilm Formation on Hydrophobic Surfaces in Marine Habitats", Biofouling. 4: 309- 318 ( 1991); J.H. Paul and Jeffrey, 'Evidence for Separate Adhesion Mechanisms for Hydrophilic and Hydrophobic Surfaces in Vibrio proteo.ytica". Appl. Environ.

Microbiol- 50: 431-437 (1985); W.K. Whitekettle, 'Effects of Surface- Active Chemicals on Microbial Adhesion", Jour. Indust. Micrbiol .. 7: 105-116 (1991); H.F. Ridgeway et al., "Bacterial Adhesion and Fouling of Reverse Osmosis Membranes", Journal AWWA, July, 1985, pp. 97-106; J. Olsson et al., "Surface Modification of Hydroxyapatite to Avoid Bacterial Adhesion", Colloid Polym. Sci.. 269 (12): 1295-

1302 ( 1991 ). SUMMARY OF THE INVENTION

The present invention discloses compositions and methods of use for inhibiting the formation of deposits of microorganisms and other matter on surfaces in contact with aqueous systems. In part, the present invention provides a method for inhibiting the microbial colonization of a surface in contact with an aqueous system by adding to the aqueous system at least one compound that will inhibit adhesion of microorganisms. The method more specifically includes adding to the system an effective amount of at least one compound having repeating ethylene oxide units. Compounds useful in the method include, for example, an ethoxylated nonionic surfactant and, more particularly, may be selected from the following: block copolymers of repeating ethylene oxide and repeating propylene oxide units; polysiloxanes including pendent polyethylene oxide grafts; alcohol ethoxylates including hydrophilic head groups and hydrophobic tail groups; and sorbitan monooleates including about 20 ethylene oxide units.

The block copolymers useful in the foregoing method of the present invention may be selected from, for example, block copolymers including first and second blocks of repeating ethylene oxide units and a block of propylene oxide units interposed between the first and second blocks of repeating ethylene oxide units. Such block copolymers may have the general structure (I):

CH₃ I (I)

HO - (CHXH₂OV(CHXHO)_v-(CHXH₂O)_x-H

wherein x and y are each independently 5-1000. Thus, the two "x" values in the above structure (I) need not be identical. The block copolymers of the above structure (I) may include from 20% to 80% ethylene oxide (EO) units by weight and have a molecular weight in the range from 2000 to 20,000. Additional examples of block copolymers useful in the foregoing method of the present invention include those wherein the copolymers include first and second blocks of repeating propylene oxide units and a block of repeating ethylene oxide units interposed between the first and second blocks of repeating propylene oxide units. Such block copolymers may have the general structure (II):

I I <π)

HO - (CH₂CHO)_x-(CHXH₂O)_y-(CHXHO)_x -H

wherein x and y are each independently numbers in the inclusive range 5 to 1,000 Thus, the two "x" values in the above structure (II) need not be identical. The block copolymers of the above structure (II) may include from 20% to 80% ethylene oxide (EO) units by weight and have a molecular weight in the range from 2000 to 10,000. In the case of either the EO-PO-EO block copolymer (I) or the PO-EO-PO block copolymer (II), it is preferred that the hydrophilic-lipophilic balance (HLB) be in the inclusive range of from 7 to 24. The foregoing method of the present invention preferably includes adding at least 0.25 ppm (parts per million, by weight) of at least one ethylene oxide-containing compound to the aqueous system containing the surface, and more preferably includes adding to the system at least 50 ppm. It is contemplated that the foregoing method may be used to inhibit adherence of microorganisms in any system wherein a surface is in contact with an aqueous system. Examples of specific applications of the method include use in process and cooling water systems, pulping systems, and papermaking systems.

The present invention also is directed to compositions and a method of using those compositions to reduce the formation of deposits of water-borne materials such as, for example, silt and microorganisms, on surfaces in contact with aqueous systems. The method comprises applying to the particular surface an amount of a multi- component composition comprising at least a polyoxypropylene-polyoxyethylene block copolymer and a biocide. Examples of the possible biocide component of the composition include glutaraldehyde, quaternary ammonium compounds, isothiazoϋne, carbamates, dibromonitrilopropionamide. and dodecylguanidine hydrochloride. The multi-component composition may also include a dispersant such as, for example, one of the anionic polyelectrolyte dispersents conventionaUy used to disperse minerals such as clay ("clay dispersants"), e.g., an acrylic acid/AMPS copolymer. Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a schematic view of the apparatus used by the present inventors to perform the Recirculating Water System (RWS) experiments discussed below; FIGURE 2 is a plot showing the effect of addition of Pluronic P103 surfactant on bacterial adherence on PVC exposed to treatment for 24 hours as measured by colony forming units per square centimeter (cfu/cnr), each bar showing results from a single RWS run, and standard errors shown by brackets;

FIGURE 3 is a plot showing reduction in bacterial adherence onto PVC in Lab RWS as measured by cfu/cnr, wherein bars represent the mean reduction for two Lab

RWS treated with 10 mg/L Pluronic PI 03 surfactant, and wherein brackets are standard errors;

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SUBST1TUTE SHEET (RULE 26) FIGURE 4 is a plot showing the effect of presoaking PVC substrata in a 0.1% solution of Pluronic P103 surfactant and then placing in a Lab RWS treated with 10 mg/L Pluronic PI 03 for 24 hours, as measured by cfu/cnr, and wherein each bar represents the percent reduction in bacterial adherence, brackets are standard errors, and 50 mg/L clay was added for Lab RWS treated with clay;

FIGURE 5 is a plot showing the reduction in bacterial adherence onto PVC in Field RWS experiments as measured in cfu/cnr. and wherein bars represent the mean reduction in single Field RWS treated with 50 mg L Pluronic PI 03 surfactant, and brackets are standard errors; FIGURE 6 is a plot showing the reduction in bacterial adherence onto PVC in

Field RWS treated with d^erent surfactants as measured by cfu/cnr, and wherein bars represent the mean reduction in duplicate RWS, and brackets are standard errors;

FIGURES 7 and 8 are graphs depicting the weight of deposit (mg deposit) per gram of PVC fill as a function of the experimental treatment of the invention used; and FIGURE 9 is a plot showing the linear regression of the relationship between

ATP accumulation and deposit accumulation onto PVC fill material exposed to Monongahela River water for 8 weeks. DETAILED DESCRIPTION OF THE INVENTION Bacterial Adherence Studies and Conclusions The inventors initiated laboratory and field studies to investigate novel treatments to minimize or control biofilms in aqueous systems. The focus of the work was on treatments which would minimize the adhesion of bacteria to surfaces, a process which will lead to biofilm development. We have found that surfactants have shown some efficacy in minimizing bacterial adherence in aquatic systems, and were investigated using several different types of assays. Results using a simple screening technique indicated that the nonionic surfactants worked more effectively than anionic or cationic surfactants. We have found that those products containing ethylene oxide and propylene oxide units (EO/PO block copolymers) work best in minimizing adherence. These products were then examined in lab systems containing microbial consortia and continuously flowing conditions, where threshold limits for surfactant performance under these conditions were determined. Evaluations under more complex conditions, at a power plant site, using plant recirculating cooling water, determined threshold performance for reduction in adherence in these systems. Comparison of data from all these studies enabled determination of a relationship between lab and field performance. Treatment with 0.25 ppm of product yielded >95% reduction in adherence in the screening assay. Approximately 40 times this amount ( 10 ppm) was required to provide significant reduction in lab recirculating water systems and 200 times tlύs amount (50 ppm) was required for significant minimization under field conditions. Studies investigating mechanisms of surfactant efficacy indicated that surfactant molecules acted primarily by alteration of substratum surface hydrophobicity rather than by alteration of cell surface charge. I. Materials and Methods a. Apparatus/Testing Protocol

Three different apparatus designs were used. The 96 Well plate/urease assay system (designated screening assay) was used for preliminary screening of surfactants. Those surfactants which performed well in the initial screen were then evaluated under more complex, dynaimc conditions in recirculating water systems (Lab RWS). Finally, a select number of surfactants were evaluated under actual cooling systems conditions, with an apparatus similar to the Lab RWS (designated Field RWS). These field studies were performed at a fossil fuel power plant in Pennsylvania using plant recirculating cooling water (river water makeup). b. Laboratory Screening Assay

A culture ofKlebsiella aerogenes (wild type isolate) was restreaked on a fresh plate of Standard Methods Agar. This plate was incubated for 24 hours at 37° C. 150 ml of sterile Trypticase Soy Broth contained in a tissue culture flask was then inoculated with a swab which had been streaked across the plate. This flask was then placed into a 37° C water bath and shaken at 80 rpm overnight ( 17 hours). As a rule, the culture was inoculated around 4:30 P.M. and removed approximately 9:00 A.M. the next morning. Thirty ml of this culture was then added to a sterile centrifuge tube and centrifuged at 70 m for 30 minutes to concentrate the bacterial cells and achieve a pellet with clear supernatant. The supernatant was discarded using a pipet, and the pellet washed twice with sterile phosphate buffered water (pH 7.2). It was spun down at 70 rpm for 30 minutes, vortexing the cells after each addition of buffer. Approximately 20 ml of sterile phosphate buffered water was added to the tube. The optical density (OD₆₀₀) was measured in a Bausch and Lomb Spec 21 spectrophotometer to determine the starting inoculum cell concentration. The OD^ should be around 0.45 to 0.55, which equates to approximately 10⁸ cells per ml. Generally, a plate count was also run on this cell suspension using Standard Methods Agar.

A Corning 96- Well, round bottom, tissue culture treated polystyrene plate was used for the assay. While the culture was spinning down, individual treatments were prepared. Triplicate treatments for each dosage concentration were run. Treatments were added to each well prior to inoculation. After treatments were added, sterile phosphate buffered water (pH 7.2), followed by the bacterial culture were added to each well (except the negative control). Each well was then mixed, using the mix function on the automatic micropipettor (Matrix Technologies, Lowell, MA). Once mixed, the plate was incubated at 37° C for 24 horn's.

After the 24-hour incubation, a tube of sterile urease substrate reagent was removed from refrigeration (Cat. no. US101U, Chemicon International, Inc.,

Temecula, CA). The Plate Reader (Dynatech MR5000 Automatic Microplate Reader, Dynatech, Chantilly, VA) was programmed for 51.7° C, equivalent to 37° C in the microwells. The plate was then removed from the incubator. The liquid was removed from each well by aspiration with a Pasteur pipette. After aspiration, sterile phosphate-buffered water was added to each well and aspirated again. This procedure was repeated thrice (a total of four washes). This step will remove any cells which are nonadherent on the well surface. The urease substrate reagent was then added to each well. The plate was then placed into the Dynatech Plate Reader and a colorimetric method developed to quantify presence of urease within bacterial cells was run. c. Lab and Field RWS Design

The schematic of the apparatus used for both Lab and Field RWS is shown in Figure 1. It was comprised of a 20-liter volume polycarbonate tank (10) which contains a small polycarbonate tower (12). Water was pumped by recirculating pump (15) from the 8-liter volume sump (14) through a Biofilm Sampling Device (BSD) (16) at 1.5 gallons per minute (which equates to 2.5 linear feet per second) then back over the tower (12). The BSD ( 16) is shown enlarged in Figure 1 and was installed in water recirculating lines ( 18) in the position indicated. The tank (10) also included

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SUBST1TUTE SHEET RULE 26 blowdown (20) at its 8-liter mark. The tower ( 12) was constructed so that the water flows onto a "deck" (22) containing evenly spaced small holes, down through a fill pack of PVC fill material and down across slats (24), each of which contained one or more fill pieces (26). A slat (24) and one fill piece (26) contained thereby is shown in a relative enlarged view in the circled portion of Figure 1. The fill pieces (26) were made of PVC material (obtained from Munters Corporation, Fort Myers, Florida) and were attached to the slats (24) using stainless steel screws (28). The Biofilm Sampling Device (16) contained multiple removable cylinders (30) (9/16 inch [14mm] I.D., 13/16 inch [20mm] O.D., 1/2 inch [13mm] long) constructed of CPVC material. Examination of the C-PVC material using Electron Spectroscopy for Chemical

Analysis (ESCA) indicated that the measurable elemental components were: carbon (72.77%), oxygen (13.30%). nitrogen (0.11%), silicon (5.32%), chlorine (7.16%), and sodium ( 1.34%). Cylinders (30) were washed in a detergent solution, then rinsed in isopropanol and sterile phosphate-buffered water prior to installation in the BSD component ( 16) of the Recirculating Water System. The flow rate through the BSD

(16) was controlled by a flowmeter. Makeup water containing treatments was fed by means of Masterflex pumps ( 19) (Cole Partner Co., Niles, IL). The RWS has a retention time of 48 hours. Average water temperature was 30° C for lab RWS and 26° C for Field RWS. Lab RWS were inoculated as follows. Ten grams of algal mat material collected from a fouled, untreated Lab RWS was added to 100 ml Butterfield Buffer in a Waring Blender, suspended by mixing one minute on the "mix" function, then 20 ml was added to each Lab RWS. This algal mat was shown to contain Phormidium spp.. Oscillatoria spp.. Anabaena spp.. and diatoms as the primary algal components. Nine separate bacterial cultures, originally isolated from untreated Lab RWS biofilms on

R2A medium, were purified, identified using the fatty acid profile analysis procedure, and frozen at -70° C. Each of these cultures was inoculated into R2A Broth, grown up at 30° C to turbidity, and then 1 ml from each was added to each LRWS. The organisms identified were as follows: fiaαUus subtilis. Bacillus amyloliquefacians. Bacillus cereus. Pseudomonas saccharophila. with the remainder being unmatched gram negative organisms. Field RWS used indigenous microorganisms: therefore, they were not inoculated. d. Bulk Water Measurements

Water chemistry measured in Lab RWS studies is shown in Table 1. This water was municipal tap water dechlorinated with 18 mg/l sodium thiosulfate. Table 2 shows water chemistry for Field RWS, made up with plant recirculating cooling water. Table 3 shows bulk water plate counts for both Lab and Field RWS.

Table 1: Water Chemistry of Lab RWS Using Dechlorinated Tap Water

Analvte Cone* Analvte Cone* pH (a), 25C 8.0 units Magnesium 9.0 mg/L

M Alkalinity 48 mg/L CaCO3 Sodium 31 mg/L

Conductivity 410 umhos Potassium 2.0 mε/L

Suspended Solids Not Run Iron <0.05 mg L

HCO3 59 mg/L Copper <0.05 mg L

Chloride 25 mg L Manganese <0.05 mg L

Nitrite <5 mg/L Aluminum <0.1 mg L

Nitrate <2 mg/L Zinc 0.05 mg/L

Orthophosphate <2 mg L Nickel <0.05 mg L

Sulfate 95 mg/L Chromium <0.05 mg/L

Calcium 36 mg/L

* Each value is the result of measurement on one sample.

Table 2: Water Chemistry of Field RWS Using Power Plant Recirculating Cooling Water

Analyte Cone* Analvte Cone* pH (a), 25C 7.6 units Magnesium 32 mg/L

M Alkalinity 68 mg/L CaCO3 Sodium 55 mg/L

Conductivity HOO umhos Potassium 6.5 mg/L

Suspended Solids 5 mg L Iron 0.1 mg/L

HCO3 83 mg/L Copper <0.05 mg/L

Chloride 28 mg/L Manganese <0.05 mg/L

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SUBST1TUTE SHEET (RULE 26 98/13305

Nitrite '10 mg L Aluminum <0.3 mg/L

Nitrate <32 mg/L Zinc <0.05 mg/L

Orthophosphate <4 mε/L Nickel <0.05 mg/L

Sulfate 420 mg/L Chromium <0.05 mg/L

Calcium 140 mg/L

* Each value is mean of 3 samples.

Table 3: Average Heterotropliic Plate Count Data from Untreated Lab and Field Recirculating Water Systems

CFU/mL x 10⁴

Makeup Water Source Mean S.D. no. samples

Dechlorinated Tap Water (Lab) 3.63 1.56 12

Plant Retire. Water (Field) 30.3 13.5 6

e. Biofilm Sampling and Measurements in LWRS

For cylinder samples, the submersible recirculating water pump (Figure 1 ) was turned off to stop flow through the BSD (16). .Alcohol sterilized pliers were then used to remove cylinders from the BSD. Each cylinder was first rinsed gently in sterile phosphate buffered water to remove reversibly attached cells prior to placing it into a sterile glass tube which contained homogenization solution and 3 mm glass beads. This homogenization solution contained peptone-20 grams, Zwittergent-0.0067 grams (Calbiochem, La Jolla, CA), ethylenebis (oxyethylenentrilo) tetraacetic acid (EGTA)- 7.6 grams, tris (hydroxymethyl) aminomethane (Tris Buffer)-24.2 grams, and deionized water-200 ml, adjusted with 1:1 HC1 to pH 7 and autoclaved, after which it was diluted 1: 10 with sterile Butterfield Buffer, pH 7.2. The tube containing the cylinder was then vortexed at a speed of 10 on a Vortex Genie Mixer (Fisher Scientific, Pittsburgh, PA) for one minute. This "biofilm suspension" was then diluted and pour plated onto R2A medium (Difco Laboratories. Detroit, MI) and incubated for

48 hours at 30° C and counted. Counts were based on number of colony forming units 98/13305

per total cylinder surface area and recorded as biofilm plate counts. For each experiment run, an untreated RWS (either Lab or Field) was run alongside the treated system. Percent reduction in bacterial adherence was calculated by the formula:

A - B X 100 = % Reduction A where: A = biofilm plate count of the treated RWS

B = biofilm plate count of the untreated RWS

For field sampling, cylinders (30) were removed from the BSD ( 16) and placed into sterile PVC shipping tubes which were filled with sterile Butterfield Buffer. These were then transported to the lab where they were processed within 24 hours. Cylinders collected in the field were not rinsed in Butterfield Buffer prior to analysis since shipment in this solution was considered equivalent to rinsing. f. Test Solutions and Treatments

All surfactants used in these test procedures, shown b Tables 4, 5, and 6 below, were made up b deionized water based on product weight, not on active basis. For treatment studies b lab RWS, surfactants were fed contbuously bto the LRWS using Masterflex pumps. For studies b which clay was added to Lab RWS, kaolinite and bentonite clays were mixed b deionized water b a proportion of 0.041 grams bentonite (Calgon Coagulant Aid CA36 Specialty Clay, Calgon Corp., Pittsburgh, PA, USA) to 0.097 grams kaolinite (Engelhard Ultra Gloss 90, Coatmg Grade Kaolinite, Engelhard Corporation. Edison. NJ, USA) per liter. This slurry was then pumped contbuously bto the LRWS sump at a rate of 2.0 ml per mbute which, when diluted with makeup water (bcludbg treatment) equated to a final clay concentration of approximately 50 mg/L.

Table 4 : Nonionic Surfactants

Evaluated for Reduction in Bacterial Adherence

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Ethylene oxide Pluronic L64 EO-PO-EO block BASF 12-18

Propylene oxide copolymer, high EO

Copolvmer content, MW 2900

Ethylene oxide Pluronic L62D EO-PO-EO block BASF 1-7

Propylene oxide copolymer, high PO

Copolvmer content. MW 2400

Ethylene oxide Pluronic PI 03 EO-PO-EO block BASF 7-12

Propylene oxide copolymer, high EO

Copolvmer content, MW 5000

Table 5: Anionic Surfactants Evaluated for Reduction in Bacterial Adherence

Chemical Name Trade Name Supplier Functional Group

Distilled Tall Oil N/A N/A Unsaturated Alkvlene carboxylate

Dusobutyl sodium Monawet MB-45 Mona Sulfosuccinic acid, sodium sulfosuccinate salt

Naphthalene sulfonate Petro 22 Witco Corp Mixture of naphthalene formaldehyde sulfonate- based surfactants condensate

Naphthalene sulfonate Petro Dispersant 425 Witco Corp Sulfonated naphthalene/ formaldehyde formaldehyde, low condensate molecular weight

AJpha-oiefin sulfonates Witconate AOS Witco Corp Sulfonated alkvienes

Linear alkylaryl sodium Witconate 1260 Witco Corp Sulfonated alkylaryl sulfonate

Phosphorylated alkyl, Tπton QS-44 Union Carbide Alkyl surfactant, acid form phosphated form

Modified sulfonated Dynasperse A Lignotech Sulfonated lignin lignin USA formaldehyde condensate Table 6: .Amphoteric/Cationic Surfactants Evaluated for Reduction b Bacterial Adherence

Chemical Name Trade Name Supplier

C, Dimethvl a¹™' ^oxid Barlox 12 Lonza

Cocoamine oxide Mackamine CO Mclntvre

Tallow hvdroxvethvl unidazoline Varine T Sherex

g. los rwDgg ation

Zeta potential measurements were determbed usbg a Zeta Sizer 3 (Malvern Instruments Inc., Southborough, MA). Contact angle measurements were determbed usbg a Kruss Processor Tensiometer K-12 (Kruss Instruments. Charlotte. NC). In both cases, measurements were made according to manufacturer's bstructions. Electron Spectroscopy for Chemical Analysis (ESCA) was performed usbg a Physics Electronics Laboratories PH3-5600 ESCA spectrometer. π. Experimental esults a. Screening Assay Results

Cationic, anionic, nonionic and amphoteric surfactants were all evaluated b the screening assay. These surfactants were selected to cover the broadest range possible of chemical structures, type of head/tail groups, ionic charge, and water solubility. Ethoxylated surfactants were among the surfactants considered, as well as other possible bacterial adherence reduction mechanisms, bcludbg dispersion by anionic agents or partitionbg via hydrophobic (or poorly water soluble) agents. Tables 7 and 8 present results of these evaluations. Each product was tested at decreasbg concentrations of surfactant until a dosage was found which did not prevent at least 90% of bacteria from adhering to the substratum. At 0.25 ppm product, there were 11 of the origbal 32 surfactants which provided greater than 90% reduction b adherence.

These were the products which were further evaluated under more complex and dynamic conditions b Lab and Field RWS. /13305

Table 7: Percent Reduction b Bacterial

Adherence onto Tissue Culture Treated Polystyrene

Plates After 24 Hours' Exposure to Different Surfactants

Surfactant 2.0 ppm 1.0 ppm 0.5 ppm 0.25 ppm

Barlox 12 20.7 0 Not Run Not Run

Mackamme CO 32.2 47.5 6.08 Not Run

Varine T 92.3 99.3 98.8 12.5

Silwet 98.9 97.7 99.8 97.1

Igepal 520 92.5 96.1 38.4 Not Run

Igepal 620 99.3 97.7 51.6 Not Run

Macol NP4 21.4 91.3 6.42 Not Run

Neodol 25-12 99.7 98.4 98.6 97.0

Neodol 25-7 99.9 100 98.7 49.5

Neodol 91-2.5 87.1 4.98 Not Run Not Run

Pegol F68 99.9 98.3 70.6 Not Run

Pluronic 17R8 99.7 95.5 98.4 98.9

Pluronic 25R2 98.4 98.3 98.8 94.4

Pluronic 25 R4 95.6 98.6 97.7 98.8

Pluronic 25 R8 98.9 98.6 99.7 99.0

Pluronic F 108 98.8 97.9 96.4 99.8

Table 8: Percent Reduction b Bacterial

Adherence onto Tissue Culture Treated Polystyrene

Plates After 24 Hours' Exposure to Different Surfactants

Surfactant 2.0 ppm 1.0 ppm 0.5 ppm 0.25 ppm

Pluronic L64 99.6 97.2 98.6 99.5

Pluronic L62D 97.8 95.8 87.1 Not Run

Pluronic PI 03 99.7 100 99.4 99.7

Emsorb 6901 93.1 99.2 63.2 Not Run

T-MAZ 80 98.2 99.1 99.4 95.7 Tween 80 Not Run 100 98.8 99.4

Sokalan HP53 100 100 64.1 Not Run

Tall Oil Not Run Not Run 0 Not Run

Monawet 82.6 83.8 0.51 Not Run

Petro Disp. 425 41.9 62.1 0 Not Run

Witconate 1260 Not Run Not Run 11.6 Not Run

Witconate AOS 23.4 31.3 Not Run Not Run

Triton QS44 0 Not Run Not Run Not Run

Petro 22 Not Run Not Run 9.9 Not Run

GIvcoside 31.8 85.5 6.4 Not Run

Lignosulfonate 62.9 77.3 5.2 Not Run

b. Lab RWS Results

Sbce the Pluronic EO PO block copolymer surfactants showed greatest reduction b adherence b the screening assay, these products were bvestigated further b Lab RWS. Prelimbary experiments (data not shown) bdicated that a higher dosage of surfactant was required for minimization to occur under the more complex and dynamic conditions found b the Lab RWS. Figure 2 compares biofilm plate count data for both treated and untreated CPVC cylbders. PI 03 surfactant was fed contbuously to each system at a dosage of 10 mg/L and samples were collected 24 hours after exposure to the treatment. The figure shows bdividual measurements for each of six RWS. Note the mean values for both treated and untreated systems. These mean values were statistically different at an alpha value of 0.05 usbg analysis of variance. Figure 3 presents data for reduction b adherence when treated with 10 mg/l P103 surfactant over a period of 40 days. The data shows that the surfactant was most effective over the first two weeks of exposure after which efficacy declbes.

There was virtually no effect after 40 days' exposure.

Figure 4 shows results of experiments run to exambe the effect of both clay feed and presoakbg of the substrata b a 0.1% solution of the PI 03 surfactant for 18 hours prior to exposure. Presoaked and non-soaked CPVC cylbders were bstaUed b Lab RWS and exposed to the test conditions for 24 hours. In treated systems, PI 03 surfactant was also fed at a contbuous concentration of 10 mg/L. The data shows that whether or not the test systems contabed added clay, presoakbg provided a significant advantage. The effect of presoakbg was similar regardless of whether clay was present. However, when cylbders were not presoaked. the addition of clay resulted b greater bacterial adherence (less reduction). c. Field RWS Results

Figure 5 shows percent reduction b adherence when PVC substrata were exposed to 50 mg/L P103 surfactant over an extended time bterval b a Field RWS made up with plant recirculatbg coolbg water. The surfactant reduced adherence up to 14 days after which the effect was diminished. This is a similar pattern to what was observed b lab studies (Figure 3) and bdicates that the wbdow of greatest efficacy b terms of minimization is approximately 30 days. Figure 6 shows a comparison between PI 03, Igepal CO-620, and Tween 80 surfactants. P103 outperformed the other two surfactants, both of which had minimal effect on reduction. d. Studies Investigat e Mechanisms of Surfactant Efficacy

Table 9 presents data showbg the effect of PI 03 surfactant treatment on Zeta Potential of planktonic ceUs. For this experiment, four samples of water from a Lab RWS were collected. Two of these samples were further boculated with cultures of bacteria (from a streak across an R2A plate count plate) b order to bcrease the number of cells b the sample. PI 03 surfactant (at 10 mg L) was added to one boculated and one unboculated sample. Zeta Potential was determbed on each sample. Results show first of all that Zeta Potential was much greater (larger negative number) b samples that were boculated. Number of cells obviously had a significant effect on this measurement. Secondly, addition of P103 made no difference b Zeta Potential, whether or not the systems were boculated.

-18-

TΪTUTE SHEET R L Table 9: Effect on Pluronic PI 03 Surfactant on Zeta Potential

Zeta Zeta Potential Potential

Sample Identification

Mean S.D.

Untreated. Inoculated - 23.0 1.5

PI 03 Treated. Inoculated - 22.2 1.5

Untreated, Not Inoculated - 6.77 1.4

PI 03 Treated. Not Inoculated - 7.13 1.5

Table 10 shows the effect of P103 surfactant on contact angle of PVC material. For this experiment, strips of PVC were placed bto contabers containing either deionized (DI) water or water coUected from a lab recirculatbg water system (LRWS). To one DI water and one LRWS sample, 10 mg/L P103 was added and dynamic contact angle was determbed. The results show that contact angle dropped dramaticaUy when the PVC fill piece was exposed to the surfactant (98.9 to 74.5 and 90.0 to 66.1 degrees) bdicatbg that the surfactant is altering the surface properties of the PVC.

Table 10: Effect of Pluronic P103 Surfactan nn Dynamic Contact Angle of PVC

Sample Identification Advancing Contact Angle

PVC Fill/DI Water 98.9

PVC Fill/P103 b DI Water 74.5

PVC Fill/LRWS 90.0

PVC FU1/P103 b LRWS 66.1

HI. Discussion

The purpose of this work was twofold: (1) examώe the effect of surfactants on bacterial adherence and (2) determine whether lab screening assays will predict performance of a treatment under more complex conditions (Lab and Field RWS). In order to address number 1, it was necessary to start with a large spectrum of surfactants, evaluate eac for effect on adherence, and determbe whether these products could be grouped according to ability to reduce adherence. The materials that worked weU b the screening assay are structurally similar b one distbct feature, namely, the presence of ethylene oxide (EO) units. It is evident from the bventors' experiments that the more EO units, the better the materials perform b the assay.

The Silwet sample is a polysiloxane with pendent PEO grafts. It is believed that both components, the PEO and the silicone, will lower surface tension and alter the nature of biobterfaces. The Neodol 25-12 surfactant is a linear alcohol ethoxylate having a hydrophilic head group of about 12 EO units, with a hydrophobic tail of 12- 15 carbons. The tail group, though considered linear, is sometimes branched with methyl groups. Apparently, higher EO levels are needed to give acceptable performance, notbg that Neodol 25-7, with an average of 7 EO, and Neodol 91-2.5, with a shorter tail and an average of only 2.5 EO, did not perform as well.

The Pluronics are block copolymers of ethylene oxide (EO) and propylene oxide (PO) segments having the general structure.

CH₃

I

HO - (CH H_:O)_x-(CH,CHO\-(CH₂CH₂O)_x-H EO - PO EO The Pluronic 17R8, 25R2, 25R4. and 25R8 are PO-EO-PO blocks, while the

F68, F108, L62D, L64, and PI 03 are EO-PO-EO blocks. Among the first type, the higher HLB samples were slightly better, with the 25R2 (HLB 1-7) the poorest, though givbg acceptable performance at 94.4% reduction. The second type of material (EO-PO-EO) seems to be better overall. Here, HLB seems to have a stronger effect, notbg that L62D does not work and has a very low HLB. In general, an HLB of 7 or greater seems to be desirable. For ethoxylated nonionic surfactants, higher HLB values give greater water solubility. The results bdicate that higher HLB surfactants are more effective b reducbg bacterial adherence. The reason for the improved performance is believed to probably be twofold. First, sbce these surfactants have higher EO content, they will more effectively reduce bacterial adherence onto hydrophobic materials. Second, they are more water soluble, allowbg more efficient dispersal b aqueous systems.

The sorbitan monooleate surfactants, which are modified with about 20 PEO units, also worked well. These types of materials have found use traditionaUy as dispersants and wettbg agents. This material has an bterestbg structure for this type of adherence assay due to the presence of its sugar or polyol component b combbation with its PEO component. Either of these or both may contribute to the observed performance.

The anionic and amphoteric surfactants showed no activity b this assay. These materials are not believed to contab EO or other functional groups which would readily deter adherence. Many of these are used as dispersbg agents, but it is apparent from the bventors' results that there is no dispersancy mechanism operable b this system. That is, the microorganisms are not deterred to any extent by the presence of anionic or amphoteric dispersbg agents. The cationic surfactant Varine T showed good activity, but this may be due to some biocidal activity as well as adherence reduction properties. Both mechanisms are operable and cannot be partitioned from this study.

Because screenbg studies pobted to the Pluronic products as bebg as or more effective than other surfactants, the bventors' further work then focused on these products. Sbce the surfactant PI 03 provided exceUent reduction b adherence

(99.7%), this product was used for testbg b Recirculatbg Water Systems both b the lab and field. The RWS design aUows for examination of surfactant performance under dynamic (flowing) conditions and b a milieu much more complex than for the screenbg assay, which is essentially the surfactant plus sterile phosphate buffered water. A consortia of microorganisms is boculated bto the Lab RWS, exposed to light and the low levels of organic and borganic nutrients present b the tap water and from the growth of the algae b the system. It would be expected that a higher dosage of surfactant would be required to obtab a similar effect b terms of bacterial adhesion. This was the case: approximately 40 times the dosage was required to significantly minimize adhesion over a 24 hour period, and even then the percent reduction was less than 90%.

-21-

SUBSTΓΓUTE SHEET (RULE 26) Over an extended period of exposure, it was found that the surfactant was most effective initially, during the first two weeks of exposure. Between 3 and 4 weeks exposure, adherence reduction became bsignificant. The reduction b efficacy over time may be due to the surfactant film abrading off or biodegradbg, or due to some bacteria breaching the surfactant barrier and colonizbg the surface. Another explanation is that the surfactant bteracts with the surface, but does not fully cover and protect the entire surface - there are holes which aUow for bacterial adhesion to the surface. These cells then multiply and develop biofilms, unaffected by the surfactant. Ultimately, the treated systems contab substrata which are colonized to the same extent as the untreated systems. This same phenomenon was observed b field studies (Figure 5). Sbce data shown b Figure 4 bdicated that pre-coatbg the substratum surface with surfactant prior to exposure resulted b greater reduction b adhesion, it may be beneficial to pretreat new or clean fill material with PI 03.

The fact that addition of clay to the Lab RWS affected the ability of the surfactant to reduce adhesion (Figure 4), bdicates some type of bteraction of the clay particles with either the surfactant, the cells, or the substratum surface. It is known that clay particles will bteract with bacterial ceUs and extracellular polymers and so it is possible that a certab percentage of cells associated with clay particles might adhere differently than unattached ceUs. It is not clear from the literature whether these specific surfactants would bteract with clay particles, but polymers, especially anionic and cationic polymers are commonly used as clay dispersants and so it is quite possible the nonionic products might perform similarly.

Experiments were run at a field site b order to evaluate the surfactant under more rigorous, highly variable conditions. Data shown b Tables 1 through 3 compare water chemistry and bulk water plate counts for lab and field RWS. Primary measured differences between these two systems were that field systems tended to have higher and more variable bulk water bacterial counts, higher suspended solids, and higher conductivity. Suspended solids were also higher b the Field RWS (Lab RWS suspended solids were not measured because turbidity appeared very low). Water temperatures tended to be slightly lower for the field bstaϋation, primarily because of the location of the test systems b the power plant. A higher concentration (50 mg/L) of PI 03 was run to achieve significant reduction b bacterial adherence (Fig. 5 and 6). The observed differences between the two systems dicate that conductivity, suspended solids, or planktonic bacterial counts may have been responsible for this difference. Both the Igepal CO620 and Tween 80 surfactants were evaluated b the Field

RWS and neither one had a significant effect on adherence. The Tween 80, which worked quite well b the screenbg assay, worked slightly better than the IgepaL which was shown to be beffective b the screenbg assay. This pobts out that results obtabed from screenbg tests may not predict performance of the nonionic surfactants b more complex systems. In the case of the Pluronics and for the Igepal the screenbg was predictive, but b the case of the Tween 80 it was not.

Mechanism of action of the surfactants b limitbg bacterial adhesion was bvestigated further usbg two different techniques. Zeta potential measurements bdicated that cell surface charge was unaffected by the addition of the surfactant (Table 9). This mdicates that the surfactant must be altering the surface of the substratum rather than the surface of the cells. This hypothesis was confirmed by experiments usbg a Kruss Surface Tensiometer for measurement of contact angle. The data b Table 10 shows that contact angle was significantly lowered after treatment with the surfactant. The surfactant was wettbg the surface and causbg it to become more hydrophilic.

VirtuaUy any process or coolbg water system subject to microbial fouling and biofilm formation could potentiaUy benefit from treatment with PI 03 surfactant. Our findings have shown a clear benefit b reducbg bacterial adhesion onto pvc surfaces b recirculatbg coolbg water. It is also expected that PI 03 would reduce or control microbial adhesion onto materials other than polyvbyl chloride. For example, treatment of recirculatbg coolbg water systems with PI 03 might be effective b reducbg microbial adhesion and biofilm formation onto heat exchanger and system pipbg surfaces, as well as aU coolbg tower fill material surfaces. Other examples where applications of these surfactants could potentiaUy reduce or control microbial adhesion follow. In pulp and paper manufacturing, microbial fouling of surfaces (and slime formation) may occur wherever these surfaces come bto contact with microorganisms. This would bclude the thin stock loop systems, Whitewater, freshwater supply tanks and pipbg, and aU showers bcluding those which use either freshwater, recirculated water, or saveaU reclaimed water. Felts are subject to slime deposition, which leads to plugging of the felts, and addition of these surfactants to the water used for washing the felts might be effective. In general the treatment of any aqueous system b a papeπniU with P103 surfactant could potentiaUy aUeviate or minimize microbial foulbg.

Pluronic surfactants are already used b spray washers used for metal cleanbg and surface finishing as antifoams. They may also provide an additional benefit by reducbg microbial adhesion. In the manufacture of ceramics and sanitary ware, clay and other inorganics are molded b a water-borne process, foUowed by heatbg and other final steps. The PI 03 may assist b this process to prevent microbial adhesion. In clay slurries which contab dispersants, the PI 03 may act both as an agent to reduce microbial adhesion and as an optical brightening agent. P103 may be useful b mouthwashes. sbce the PEO surfactants are currently used for this application. The PI 03 surfactants would provide the benefit of providing an effective product with less foam than other PEO surfactants. The use of PI 03 also may inhibit the foulbg of water craft, ships, and other structures which reside b water, where it is necessary to prevent attachment of microorganisms. Multi-Component Surfactant/Biocide Studies and Conclusions

The foregobg studies were initiated to bvestigate the effect of surfactants on bacterial adhesion. The studies^' purpose was to determbe whether and to what extent surfactants might minimize bacterial adherence onto PVC material. The ultimate btent was to provide the first component of a treatment scheme to minimize fiU foulbg by minimizing the microbial component. As discussed above, the studies' results bdicate that nonionic surfactants of the EO PO configuration were effective b minimi ring adherence b both lab systems and under field conditions.

PVC high efficiency coolbg tower fiU material has been shown to foul rapidly with water-bome sϋt and microorganisms. The foulbg deposits formed are complex and difficult to either prevent or remove. The present bventors hypothesized that deposition with clay/microorganisms might be minimized if a combbation of clay dispersant-nonionic surfactant-biocide was utilized. Results, detailed below, bdicated that dispersant/surfactant combbations were beffective for controUbg the foulbg deposition onto nonfouled PVC fiU material when exposed to natural water-borne silts from a fresh water river. However, the same results bdicated that an EO PO block copolymer nonionic surfactant alone without the dispersant but b combbation with a biocide is effective b reduction of deposition onto PVC material under the same conditions. The results also showed that there was no relationship between biofilm accumulation and total deposit accumulation. The bventors hypothesized that surfactants b combbation with clay dispersants and biocides might be effective b reducbg total foulbg accumulation. In this case, the dispersant could reduce the rate of panicle deposition onto the biofouled surface, and the biocide would reduce the number of planktonic organisms and therefore the rate of biofilm formation. An experimental design was then implemented b order to test this hypothesis. The clay dispersant used was an acrylic acid/AMPS copolymer. The biocide used was glutaraldehyde. In addition to the EO/PO surfactants, an additional nonionic surfactant mixture b combbation with the clay dispersant was also tested.

The site chosen for the experimental work was a power plant located on the Monongahela River b Pennsylvania. The plant uses the river as makeup water for its recirculatbg coolbg water. Because it is a surface water source, it would be expected to carry a variable silt load, dependbg upon season and weather related run-off events. Exambation of many samples of fouled PVC fiU material from coolbg towers b the United States by Calgon laboratories revealed that those plants receivbg surface water makeup from fresh water rivers b the eastern/southeastern U.S. contabed a significant clay component. Experiments were then designed to expose PVC material to a side stream of Monongahela River water during Summer and FaU months, when sUt loadbg and biofoulbg would be expected to peak.

-25-

SUBST1TUTE SHEET (RULE 26) I. Materials and Methods a. Apparatus and Testbg Protocol

The recirculatbg water system (RWS) apparatus used for aU experiments is shown b Figure 1 and is described above b connection with the bacterial adherence studies. Recirculatbg water systems were bstaUed at the power plant site. Each system contabed 8 liters of Monongahela River water which was contbuously added to provide a retention time of 48 hours. Water temperatures b the RWS averaged about 30° C. The flow of recirculated water over the mini-tower b the RWS was controUed by a screw clamp so that each RWS had a similar flow over the exposed fiU pieces. PVC fiU material was obtabed from Munters Corporation (Fort Myers, FL).

Fill pieces were cut, rinsed b ethanol then b Butterfield Buffer (pH 7.2) prior to bstaUation. b. Test Solutions and Treatments

AU stock solutions were made up b deionized water and concentrations were based on a product weight, not on an active basis. The biocide used b this case was

45% active glutaraldehyde. Acryhc Acid AMPS was a combbation of acrylic acid (60%) and 2-acrylamido-2-methylpropylsulfonic acid (40%). For this work, a 28% active solution was used. The nonionic surfactant blend was comprised of the followbg components: 14.55% nonyl-phenoxy-polyethanol, 14.55% polyoxypropylene-polyoxyethylene block copolymer, 1.99% low molecular weight copolymer, and 0.49% 3-5 dimethyl-2H- 1.3,5-thiadiazbe-2-thione. 21% salt. The EO PO surfactant was a polyoxypropylene-polyoxyethylene block copolymer obtabed from BASF Corporation, Parsippany, N.J. In the experimental data discussed below, a BASF Pluronic PI 03 EO/PO block co-polymer surfactant was used. The Pluronic co- polymers are discussed in detail above b connection with the bacterial adherence studies. Makeup to each recirculatbg water system was pumped contbuously usbg a Masterflex pump (Cole Partner. NUes, IL). Surfactant and dispersant solutions were made up b 20 liter Nalgene carboys by addbg stock solutions to the makeup water. The biocide was added directly bto the sump of each RWS at a concentration of 60 mg/L (product basis). The EO/PO surfactant and the nonionic surfactant blend products were added at a concentration of 10 mg L (as product), and the acryhc acid/AMPS at a concentration of 30 mg/L (as the 28% product). A chemical analysis of the water coUected from the sump of the RWS is shown b Table 11. Planktonic heterotrophic plate counts on water coUected from the RWS averaged 2 X 10⁶ cfii/ml for the first eight week experiment and approximately 1 X 10⁵ cfii ml for the second. The untreated RWS tended to have somewhat lower plate counts than the treated systems though not significantly so. Otherwise, treatments had no obvious effect on the counts.

Table 11. Chemical Analysis of Water CoUected from Recirculating Water Systems

Supplied with Monongahela River Water.

c. Biofilm and Deposit Sampling and Analysis

PVC fill pieces were coUected and analyzed for biofilm parameters. Figure 1 shows emplacement of fiU pieces (26) b the RWS. Samples were coUected

¹ All analyte concentrations in mg L with the following exceptions pH = units, alkalinity = mg L as

CaC03, conductivity = utnhos/cm

Results are for a single sample collected from the Recirculating Water System sump after 8 weeks exposure to the treatment. After the exposure btervaL 3 fill pieces, each taken from a different level b the mini- coolbg tower, were removed and processed. For biofilm analysis, fiU sample biofilms were analyzed for ATP by placbg fiU pieces bto sterile glass tubes containing a homogenization solution and vortexed on a Vortex Genie Mixer (Fisher Scientific, Pittsburgh, PA) at a settbg of 10 for 1 mbute. For

ATP determbation an aliquot of this biofilm suspension was extracted b boilbg Tris Buffer (2.43 grams per Liter) for 5 minutes, then combbed with HEPES buffer (Turner Design, Sunnyvale. CA) and Lucifer /Lucif erase (Turner Design, Sunnyvale, CA) to determbe relative Ught output. Relative Ught units were then calibrated agabst a 4 X lO^*4 μg external ATP standard (Turner Design, Sunnyvale, CA).

Fill samples were also analyzed for deposit weight as foUows. Each fiU piece was removed from the RWS. dried at 105° C overnight, cooled b a desiccator and weighed. It was then washed b a detergent solution, rinsed b deionized water and redried for several hours at 105° C. The fiU piece was then weighed. The difference between weights was determbed to be the deposit weight.

Weights were calculated per gram of clean fiU weight.

II. Experimental Results a. Effect of Treatments on Deposit Formation

Figures 7 and 8 show the affect of treatments on deposit formation. Data shown b Figure 7 were coUected during an eight week exposure period b July and

August, while data shown b Figure 8 were coUected during an eight week exposure b October and November. Each bar represents results for a separate RWS. AA/AMPS was the acrylic acid/AMPS copolymer, NSB the nonionic surfactant blend, EO/PO the EO/PO block copolymer treatment. All treated systems were also treated with the glutaraldehyde biocide. Analysis of variance of the data bdicates that the EO/PO surfactant plus 45% glutaraldehyde biocide treatment provided a statisticaUy significant reduction b foulbg deposition (alpha 0.05). None of the other treatments provided a statisticaUy significant reduction b deposit formation.

-28- b. Effect of Treatment? on Riflf-lm nπηπTΪoπ

Tables 12 and 13 below show the effect of treatments on biofilm ATP concentrations. As b Figures 7 and 8, these data represent results from two separate experiments. Data shown b Table 12 were coUected at the completion of an experiment run b July and August; data b Table 13 from an experiment performed b October and November. The data was highly variable and did not bdicate that any of the treatments provided a significant reduction b biofilm ATP levels.

Table 12: Biofilm Formation Onto PVC FiU As a Function of Treatment b

Monongahela River Water for 8 Weeks Exposure.

¹ 45% active glutaraldehyde biocide was added on an intermittent basis each Monday,

Wednesday, and Friday at 60 ppm to all treated systems.

- Acrylic Acid/AMPS copolymer.

³ Acrylic Acid/AMPS copolymer plus nonionic surfactant blend.

⁴ N for ail determinations was 3.

-29-

SUBST1TUTE SHEET (RULE 26) Table 13: Biofilm Formation Onto PVC FUl As a Function of Treatment b Monongahela River Water for 8 Weeks Exposure.

¹ 45% active glutaraldehyde biocide was added on an btermittent basis each Monday, Wednesday, and Friday at 60 ppm to aU treated systems.

² The EO/PO surfactant Pluronic P 103.

^J EO/PO surfactant plus the acrylic acid/AMPS copolymer.

⁴ The nonionic surfactant blend plus acrylic acid/AMPS copolymer.

N for aU determbations was 3.

c.

Figure 9 shows the lbear regression of the relationship between ATP accumulation and deposit accumulation on fill material. The R square value of 0.023 bdicates essentiaUy no correlation between these two measured variables. m. Discussion

The bventors' origbal hypothesis that a dispersant/surfactant/biocide combbation would reduce the extent of deposit formation on fiU surfaces cannot be supported by the above results. In the case where the acryUc acid/AMPS copolymer was combbed with either the combbation nonionic surfactant (Fig. 7, 8) or the EO/PO surfactant (Fig. 8) and the glutaraldehyde biocide, there was not significant reduction b deposit accumulation. The contrary was true for the systems treated with the EO/PO surfactant plus biocide (Figure 9). These results show a 69% reduction b deposit formation compared to the untreated control systems. It appears that the EO/PO surfactant may be exhibitbg dispersant properties, sbce there was no measured reduction b microbial adhesion by the treatment. The data presented here demonstrate that treatment with the surfactant does b fact reduce sUt accumulation onto the PVC surfaces.

Because biofilm formation is an btegral aspect of foulbg deposition, it is beUeved that a treatment program should also address this aspect. Eager et al., "Glutaraldehyde: Factors Important for Microbiological Efficacy", Third Conference on .Progress In Chemical Disinfection, April 3-5, 1986, Bbghamton. New York presented data bdicatbg that much higher levels of glutaraldehyde are required to minimize or control biofilm formation (no effect level of 20 ppm active) than for planktonic bacteria. On a product basis, this will equate to about 44 mg/L (45% active glutaraldehyde). However, their data measured glucose uptake of bacteria b biofilms as a function of treatment. Dosage required to minimize bacterial adherence may be much higher. The results from this study show that the glutaraldehyde biocide, at the concentration/dosage used, was beffective b reducbg biofilm accumulation onto the PVC fiU surfaces. Even though the dosage used may be considered adequate for control of planktonic bacteria, it appears that the accumulated biotic and abiotic components of the biofilm limited the efficacy of this product. The EO/PO surfactant dosage b this study (10 mg L) was below the level shown to be effective b eariier studies with surfactant alone for prevention of bacterial adhesion, which showed that between 30 and 50 mg/L were requires, and that this reduction b adhesion was beneficial for only the first approximately thirty days of exposure. Results of the present study support those conclusions. Biofilm ATP concentrations were unaffected by the treatment b the present bventors' studies, even with the supplemental biocide. It would appear that the effect of the EO/PO surfactant b reducbg deposit accumulation is not due primarily to an effect on bacterial adhesion but rather to the control of clay deposition, either by dispersbg that clay prior to association with the biofilms. or somehow reducbg the efficiency with which it sticks to the biofilm surface.

Though this treatment has been shown to be effective for a very specific appUcation, it would be expected to work equaUy weU wherever there is a need to limit the biύldup of sUt/clay deposits on surfaces b bdustrial processes. PotentiaUy this might bclude appUcations b the foUowbg bdustries/applications: paper process, recirculatbg and once through coolbg, surface treatment, food and beverage processes, pasteurizers, preservation of water-based pabts, and b the processbg of clay slurries. In aU these aforementioned examples, microorganisms are known to adhere firmly to surfaces and are recalcitrant to biocides. Clay particles, either btroduced by the process or as a contambant. could potentiaUy stick to the conditioned surfaces.

In the paper process area, as paper miUs bcreasbgly utilize recycUng for environmental and economic reasons, there is a greater need to control the rate of solids deposition. Slime formation b paper machbe fines would be expected to provide sites for abiotic particle association, bcluding silt and clay. This treatment could potentiaUy minimize this problem. Treatbg the water used for washbg felts b papermiUs is another possible appUcation. sbce slime deposit on the felts commonly contab both microorganisms and borganic components.

In water-based pabts, which contab clay fiUers and other borganics, biocides are required as preservatives. The surfactant-biocide combbation could potentiaUy be more effective than biocide alone. In plastics or composites which contab clay fillers, this combbation could serve as a processbg aid to control deposits and microbial growth.

Spray washers used for metal cleanbg and surface finishing may have resultbg buildup of soU deposits and bacterial growth. Household and bdustrial washers may have a similar buildup. The surfactant-biocide combbation may help to control this problem b each of these systems.

In the manufacture of ceramics and sanitary ware, clay and other borganics are molded b a water-borne process, foUowed by heatbg and other final steps. Clay is also used as a fiUer b plastics or coπφosites. In both cases, the use of the surfactant b combbation with the biocide may serve as a processbg aid to control deposition and microbial growth. These surfactants combbed with appropriate biocides might be useful as dental antiplaque agents, where bacterial growth and borganic deposits form on dental surfaces. Alternatively, these may be useful b denture adhesives, which are water-borne materials often containing borganic fiUers like clay. FbaUy, this technology may inhibit foulbg of water craft, ships, or other structures which reside b water, where it is necessary to prevent attachment of organisms.

It would be expected that other biocides besides glutaraldehyde might work equaUy weU b these applications, when used at concentrations adequate to kill biofilm bacteria. Such biocides might bclude quaternary ammonium compounds, isothiazolbe. carbamates, DBNPA (dibromonitrilopropionamide), or dodecylguanidbe hydrochloride (DGH). As welL nonionic surfactants of the EO/PO configuration other than Pluronic PI 03 would be expected to act b similar fashion.

Claims

1. A method for inhibitbg the adhesion of micro-organisms to a surface b contact with an aqueous system, the method comprisbg addbg to the surface or the system an effective amount of at least one compound comprisbg repeatbg ethylene oxide units.

2. A method according to claim 1 and whereb at least 50 ppm of said at least one compound is added to the aqueous system.

3. A method accordbg to claim 1 or claim 2 and whereb said at least one compound is selected from a polypropylene oxide-polyethylene oxide block copolymer, a polysUoxane bcludbg pendent polyethylene oxide grafts, an alcohol ethoxylate bclud g a hydrophilic head group and a hydrophobic tail group, and a sorbitan monooleate bcludbg from 15 to 25 ethylene oxide units.

4. A method accordbg to claim 3 and whereb said at least one compound is said polypropylene oxide-polyethylene oxide block copolymer, and has a structure selected from

CH₃

I

HO-(CH₂CH₂O)_)tI-(CH₂CHO)_y-(CH₂CH₂O)_x2-H

and

CH₃ CH₃

HO-(CH₂CHO)_xl-(CH₂CH₂O)_y-(CH₂CHO)_x2-H

whereb xl, x2, and y are each bdependently numbers b the bclusive range 5 to 1,000 and whereb said block copolymer comprises from 20% to 80% ethylene oxide units by weight.

5. A method accordbg to claim 4 whereb said block copolymer has a molecular weight b the range from 2,000 to 20,000.

6. A method according to claim 1 whereb the said micro-organisms comprise bacteria.

7. A method accordbg to any one of the precedbg claims and whereb the method bcludes addbg to the aqueous system b addition to the said compound a biocide thereby reducbg the formation of deposits on the said surface.

8. A method according to claim 7 and whereb the said com ound is a polyoxypropylene-polyoxyethylene block copolymer which is added to said aqueous system b an amount between 30 mg/L and 50 mg L.

9. A method accordbg to claim 7 or claim 8 and whereb said biocide comprises at least one compound selected from gluteraldehyde, quaternary ammonium compounds, isothiazolbe, carbamates, dibromonitrilopropionamide, and dodecylguanidbe hydrochloride.

10. A method accordbg to claim 9 and whereb said biocide is gluteraldehyde and at least 60 ppm of said gluteraldehyde is added to the aqueous system.

11. A method accordbg to any one of claims 1 to 10 and whereb the aqueous system is selected from papers miUs, paper processbg systems, recirculatbg and once- through water coolbg systems, food and beverage processbg systems, pasteurisbg systems, clay slurry processbg systems, and systems for manufacturing ceramics, sanitary ware, plastics, and composites.

12. A method accordbg to any of claims 7 to 10 and whereb the deposits comprise at least one selected from water-borne silt, water-borne clay, and water- borne micro-organisms.

13. A method accordbg to any one of claims 1 to 12 and whereb the surface comprises polyvbyl chloride.

14. A composition for appUcation to a surface b contact with an aqueous system to reduce formation of deposits on the surface, the composition comprisbg a polyoxypropylene-polyoxyethylene block copolymer and a biocide.

15. A composition according to claim 14 and whereb said biocide comprises at least one compound selected from gluteraldehyde, quaternary ammonium compounds, isotliiazol e, carbamates, dibromonitrilopropionamide. and dodecylguanidbe hydrochloride.

16. A composition according to claim 14 or claim 15 and also bcludbg an anionic polyelectrolyte dispersant.