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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1984, p. 395-403 Vol. 48, No. 3 0099-2240/84/080395-09$02.00/0 Copyright ©D 1984, American Society for Microbiology Evaluation of Cleaning Strategies for Removal of Biofilms from Reverse-Osmosis Membranes C. WHITTAKER,1 H. RIDGWAY,2 AND B. H. OLSON'* Environmental Analysis, Program in Social Ecology, Univ'ersity of California, Irvine, California 927171 and Water Factory 21, Orange County Water District, Fountain Valley, California 927082 Received 29 February 1984/Accepted 30 May 1984

An evaluation was made of the efficiency of five classes of chemical cleaning agents for removing biofilm from spirally wound cellulose acetate reverse-osmosis membranes receiving influent with high or low levels of combined chlorine. Each cleaning regimen utilized one or more of the following types of chemical: (i) and , (ii) chaotropic agents, (iii) bactericidal agents, (iv) enzymes, and (v) antiprecipi- tants. Cleaning efficiency was tested in the laboratory on membrane material removed from operations at various intervals (2 to 74 days). Cleaning effectiveness was evaluated against nontreated control membranes and was scored by scanning electron microscopy and enumeration of surviving bacteria after treatment of the membranes. The combinations of classes which were most effective in biofilm removal were the anionic and combination and combinations involving enzyme-containing preparations. Membranes receiving influent with high levels of combined chlorine were easier to clean but more susceptible to structural damage from prolonged exposure to combined chlorine. No treatment or combination of treatments was completely effective or effective at all stages of biofilm development.

The development and use of the cellulose acetate reverse- removal of accumulated materials from pipes and reservoirs. osmosis (RO) membrane have expanded greatly over the Cleaning cellulose acetate RO membranes is similar in past 30 years. The first practical and efficient membrane was approach to removing biofilms from outer surfaces, but it is developed by S. Loeb and S. Sourirajan in the early 1960s (8; more complex. The integrity of the cellulose acetate mem- U.S. patents 3, 133, 132 and 3, 133, 137, 1964). It was brane is easily destroyed by strong chemical oxidants, improved dramatically by Loeb and Manjikian in 1967 (7) enzymes, pH shifts, and temperature fluctuations. The de- and has since been incorporated into several industrial sign configuration of spirally wound membranes makes processes, the most common being the demineralization and physical methods of cleaning ineffectual or impossible. Pre- purification of water from brackish and wastewater sources viously, the fouling layer of RO membranes has been consid- (7). Several physical arrangements of the membrane unit ered to be primarily inorganic in composition (16-18). Re- structure have been designed to maximize the surface area cent work (3, 12), however, has shown that the fouling layer per unit of volume (packing density) and to maintain high may be either microbial or inorganic and that the extent to pressure across the membrane (1). The spirally wound which either type of material predominates is the result of membrane configuration (Fig. 1) has the advantage of a high such interacting factors as the chemical quality of the water, surface area per unit of volume, it also has the disadvantage the microbial quality of the water, and the presence and of relatively high pressure losses across the membrane and amounts of calcium and magnesium . Microorganisms difficulty in handling large concentrations of solids. An composing the biofilm may accumulate owing to the adhe- additional disadvantage is the difficulty in cleaning modules sion of organisms present in the feedwater or to the prolifera- of this type of design owing to inefficiency in recirculating tion of a few opportunistic organisms at the membrane the concentrate and subsequent resuspension of the fouling surface. Some of the fouling bacteria involved in the earliest layer (4). stages of biofilm formation have been shown to exhibit a Biofouling of surfaces over or through which water must marked affinity for the cellulose acetate membrane surfaces pass has been of major economic importance because the (13). The differences in the accumulation mechanisms in the formation of microbial biofilms can decrease water velocity, formation of a biofilm may be important in determining the clog pipes, increase energy utilization, and decrease the best way to clean membranes or maintain their efficiency efficiency of operations. Biofouling has been a major prob- with bacteriostatic or other agents. lem in cooling systems for many years. Research on the This study examined a variety of compounds by utilizing a fouling of cooling units has led to extensive characterization screening procedure to determine appropriate and potential- of fouling layers on pipe and tank surfaces and other ly effective classes of chemicals which could be employed in segments of each system (9, 14,15). The growth of microor- cleaning spirally wound cellulose acetate RO membranes ganisms and the precipitation of inorganic chemical constitu- (Table 1). Chemical cleaning agents and combinations of ents have been recognized as the major constituents of these these agents were classified as (i) surfactants and detergents, fouling layers. To combat biofouling on surfaces of this type, which neutralize charged colloidal particles and resolubilize bactericidal agents as well as various dispersants and surfac- or resuspend them; (ii) chaotropic agents, which denature tants have been successfully applied in numerous laboratory and therefore readily solubilize organic constitu- and field trials (9, 14). ents; (iii) bactericides, which kill microorganisms through Cleaning cooling systems requires physical or chemical or dissolution; (iv) enzymes, which hydrolyze the proteinaceous and glycoprotein exopolymers surrounding * Corresponding author. microorganisms; and (v) antiprecipitants, which remove 395 396 WHITTAKER, RIDGWAY, AND OLSON APPL. ENVIRON. MICROBIOL.

MODULE SEAL (SEALS AGAINST THE INSIDE WALL OF A PRESSURE /VESSEL TO FORCE THE FEED SOLUTION THROUGH THE MODULE) / v PERMEATE COLLECTION HOLES

FEED SOLUTION

FEED CHANNEL FEED SOLUTION it- SPACER ;D - MEMBRANE PERMEATE COLLECTION 9- MATEfRlAL

>E- MEMBRANE > FEED CHANNEL - SPACER PERMEATE FLOW (AFTER PASSAGE THROUGH MEMBRANE INTO PERMEATE COLLECTION MATERIAL)

FIG. 1. Interior structure of a spirally wound RO membrane (13). metals and other precipitated ions from the layer of fouling. moved, the layers of the membrane were carefully unwound This experimental procedure allowed the screening of a large and spread upon a sterile surface. A measured section of the number of possible cleaning agents and considered the membrane (600 cm2) was scraped with a sterile razor blade chemical principle or mechanism of the selected cleaning to remove the adhering biofilm. This fouling layer was compound. In this way, classes of chemicals or combina- tions of classes were studied, and their effectiveness was generalized to other compounds of the same class. Visual inspection by scanning electron microscopy (SEM) and TABLE 1. Classes of compounds used in cleaning treatments for counting of the bacterial population remaining on the mem- RO membranes brane were used to assess the efficiency of the treatment in Class Compounds" the removal of biofilm. Antiprecipitants ...... EDTA (disodium ) STP MATERIALS AND METHODS TSP

Sampling. Miniature spirally wound RO membrane units Bactericides...... MBTC (diameter, 6.35 cm) were provided by Water Factory 21, GuHCI Fountain Valley, Calif. This facility has been described in a Urea previous paper (12). These units were put into service in an ZDDC on-site testing module which received the same water as the CTAB full-scale (diameter, 20.3 cm) counterparts to simulate actual Detergents-sequestrants ...... Triton X-100 operating conditions. Six membranes units in the module Biz" were available: four received water containing 3 to 5 mg of CTAB combined chlorine per liter (designated hereafter as the low- TSP chlorine [LC] membranes), and two received water with a tripolyphosphate combined chlorine concentration of 15 to 20 mg/liter (high- SDS chlorine [HC] membranes). The feedwater entering the test modules was secondary (activated sludge) treated municipal Enzymes ...... Trypsin Protease wastewater from the Orange County Sanitation District Thermolysin facility. This water was treated by lime clarification, ammo- Papain nia air stripping, recarbonation, and multimedia filtration Esterase before RO. The membrane units were removed from the test Pancreatin module and replaced by new units at each sampling time Biz (Table 2). Membrane units were removed at intervals span- ning 3 to 74 days. During each sampling, the membrane unit Chaotropic-denaturing agents ...... Urea was removed from the module, placed directly on ice, and GuHC transported to the laboratory (elapsed time between removal SDS of the unit and processing was less than 2 h). At the " Abbreviations: CTAB, cetyltrimethylammonium bromide, GuHCI. guani- laboratory, the spun-glass protective coating was removed. dine hydrochloride; MBTC. methylene bisthiocyanate: SDS. sodium dodecyl ; STP, sodium triphosphate: TSP. ; ZDDC. zinc At this point, aseptic conditions were introduced and main- dimethyidithiocarbamate. tained throughout the rest of the processing. ' Biz is the trade name for a laundry presoaking containing broad- Processing. After the spun-glass covering had been re- spectrum enzymes and bleaching compounds. VOL. 48. 1984 REMOVAL OF BIOFILM FROM RO MEMBRANES 397

TABLE 2. Sampling schedule of RO membrane units TABLE 3. Chemical compounds and dosages used to remove Days elapsed at time of sampling" biofilm from RO membranes Membrane unit unit Replacement unit Treat- Exposure Initial Dose time ment no. Chemical(s) 1 3 43 (min) 2 5 57 1 Esterase 10 ,ug/ml 30 3 11 EDTA 311 FLl/ml (10 mM) 4 20 Sb 3 22 Trypsin 100 ,ug/ml 30 6" 11 63 Triton X-100 1 Ill/ml Values show days elapsed after the beginning of the experiment. After each membrane unit was removed, it was replaced by a new unit. " Received 15 to 20 mg of combined chlorine per liter in feedwater. 2 EDTA 311 ,ul/ml (10 mM) 60 Urea 120 mg/ml (2 M) TSP 10 mg/ml suspended in 10 ml of 100 mM phosphate buffer. pH 7.1. Appropriate serial dilutions were spread plated onto m- 4 GuHCI 120 mg/ml (2 M) 60 standard plate count (m-SPC) agar (16) plates to determine CTAB 10 mg/ml bacterial counts. Of the remaining membranes, two strips (1 12 were in sterile screw-cap one for by cm) placed tubes, 5 Urea 120 mg/ml (2 M) 60 experimental cleaning treatment and one for a control. The SDS 10 mg/ml control section received 100 mM phosphate buffer. pH 7.0. Experimental treatments and dosages are summarized in Table 3. The tubes were sealed, placed horizontally in a 6 Urea 120 mg/ml (2 M) 60 rack, and shaken at 110 rpm in a Lab-line Orbit Environ- CTAB 10 mg/ml Shaker at 35°C for 1 h. The treated and control strips were then removed from the tubes and were sectioned for ATP 7 Papain 100 ,ug/ml 30 analysis, analysis, bacterial counting, and SEM. Triton X-100 1 ,ul/ml Bacterial counts and isolates. Serial dilutions of the mem- EDTA 311 p.1/ml (10 mM) brane scrapings in phosphate buffer were spread plated onto m-SPC agar. After 5 to 7 days of incubation at 28°C, colonies Protease 100 ,ug/ml 30 were counted. All plates containing 40 colonies or fewer Triton X-100 1 IlU/mI were selected for identification. On plates with more than 40 EDTA 311 [LI/ml (10 mM) bacterial colonies, 40 colonies were randomly selected from the plate. Colonies were purified and stored on m-SPC agar 8 Biz 10 mg/ml 6() slants at 4°C until identification. Identification of the isolates was accomplished by the three-tube Lassen method for gram-negative rods (6) and the acid-alcohol fast determina- 9 Pancreatin 100 p.g/ml 60 tion for mycobacteria with additional tests as recommended Triton X-100 1 p.1/ml by Berge 's Maniual of Deterininativse Bacteriologv (2) for EDTA 311 ,ul/ml (10 mM) gram-positive bacteria. The 1-cm2 section removed from each strip was vortexed 10 Thermolysin 100 ,ug/ml 60 vigorously for 1 min in 5 ml of m-SPC broth to remove the Triton X-100 1 ,ul/ml bacterial film. Serial dilutions were then plated onto m-SPC EDTA 311 ,ul/ml (10 mM) agar plates. CFU were counted after 5 to 7 days at 28°C, and 15 colonies were picked, isolated, and identified as described above. 11 GuHCI 2 M 30 CTAB 10 mg/ml SEM. The pieces of membrane to be studied by SEM were Protease 100 p.g/ml placed in 5 ml of 2.5% glutaraldehyde in phosphate buffer, pH 7.0, and were refrigerated overnight. The membranes Papain 100 ,ug/ml 30 were then dehydrated in a series of ethanol solutions: 30, 50, Esterase 10 p.g/ml 75, 85, 95, and 100% ethanol (the last twice), ethanol-Freon EDTA 311 p.l/ml (10 mM) 113 (1:1), and 100% Freon 113 (twice). The samples were then air dried and mounted on aluminum stubs with a collodial silver adhesive. The stubs were then coated with 12 CTAB 10 mg/ml 60 gold-palladium (60:40) and viewed on a Hitachi model SU 500 scanning at 15 kev with a working 13 SDS 10 mg/ml 60 distance of 10 mm and a stage tilt angle of 00. Black and white photographs were made with Polaroid P/N55 film. The effectiveness of each cleaning treatment was scored semi- 14 Triton X-100 10 mg/ml 60 quantitatively according to the presence and appearance of the biofilm. A score of 0 was given if no removal was evident 15 MBTC 10 mg/ml 60 (compared with an untreated control), and a score of 4 was CTAB 10 mg/ml assigned if removal was complete. For the criteria utilized for scoring by SEM, see Table 4, footnote b. Chemical cleaning treatments. The chemicals used in each Continiued on following page 398 WHITTAKER, RIDGWAY, AND OLSON APPL. ENVIRON. MICROBIOL.

TABLE 3-Continued of the 24 treatments and doses applied are presented in Table Exposure 3. Doses of previously investigated chemicals were chosen Treat- Chemical(s) Dose time ment no. (min) to approximate or exceed values reported in the literature (7, 11, 19). All of the chemicals were dissolved in 100 mM phosphate buffer, pH 7.0. The control consisted of a strip of 16 ZDDC 10 mg/ml 60 treated in the same manner as test membranes Triton X-100 0-1 ,V/mi the membrane but exposed to 100 mM phosphate buffer, pH 7.0, alone. This was used to control for the physical action of the liquid 17 STP 10 mg/ml 60 on the membrane. EDTA 10 mg/ml TSP 10 mg/ml RESULTS Observation of the membranes by SEM provided the most 18 EDTA 311 RI/ml (10 mM) 60 direct evidence for biofilm removal. In Table 4, results of SEM scores of the treated membranes are presented in ranked order, as described above, indicating the amount of 19 Trypsin 100 Rg/ml 60 EDTA 311 ,u/ml (10 mM) biofilm removed compared with that removed from a treated Triton X-100 1 ±1/ml control. The control corrected for the removal of any biofim by mechanical effects during the treatment period. Table 4 shows the scores of the treated LC and HC membranes and 20 Esterase 10 ,g/ml 60 the mean score for all days sampled for each treatment. EDTA 311 ±1/ml (10 mM) Finally, the overall score for each treatment was calculated Triton X-100 1 Rl/ml as follows: n n z XLC + z XHC 21 Urea 120mg/ml (2 M) 60 Overall score = Xtotal = i=1 i=1 nLC + nHC 22 TSP 10 mg/ml 60 Overall rankings of the treated membranes indicated that treatments with a mean score of -2.5 were very good 23 Pancreatin 100 ,ug/ml 30 cleaners. These treatments included esterase-EDTA/trypsin- EDTA 311 RI/ml (10 mM) Triton (i.e., treatment with esterase-EDTA followed by Triton X-100 1 RI/ml trypsin-Triton), urea-sodium dodecyl sulfate (SDS), papaih- Triton-EDTA/protease-Triton-EDTA, Biz, and pancreatin- CTAB 10 mg/ml 30 Triton-EDTA. Cleaners with an overall score of 2.0 to 2.5 were considered to be good and included SDS, sodium triphosphate (STP)-EDTA-trisodium phosphate (TSP), tryp- Control Potassium phosphate 17.4 mg/ml (100 mM) 60 sin-EDTA-Triton, and Triton. The remainder of the treat- (pH 7.0) ments (poor cleaners) were consistently unable to reduce the biofilm by 50% and are not included in Table 4.

TABLE 4. SEM evaluation of biofilm reduction by various cleaning agents on RO membranes receiving HC or LC doses of feedwater Cleaning effectivenessb Treatment' LC membranes on day: HC membranes on day: Overall' 3 5 11 20 43 57 Mean 3 11 22 63 Mean Urea-SDS 2+ 3+ 3+ 3+ 2+ 3+ 2.7 3+ 3+ 4+ 4+ 3.5 3.0 Biz 3+ - 4+ 3+ 2+ 1+ 2.6 3+ 4+ 3+ 4+ 3.5 3.0 Esterase-EDTA/ 2+ 2+ 3+ 2+ 2+ 2+ 2.2 3+ 3+ 3+ 4+ 3.2 2.6 trypsin-Triton Pancreatin-Triton-EDTA 2+ - 2+ 3+ 2+ 1+ 2.0 3+ 3+ 3+ 4+ 3.2 2.6 Papain-Triton-EDTA/ 2+ 3+ 3+ 2+ 2+ 1+ 2.2 2+ 3+ 3+ 4+ 3.0 2.5 protease-Triton-EDTA SDS - 3+ 1+ 1+ 1+ 2.0 3+ 3+ 4+ 3.3 2.3 Triton - 2+ 0 - 1+ 1.0 - 2+ 3+ 4+ 3.0 2.0 STP-EDTA-TSP - - - 1+ 1+ 1.0 - - 4+ 4.0 2.0 Tiypsin-EDTA-Triton - - 1+ 1+ 1.0 4+ 4.0 2.0 Pancreatin-EDTA/Triton-CTAB - 2+ 1+ 1.5 3+ 3.0 2.0 aTreatments with an overall rank of <2.0 are not included. b Scores, based on comparison with treated control, were assigned as follows. 0, No removal of biofilm apparent; multilayer biofilm (MLB, two or more cell layers thick) and single-layer biofilm (SLB) both present. 1+, Poor biofilm renmoval, usually less than 20 to 30%; MLB and SLB both present. 2+, Fair biofilm re- moval, usually 50 to 70%; MLB absent, SLB present. 3+, Good biofilm removal, usually 70 to 80%; MLB absent, SLB present. 4+, Excellent biofilm removal, usually 90 to 100%o; a few scattered cells might remain, but there were no patches; no MLB or SLB present.-, No sample was treated with the indicated cleaning agent. c Overall score was determined by the formula given in Results. VOL. 48, 1984 REMOVAL OF BIOFILM FROM RO MEMBRANES 399

Representative SEM micrographs in Fig. 2 and 3 show the pancreatin-Triton-EDTA treatment (Fig. 2g and 3g). The HC relative rankings of selected treatments compared with the membranes appeared to be cleaned with higher efficiency in control. The biofilm formed a confluent layer over the nearly all cases. This variability was demonstrated at other membrane on both the HC and LC controls. The micro- sampling times with several treatments (Table 4). graphs depict samples from the LC membrane (Fig. 2) and A number of the experimental cleaning formulations ap- the HC membrane (Fig. 3) after 11 days of operation. In peared to exhibit bactericidal activity. For several of the these figures, the range of cleaning effectiveness is demon- enzymatic cleaning treatments, a problem with the sterility strated from no differences from the control (Fig. 2h and 3h) of the solutions made counts of CFU inaccurate, and some to >90% removal of biofilm (Fig. 2f and 3f0. treatment schemas, such as SDS and Triton, were initiated The various chemical strategies and combinations of strat- after the study had begun. egies illustrated in Fig. 2 and 3 included enzyme-- Table 5 shows the percentage of reduction of numbers of chelator combinations (b, e, and g), denaturant-detergent bacteria compared with the control for the compounds found combinations (c and d), an enzyme-detergent combination (f) to have a rating of .2.0 in the SEM evaluation. The urea- and a biocide-detergent combination (h). The methylene SDS combination was the most effective as a bactericide and bisthiocyanate (MBTC)-cetyltrimethylammonium bromide was also a successful cleaning treatment. Biz consistently (CTAB) treatment (Fig. 2h and 3h), representing a biocide- showed good reduction in numbers of bacteria, but many of detergent combination, did not reduce the biofilm when the other cleaning strategies produced more uneven results. compared with the treated control. On the other hand, the Some of this variability may have been due to the lack of a Biz treatment (Fig. 2f and 30) showed excellent biofilm highly reproducible method for removing all bacteria from reduction on both LC and HC membranes. Some variability the treated and control membranes. in cleaning efficiencies between the LC and HC membranes The cleaning effectiveness of the highly bactericidal com- is seen in the urea-CTAB treatment (Fig. 2d and 3d) and the pounds (>90% reduction in numbers of bacteria) is shown in

FIG. 2. Representative SEM micrographs showing the relative effectiveness of various cleaning treatments in removal of biofilm from LC RO membranes after 11 days in operation. (a) Treated control membrane, given a score of 0 (no biofilm removal). (b) Membrane treated with esterase-EDTA/trypsin-Triton and given a score of 3+ (good biofilm removal, 70 to 80% reduction). (c) Membrane treated with urea-SDS and given a score of 3+ (good biofilm removal). (d) Membrane treated with urea-CTAB and given a score of 1+ (poor biofilm removal, <20 to 30% reduction). (e) Membrane treated with papain-Triton-EDTA/protease-Triton-EDTA and given a score of 3+ (good biofilm removal). (f) Membrane treated with Biz and given a score of 4+ (excellent biofilm removal, 90 to 100% reduction). (g) Membrane treated with pancreatin- Triton-EDTA and given a score of 2+ (fair biofilm removal, -50 to 70% reduction). (h) Membrane treated with MBTC-CTAB and given a score of 0 (no biofilm removal discernible). Bars, 5 p.m. 400 WHITTAKER, RIDGWAY, AND OLSON APPL. ENVIRON. MICROBIOL.

_I _I_I _I _II _I s, _I _II _III

_x_S 11

FIG. 3. Representative SEM micrographs showing the relative effectiveness of several cleaning agents in removal of biofilm from HC RO membranes after 11 days in operation. (a) Treated control membrane, given a score of 0 (no biofilm removal). (b) Membrane treated with esterase-EDTA/trypsin-Triton and given a score of 3+ (good biofilm removal, -70 to 80%4 reduction). (c) Membrane treated with urea-SDS and given a score of 3+ (good biofilm removal). (d) Membrane treated with urea-CTAB and given a score of 2+ (fair biofilm removal, -50 to 70% reduction. (e) Membrane treated with papain-Triton-EDTA/protease-Triton-EDTA and given a score of 3+ (good biofilm removal). (f) Membrane treated with Biz and given a score of 4+ (excellent biofilm removal, 90 to 100% reduction). (g) Membrane treated with pancreatin- Triton-EDTA and given a score of 3+ (good biofilm removal). (h) Membrane treated with MBTC-CTAB and given a score of 0 (no biofilm re- moval). Bars, 5 p.m.

TABLE 5. Effects of cleaning agents on reduction of counts of viable bacteria from LC RO membranes" SEM % Reduction from control on day: Treatment score (range) 3 5 11 20 43 57 Biz >2.5 83.5 70.5 69.5 58.4 71.2 32.7 Urea-SDS 94.1 100.0 100.0 100.0 99.9 100.0 Pancreatin-Triton-EDTA NE" NE NE NE 88.9 18.3 Esterase-Triton/ NE NE NE NE 32.6 19.2 trypsin-EDTA Papain-Triton-EDTA/ 97.8 NE NE NE 69.0 92.1 protease-Triton-EDTA SDS 2.0-2.5 ND" ND 97.5 48.9 68.7 0.0 Triton ND ND 87.6 0.0 0.0 49.0 TSP-EDTA-STP ND ND ND ND 0.0 0.0 Trypsin-EDTA-Triton ND ND ND ND 31.0 0.0

' Treatments shown are those which received a score of at least 2.0 in the SEM evaluation (Table 4). There were no colonies isolated from the HC membranes until 63 days had elapsed, when 2.0 x 102 colonies per cm2 were isolated from the control membranes. Colonies were not enumerated from the treated HC membranes. *NE, Not enumerated; contamination from enzyme preparations. ND, Not determined. VOL. 48, 1984 REMOVAL OF BIOFILM FROM RO MEMBRANES 401

Table 6. Pancreatin-EDTA-Triton/CTAB showed some Other evidence that lends support to this hypothesis is that cleaning potential, and urea-SDS ranked as a very good although no bacterial colonies were isolated from the HC cleaner. For the most part, however, the highly rated membrane until 63 days had elapsed, the HC and LC bactericidal combinations (those containing CTAB or urea) membranes appeared under SEM to be equally fouled by were not very effective at removing biofilm. bacteria at each sampling time in the experiment. This Identification of the bacteria recovered from the fouled evidence suggests that chlorine-inactivated bacteria may RO membranes indicated that >95% of isolates were mem- also produce a biofouling layer on the RO membrane sur- bers of the genus Mycobacterium, an acid-fast, gram-posi- face. tive group of rod-shaped microorganisms (2). Mycobacteria Some chemicals consistently reduced the numbers of form tenacious mucoid colonies on m-SPC agar, and at least bacteria by 30 to 80%. These compounds may not act one strain has been recently demonstrated to exhibit a through bactericidal activity; the reduction in numbers may marked affinity for the cellulose acetate RO membrane be due rather to the actual removal of the bacteria from the surface (13). surface of the membrane. This effect would be preferred over bactericidal activity. Chemical treatments which exhib- ited this type of action included Biz, papain-Triton-EDTA/ DISCUSSION protease-Triton-EDTA, pancreatin-Triton-EDTA, and SDS (Table 5). Innovations in cleaning techniques for RO membranes The SEM photographs show some dramatic decreases in have been sporadic and somewhat haphazard in their devel- biofilm that were not well reflected by corresponding counts opment. Most techniques have been developed under actual of bacteria. Some of the membranes appeared virtually bare, operating conditions by simply substituting new procedures yet CFU measurement revealed that a substantial number of for old ones and passing along the insight gained. Many new bacteria remained associated with the membranes. This cleaning agents have been utilized on the assumption that the effect may be due to one of several causes. The confluency predominant fouling material is inorganic scale or precipitate of biofilm may have been so disrupted after cleaning that the and that any organic material present is primarily humic samples that were measured were too small (1 cm2) to be acids (3). representative of the whole sample. It is also possible that The most common treatments for removing the fouling the processes of fixation and dehydration for SEM may layer of RO membranes are periodic flushing with mixtures actually alter the biofilm, in this case enhancing the removal of detergents and chelating agents, changes in pH, and the of bacteria from the membrane and exaggerating the report- addition of antiprecipitants such as sodium hexametaphos- ed effectiveness of the cleaning treatments. Since SEM is the phate or citric acid (3, 10, 19). most important parameter assessed in this study, it is critical In this study, we have characterized the biofilm micro- that the limitations of this method be understood. However, scopically and bacteriologically to examine the effectiveness assuming that the effects of fixation and dehydration are of different cleaning strategies in the removal of a developing approximately equal across the treatments, SEM evaluation biofilm. Our cleaning strategies, contrary to those in previ- should still be a good measure of the relative effectiveness of ous work, have assumed that the major fouling entity is each treatment, although it may not be quantitative. bacterial in nature with its associated organic and inorganic Analysis of effective cleaning treatments. Each treatment by-products. incorporated one or several cleaning strategies. Using the The present studies indicate that the most reliable evi- criteria from Table 4 to determine the effectiveness of dence for the degree of biofilm removable was direct SEM biofilm removal, it is apparent that cleaning treatments that examination. Bacterial counts did not correlate well with the were most successful (mean score, >2.5) had constituents in visual observations via SEM. Strongly bactericidal com- each of the following categories: enzymes, antiprecipitants, pounds were not necessarily effective in removing biofouling bactericides, and denaturing agents. We have already deter- layers, and cleaning solutions that were effective in biofilm mined that bactericidal compounds are not necessarily effec- removal were not necessarily bactericidal. This dichotomy tive in biofilm removal, so the remaining constituents require of effects eliminates one of the proposed strategies as an closer examination. effective mechanism of attack on the biofilm; many bacteri- Preparations containing enzymes. The enzyme-antiprecipi- cidal compounds probably do not lyse and dissolve the tant-dispersant combination formed most of the very good fouling layers. cleaners. EDTA, a chelator, was added to the enzyme

TABLE 6. Effectiveness of cleaning combinations that reduced counts of viable bacteria by at least 90% on HC and LC RO membranes Cleaning effectiveness" Treatment LC membranes on day: HC membranes on day: Overall'' 3 5 11 20 43 57 Mean 3 11 22 63 Mean EDTA-urea-TSP 0 1+ 2+ 2+ 1+ 3+ 1.5 2+ 2+ -h 1+ 1.7 1.6 Urea-SDS 2+ 3+ 3+ 3+ 2+ 3+ 2.7 3+ 3+ 4+ 4+ 3.5 3.0 Urea-CTAB 2+ 0 1+ 1+ 0 2+ 1.0 2+ 2+ 3+ 4+ 2.7 1.7 CTAB 0 0 1+ 0 0 0.2 1+ 1+ 3+ 1+ 1.5 0.8 MBTC-CTAB - 0 1+ 0.5 - 0 1+ - 0.5 0.5 Urea - 0 2+ 1.0 2+ 2.0 1.3 Pancreatin-EDTA/ 2+ 1+ 1.5 3+ 3.0 2.0 Triton-CTAB a Measured by SEM examination with scopes assigned as explained in Table 4, footnote b. b_, No sample was treated with the indicated cleaning agent. C Overall score was determined by the formula given in Results. 402 WHITTAKER, RIDGWAY, AND OLSON APPL. ENVIRON. MICROBIOL. solutions to chelate any toxic metals that could retard highest score in the SEM evaluation. Biz is a commercial enzymatic action. Triton, a nonionic detergent, was used laundry presoaking detergent which contains broad-spec- because of its surfactant activity to facilitate penetration by trum enzymes, bleaching compounds, and detergents. The the enzyme of the biofouling layer. In these combinations, exact mechanism of action of this compound, of course, is the enzyme itself is believed to be the active agent, but not known because Biz combines so many different com- enzymes used alone did not fare as well. Perhaps this was pounds. However, this combination of enzymatic, oxidizing, because substances of such large molecular weight are and detergent compounds appears very efficient in removing unable to penetrate the biofilm effectively without the aid of biofilm, especially when the film is less extensive, as evi- surfactants of chelating agents. The enzymatic mechanism of denced by greater effectiveness at the earlier sampling times. action is the proteolytic and glycolytic (esterase) action, The major drawback in using Biz as a cleaning agent is its which disrupts the glycocalyx that probably surrounds the oxidizing properties, which endanger the integrity of the bacteria, encouraging sloughing of the material. By attacking membrane. For several days before the sampling time on day the exopolymers that are implicated in mediating bacterial 12, the ammonia concentration was accidentally lowered, attachment, it is possible to solubilize the biofilm and clean exposing one membrane unit to 15 to 20 mg of free chlorine the membrane. per liter. The membrane unit analyzed shortly after this Use of enzymes under operating conditions has two major event showed signs of membrane failure and loss in salt drawbacks, expense and lack of stability. The cost of rejection ability, and under SEM, elliptical depressions and enzymes varies widely, depending on the source of the holes appeared in the membrane. The biofilm also seemed to enzyme. Some can be quite expensive, and even crude be removed much more easily than at previous times. It is preparations of enzymes are more expensive than cleaning also evident that HC membranes were cleaned more effi- products currently used on RO membranes. Before enzymes ciently at all times than LC membranes were (Table 4). can be considered as a viable alternative to present cleaning Because chlorination destroys the cellulose acetate mem- methods, more evidence should be collected to support their brane, such oxidizing compounds must be used with care. effectiveness against biofilms. Furthermore, each biofouling Repeated use of such products as Biz may cause deteriora- problem should be analyzed to determine the nature of the tion of membrane integrity. fouling layer (bacterial, organic, or inorganic), and a cost- In summary, the strategies which were most efficient in benefit assessment should be sought. removing biofilm composed primarily of mycobacteria and Preparations containing detergents. Another successful their organic matrix were an enzyme-chelator-dispersant treatment in our schema was the urea-SDS treatment. This combination, an anionic detergent-denaturant combination, treatment combined a chaotropic and bactericidal agent, and Biz. None of these was effective at all sampling times, urea, with an anionic detergent, SDS. Some evidence has and all have some drawbacks in use, but the elimination of been collected that cationic detergents are quite effective in alternatives (such as bactericides) as effective means of solubilizing gram-negative bacteria, whereas anionic deter- biofilm removal may ultimately save costs to industrial gents have more success in solubilizing gram-positive bacte- ventures. This study indicates that research into the funda- ria (9). Furthermore, the bacteria isolated from the mem- mental mechanisms of biofouling of RO membranes is war- branes belonged almost exclusively to the genus ranted to determine which classes of compounds and en- Mycobacterium, which has a gram-positive wall structure. zymes will be most efficient in cleaning. In this manner, SDS by itself ranked as a good cleaner, although its cleaning classes of compounds can be eliminated as inappropriate, abilities were greatly enhanced when coupled with urea. and efforts can be concentrated on members of the classes of Urea by itself did not rank high as a cleaning agent, possibly compounds that are effective for the particular fouling prob- because it was applied at too low a concentration. Typically, lem at hand. urea of at least 6 M are required to solubilize concentrations ACKNOWLEDGMENTS most polypeptides (5). In other experiments, urea at concen- trations of 6 to 8 M did in fact exhibit excellent biofilm This project was funded by the Office of Water Research and removal (unpublished data). Technology contract 4107-160-81 through the Orange County Water District. The three types of detergents which were utilized-cation- The authors thank Carol Justice for her technical assistance ic (CTAB), anionic (SDS), and nonionic (Triton X-100) during the experimental phase of this project. were each tried alone and in combination with other compounds. SDS in combination with urea was the most LITERATURE CITED successful. 1. Bailey, D. A., K. Jones, and C. Mitchell. 1971. The reclamation CTAB, the cationic detergent, although an excellent bac- of water from sewage effluents by reverse osmosis. J. Water had a very low cleaning score when used alone. Pollut. Control Fed. 73:353-366. tericide, 2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's Triton, the nonionic detergent, merited a score of 2.0 and manual of determinative bacteriology. 8th ed. The Williams & was labeled a good cleaner. This was probably due to its Wilkins Co., Baltimore. surfactant activities, which were also utilized in the enzy- 3. Burns and Roe Industrial Services Corp. 1979. 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