Vol. 45, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1983, p. 418-4270099-2240/83/020418-10$02.00/0Copyright C 1983, American Society for Microbiology

Inability of Microorganisms To Degrade Cellulose AcetateReverse-Osmosis Membranest

LEIGHTON C. W. HO1* DAVID D. MARTIN,2 AND WILLIAM C. LINDEMANN'*Department of Crop and Soil Sciences, New Mexico State University, Las Cruces, New Mexico 880031; and

Planning Research Corp., Roswell, New Mexico 882012

Received 18 August 1982/Accepted 14 October 1982

Operational cellulose acetate reverse-osmosis membranes were examined forevidence of biological degradation. Numerous fungi and bacteria were isolated bydirect and enrichment techniques. When tested, most of the fungi were activecellulose degraders, but none of the bacteria were. Neither fungi nor bacteria wereable to degrade cellulose acetate membrane in vitro, although many fungi wereable to degrade cellulose acetate membrane after it had been deacetylated.Organisms did not significantly degrade powdered cellulose acetate in pure ormixed cultures as measured by reduction in acetyl content or intrinsic viscosity orproduction of reducing sugars. Organisms did not affect the performance ofcellulose triacetate fibers when incubated with them. The inability of theorganisms to degrade cellulose acetate was attributed to the high degree of acetatesubstitution of the cellulose polymer. The rate of salt rejection decline wasstrongly correlated with chlorination offeed water and inversely with densities ofmicroorganisms. These data suggest that microbial degradation of operationalcellulose acetate reverse-osmosis membranes is unlikely.

The premature failure of cellulose acetate(CA) reverse-osmosis membranes, as manifest-ed by a loss of salt rejection capacity andincrease in water flux, has often been attributedto microbial degradation (6, 7, 12, 15, 16). CA oflow to moderate degrees of acetate substitutionhas been shown to be susceptible to microbialdegradation (6, 7, 22). However, episodes ofmicrobe-induced membrane failure in operation-al reverse-osmosis modules have been poorlydocumented, and in many cases it is not clearwhether abiotic factors were involved. Little isknown about the microbial flora of operationalreverse-osmosis membranes and whether micro-organisms are in fact able to degrade the highlysubstituted CA presently used in modem mem-branes.According to the Reverse Osmosis Technical

Manual (23), cellulolytic microorganisms arecapable of degrading CA membranes, resultingin marked declines in salt rejection capacity inless than 12 h. But membrane performancedeclines, whether acute or long term, can resultfrom a variety of abiotic causes (23). If microor-ganisms are involved in membrane failure, (i) acellulolytic microflora should be present that

t Journal article 948 of the New Mexico Agricultural Ex-periment Station, Las Cruces, NM 88003.

t Present address: Department of Biology, University ofOregon, Eugene, OR 97403.

can utilize CA as a carbon and energy source,(ii) physical damage to the membrane should beattributable to microbial presence, and (iii) mi-crobial occurrence and membrane failure shouldbe positively correlated.The ability to degrade cellulose is widespread

among the higher fungi (20) and some groups ofbacteria (24), but the ability of microorganismsto degrade substituted cellulose such as CA isless well known. Reese (22) reported that cellu-lose derivatives became more resistant to enzy-matic attack with increasing degrees of acetatesubstitution. Generally one substitution per an-hydroglucose unit (degree of substitution [DS]equal to 1.0) is thought to be sufficient to conferprotection from enzymatic attack (8). However,the DS value represents an average, and a DShigher than 1.0 is required to insure at least onesubstitution per anhydroglucose unit.Reese (22) found that a number of cellulolytic

fungi were able to completely degrade a water-soluble CA with a DS of 0.76 through the actionof cellobiose octaacetase. However, this en-zyme was apparently only able to deacetylateshort oligomers of CA. Organisms possessingcellobiose octaacetase were not able to degradecellulose triacetate (CTA) with a DS of 2.86 andwere not tested for their ability to degrade CA ofintermediate degrees of substitution. Cantor andMechalas (6) demonstrated that some bacteriawere able to degrade CA membranes on nutrient

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CELLULOSE ACETATE DEGRADATION 419

agar, but the DS of the constituent CA was notstated, nor was the membrane present as thesole carbon source.CAs of modem reverse-osmosis membranes

generally have higher DS values than earliermembranes. Early membranes were commonlyconstructed of a single CA, usually CA 398-3(DS of 2.45), whereas current membranes are

generally a blend of CA (DS of 2.4 to 2.5) andCTA (DS of 2.86) according to Saltonstall (25)and King et al. (14). Some membranes are alsoconstructed solely from CTA in a hollow config-uration. Because of their high DS, it is doubtfulthat presently used CA membranes are suscepti-ble to microbial attack. However, the ability ofmicroorganisms to attack modem membranes isuncertain.

This study examines the ability of bacteria andfungi isolated from operational CA reverse-os-mosis membranes to degrade CA and modemCA and CTA membranes.

MATERIALS AND METHODS

Membrane description. Microbial samples were tak-en from two types of CA reverse-osmosis membranesat the Roswell Test Facility of the Office of WaterResearch and Technology, U.S. Department of theInterior, Roswell, N.M. Spiral wound membranesconsisted of a rectangular envelope of two separatemembranes laminated to a dacron backing material.Manufacturers of the spiral wound modules includedPurtech, Salt Lake City, Utah (membrane from Envi-rogenics Systems Co., El Monte, Calif.); HydranauticWater Systems, Santa Barbara, Calif.; and Fluid Sys-tems Div., Universal Oil Products, San Diego, Calif.Hollow fiber modules (Dow Chemical Co., WalnutCreek, Calif.) consisted of parallel hollow fibers em-bedded in resin at both ends.The membranes in this study were composed of CA,

CTA, or mixtures of CA and CTA with variousdegrees of acetate content. The Dow (15.2 by 121.9cm, CTA of 43.5 to 43.7% [wt/wt] acetyl), Hydranau-tics (21.6 by 101.6 cm, 50:50 blend of CA of 39.8%[wt/wt] acetyl and CTA of 43.5% [wt/wt] acetyl), andFluid Systems (30.5 by 152.4 cm, CA of 40.0%o [wt/wt]

acetyl) modules were commercial units tested at theRoswell Test Facility. The Purtech (6.4 by 30.5 cm,

50:50 blend of CA of 39.8% [wt/wt] acetyl and CTA of43.2% [wtlwt] acetyl) modules were small sacrificialunits placed in 10 locations within the plant to sampledifferent areas for microbial contamination. Feed wa-

ter to most modules was a total dissolved solids blend(3,000 mg/liter) of Roswell city water and brackish wellwater adjusted to a target pH of 5.5 to 5.8 with H2SO4.Other feed water pretreatments included a combina-tion of the following: filtration through a manganese

green sand bed, an activated carbon bed, or a mixedbed of both manganese green sand and activatedcarbon and chemical treatment with (NaPO3)6,NaOCl, or Na2S203.

Microbial enumeration and isolation. After variousperiods of operation (Table 1), individual filter mod-ules were opened under aseptic conditions to exposethe membrane surface. For the Dow module, smallbundles of fibers (1 to 2 cm long) were removed fromthe outside edge at the feed and product ends. Bundlesof fibers were also removed from the same locationsnear the central core of the module. For bacterialenumeration, fibers from each of the sample pointswere placed in sterile petri dishes, shaken to separatethe fibers, covered with molten (-45°C) nutrient agar(BBL Microbiology Systems, Cockeysville, Md.), andswirled to further separate the fibers and disseminateany organisms present. The bacteria plates were incu-bated at 30°C, and microbial counts were expressed asCFU per square centimeter of membrane surface.

Fungi were isolated from the hollow fiber mem-branes by placing small bundles of fibers on plates ofhalf-strength cornmeal agar (Difco Laboratories, De-troit, Mich.), 2% malt extract agar (Difco), and CAagar (6). The fungal media contained 25 mg each ofpenicillin and streptomycin per liter to inhibit bacterialgrowth. Plates were incubated for 10 to 15 days at250C.

Bacteria were enumerated from spiral wound mem-branes by swabbing 2-cm2 areas of feed and productsurfaces with sterile cotton. Swabs from the feed sideof the leaves were taken directly from the membranesurface, and swabs from the product side were takenfrom the dacron backing. The swabs were placed in 2ml of pH 7 sterile phosphate buffer and agitated on a

Vortex mixer, and 1-ml samples were used for nutrientagar pour plates. Colonies developing at 250C were

TABLE 1. Operational parameters and performance statistics of reverse-osmosis modules

Operating Salt rejection ~Mean feedDy

Module Total h of Opressratneateeto water DayslModule opertion pressure Starting Ending Decline residual Cl cuntioperaion(MPa) (%) (%) (to/day) (mgliter) Culturea

Dow I 2,723 1.72 96.10 57.62 -1.74 x 10-' 0.67 1Dow Ila 7,872 5.17 95.38 94.63 -3.09 x 10-3 0.00 NDDow lIab 1,656 1.72 95.53 92.34 -3.03 x 10-2 0.90 NDDow Ilb 4,533 1.72 96.49 95.71 -4.01 x 10-3 0.00 1Dow III 5,874 1.72 93.75 90.82 -1.10 x 10-2 0.62 11Hydranautics 898 3.03 95.05 77.93 -3.30 x 10-1 0.86 14Fluid Systems 338 2.96 92.79 88.52 -6.24 x 10-1 1.12 90

a Number of days from unit shutdown until cultures were taken. ND, Microbial cultures or salt rejectiondecline not determined.

b Unit was shut down for 1 day for the attachment of chlorination equipment and was promptly restarted.

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420 HO, MARTIN, AND LINDEMANN

counted and expressed as CFU per square centimeter.Fungi were isolated from the spiral wound membranesby placing membrane pieces (approximately 1 cm2)feed side down on agar plates as described above forthe hollow-fiber membranes.Enrichment cultures were also made from both

membrane types for bacterial and fungal isolations.The media were as described above. Membrane sam-ples were also enriched in liquid culture with the CAmedium of Cantor and Mechalas (6), basal salts-yeastextract medium (BSY) of Sinclair (27), basal medium(BM) plus 0.05% yeast extract (Difco), BM plus 0.5%reprecipitated CA (CA 398-3; Eastman Chemical Co.,Rochester, N.Y.), and Hutchinson (HUT) medium asdescribed by Rodina (24) plus 0.5% reprecipitated CA(Eastman CA 398-3). The BM contained the following(per liter of deionized water): KH2PO4, 1.0 g;(NH4)2SO4, 0.54 g; KCI, 0.5 g; MgSO4 * 7H20, C.2 g;CaCl2 * H20, 0.13 g; FeCl2, 50 mg; MnSO4 * 4H20,50 mg; ZnSO4, 44 mg; CuS04 55H20, 10 mg;NaMoO4 * 2H20, 5 mg; Na2B4O7, 1 mg; nicotinic acid,0.3 mg; folic acid, 0.3 mg; thiamine, 0.03 mg. All mediawere prepared in either distilled water or facility water(FW). The FW was used to simulate the water condi-tions within the Roswell Test Facility and included theaddition of 0.01% EDTA to minimize precipitate for-mation. If a solid medium was required, 2% purifiedagar (Difco) was added. Peptone and yeast extract,added as amendments to some enrichment cultures,were also from Difco. The enrichment flasks wereincubated at 25°C with daily swirling by hand for atleast 30 days, and samples from each flask weretransferred to nutrient agar plates (bacteria) and corn-meal and malt agar plates (fungi) for isolation andidentification.

Bacterial colonies from direct isolations, enumera-tion plates, and enrichment plates and flasks weresubcultured until pure cultures were obtained. Theisolates were screened on the basis of colony morphol-ogy on Trypticase soy agar (BBL), Gram stain, andcell morphology. Representative isolates were classi-fied according to Bergey's Manual of DeterminativeBacteriology (5) and the CRC Handbook ofMicrobiol-ogy (10) for a more detailed description of Bacillus.

Fungal colonies isolated from enrichment flaskswere identified with the aid of dissecting and com-pound microscopes. Colonies ofFusarium were distin-guished from Cephalosporium-type fungi (includingPhialophora) by their faster growth rate (greater than35 mm in 10 days) and the production of macroconidia(9). The frequency of occurrence of fungi in directisolations is expressed as the number of occurrencesper number of plates inoculated. Because more thanone fungal species appeared on a single plate, frequen-cies of individual fungi do not necessarily total to thefrequency of plates with fungi present. A single genuswas counted only once per plate.Specimens from CA membranes were examined for

membrane degradation with dissecting and compoundmicroscopes as well as by scanning electron microsco-py at the Biology Department, New Mexico StateUniversity, and at the Dow Chemical Co. facilities.Two cultures of Cellulomonas were obtained from

the American Type Culture Collection (ATCC 21399and 21681). Two isolates of bacteria from the YumaDesalting Test Facility that were capable of filter paperdegradation (S226 and S209) were provided by N. A.

APPL. ENVIRON. MICROBIOL.

Sinclair, University of Arizona. N. A. Sinclair alsosupplied a CA membrane primary enrichment culture(C257 CNL) of waters from the Mohawk-Welton Ca-nal, Yuma, Ariz., in which degradation of a CAmembrane strip was evident.

Ceilulose, CA, and CTA degradation studies. Pow-dered CA 398-3, CA 394-45, and CA 400-25 wereobtained from Eastman. The first number of the CAtype gives the acetyl content, and the second numberindicates the American Society of Testing Materialsviscosity rating. The acetyl contents of the CA used inthis study, as determined by E. H. Hill of TennesseeEastman Co., Kingsport, Tenn., were 40.00, 39.55,and 39.45% (wt/wt), respectively. The DSs of the CAs,as calculated with the formula of Tanghe et al. (30),were 2.49, 2.43, and 2.42, respectively. A reverse-osmosis membrane constructed from a blend of equalweights of CA 398-3 and CA 435-85S in a sheetconfiguration was obtained from Envirogenics Sys-tems Co., El Monte, Calif. The membrane was peeledfrom the dacron backing material before use. Reverse-osmosis membrane constructed from CTA (43.6%acetyl) in a hollow-fiber configuration was obtainedfrom Dow Chemical Co.

Several qualitative tests were used to evaluate theability of organisms to degrade cellulose and cellulosederivatives. Fungi and bacteria were tested for theability to clear a 0.25% (wt/vol) suspension of phos-phoric acid-swollen cellulose in tubes of BM agar (21,32). Depth of clearing was measured after 5 weeks.Fungi were also tested for the ability to clear pow-dered CA 398-3, phosphoric acid-treated blend mem-brane, and phosphoric acid-treated CTA hollow fiberswith the same technique. Bacteria were also tested onplates of 0.5% (wt/vol) carboxymethyl cellulose(9M8F; Hercules, Inc., Wilmington, Del.) in BM agar(13).

Fungi and bacteria were tested for their ability todegrade strips of filter paper (1 by 7.5 cm) in tubes ofHUT-FW. After 4 weeks of incubation, the bacteriawere reinoculated and enriched with yeast extract(final concentration, 0.05%) to encourage growth.Fungi were tested for the ability to degrade strips ofblend membrane in tubes of BM-FW and BSY-FW.Bacteria were tested on strips of blend membrane inHUT; as in the filter paper test, after 4 weeks,bacterial tubes were reinoculated and enriched withyeast extract. The degradation of filter paper andmembrane strips was assessed visually; loss of struc-tural integrity was evaluated by vigorously swirling thetubes on a Vortex mixer. One set of blend membranestrips in HUT-FW was sterilized with propylene oxideand inoculated with fungi. Fungi were also inoculatedonto deacetylated blend membrane strips in BM-FW.The membrane was deacetylated by soaking in equalvolumes of ethanol and ammonium hydroxide for 24 hto the point of acetone insolubility.More sensitive quantitative tests were performed on

powdered CAs inoculated with microorganisms underseveral different cultural conditions. These includedthe following.For CA 400-25, fungi were cultured on 3.5 g of CA

400-25 in 100 ml ofBM-FW for 9 weeks. Bacteria werecultured on 2.0 g of CA 400-25 in 100 ml of HUT-FWfor 8 weeks.

For CA 394-45, fungi and bacteria were cultured on1.5 g of CA 394-45 in 100 ml of BSY for 4 weeks.

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CELLULOSE ACETATE DEGRADATION 421

TABLE 2. Bacterial densities of membranes bydirect isolationBacterial density (CFU/cm2) at sample

Module locations:Feed end Middle Product end

Dow IInteriora 0 0 0Exterior" 0 0 0

Dow IIbInterior 800 1,200 1,400Exterior 730 1,410 1,270

Dow IIIInterior 0 770 NDcExterior 0 500 ND

Hydranautics ND/NDd olod ND/NDda Interior portion of fiber bundle.b Exterior portion of fiber bundle.c ND, Microbial cultures not determined.d Feed side/product (backing) side.

For CA 398-3, enrichment cultures from operationalreverse-osmosis modules were incubated for 16 weeksin 50 ml of BM with 1% CA 398-3.

Analyses on each CA consisted of the determinationof acetyl content by the solution method (2), of intrin-sic viscosity by the method of Tanghe et al. (31) withan Ostwald-Fenske viscometer, and of reducing sugarcontent in the cultural ifitrate by the ultramicro TPTZ(tripridyl-s-triazine) method of Avigad (3). The micro-bial biomass on CA 400-25 was deemed minimal andwas carried along with the sample for acetyl determi-nation. Before determination of intrinsic viscosity, thesample was weighed, dissolved in acetone, and filteredthrough a Whatman GF/D filter, a high-flow, high-loading-capacity filter with an effective retention diam-eter of 2.7 pLm. Changes in viscosity due to acetoneevaporation during filtration were regarded as minimalas samples passed through filters rapidly (less than 2s). With CA 394-45, considerable microbial biomassaccumulated because of the presence of yeast extractin the medium. Thus, weighed samples for both acetyland viscosity determinations were dissolved in ace-

tone and filtered through tared GF/D filters. Theweight of the sample was corrected for the loss offilterable solids. Loss of sample by adsorption to thesintered glass ffiter support and sides of the filtrationflask may have caused a small loss of accuracy inacetyl determination. With CA 398-3, samples weredissolved in acetone, filtered, and reprecipitated bydirecting a stream of distilled water into the acetonesolution. The reprecipitated CA was dried andweighed for analysis.The effects of microorganisms on the performance

of reverse-osmosis membranes were evaluated as fol-lows. Bundles of CTA hollow-fiber membrane 60 cmin length were disinfected in 0.5% Formalin for 1 h,rinsed in sterile distilled water for 1 h a minimum offour times, added to 100 ml ofBM in 250-ml Erlenmey-er flasks, and inoculated. Flasks were gently handswirled daily. Fiber bundles were removed at 2, 4, and8 weeks, placed in 3% glutaraldehyde (pH adjusted to6), and sent to Dow Chemical Co. for performancetesting.

Statistical treatment of the chemical analysis dataconsisted of analysis of variance followed by theStudent-Newman-Keuls multiple-comparisons test (P= 0.05) to separate significant means (29). Unreplicat-ed values treatments were compared with 95% confi-dence intervals calculated from the control mean.

RESULTS

Performance statistics of the reverse-osmosismembranes are given in Table 1. The rates ofsalt rejection decline were determined by regres-sion analysis of salt rejection over time. Regres-sion analysis of the Purtech modules was notperformed; the small membrane surface areaand low fluctuating feed water pressures of thePurtech modules contributed to suboptimumand variable salt rejection data. The rates ofdecline of salt rejection ranged from -3.09 x1i-0 to -6.24 x 10-1% per day for the largemodules. Data for normal rates of decline arelimited, but one unspecified reverse-osmosissystem operated for 2 years with a salt rejection

TABLE 3. Frequency of occurrence of fungi on CA and CTA membranes as determined by direct isolationand enrichment culture

Frequency of occurrence of fungi on membrane:

Fungus Hydra- Dow I Dow Ilb Dow IIInautics Interior' Exterior' Interior Exterior Interior Exterior

Ascomycetes 0/0' 0/0 0/0 0/0 0/0 2/0 0/0Aspergillus 0/0 0/0 0/0 0/0 2/0 0/0 0/0Aureobasidium 0/0 0/0 0/0 0/0 0/0 0/0 0/0Cladosporium 0/0 0/0 0/0 2/0 2/0 0/0 0/0Fusarium 0/0 0/0 0/0 96/67 96/100 0/0 0/0Penicillium 2/0 0/0 0/0 2/0 4/0 0/0 0/0Trichoderma 0/0 0/0 0/0 86/0 88/0 35/50 0/0Yeast 0/5 2/0 0/17 0/0 0/0 0/0 0/0

a Interior portion of the fiber bundle.b Exterior portion of the fiber bundle.c Percentage of plates which had the fungus by direct isolation/percentage of flasks which had the fungus by

enrichment culture.

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422 HO, MARTIN, AND LINDEMANN

decline of -3.65 x 1i-0 to -1.94 x 10-4% perday (23). It is unclear what sort of performancedeclines are characteristic of biological degrada-tion, because alleged episodes of biological deg-radation have been poorly documented. Howev-er, the Dow I, Hydronautics, and Fluid Systemsmodules exhibited sharp declines in salt rejec-tion capacity that were consistent with reportsof alleged membrane failure (11, 12, 23), andengineers at the Roswell Test Facility attributedthese rejection declines to biological degradation(B. Gibbons, personal communication).None of the membranes showed signs of mi-

crobial degradation when viewed under light orscanning electron microscopes. The lack of visi-ble degradation of the CA membrane contrastswith other reports (26) which show considerablemicrobial growth and considerable degradationof cellulose films submerged in marine environ-ments. Scanning electron microscopy of mem-brane surfaces revealed only scattered bacterialcells with no apparent etching, indentation of themembrane surface, or method of direct attach-ment. Only scattered hyphal filaments were ob-served, indicating that the fungi were not colo-nizing the membrane surface. Except for theFluid Systems membranes, the spiral wound andhollow-fiber membranes had no discoloration orslime indicative of microbial growth. The FluidSystems membrane had a slime on the rejectionsurface which contained motile, rod-shaped bac-teria. However, this membrane was stored in aplastic bag at 25 to 30°C for 90 days, a period oftime that exceeded the operational time. Theslime formation was believed to have occurredduring storage rather than during operation.The fungal genera isolated from reverse-os-

mosis modules and waters within the RoswellTest Facility and the numbers of different spe-cies or biovariants of the genus or species identi-fied were: Acremonium, 1; Alternaria, 1; Asper-gillus, 2; Aureobasidium, 1; Candida, 1;Cladosporium, 1; Cleistothecial ascomycetes, 1;Fusarium, 6; Geotrichum, 1; Mucorales, 1; My-celia Sterilia, 1; Penicillium, 4; Phialophora, 3;Rhodotorula, 1; and Trichoderma, 1. The bacte-rial genera isolated and the number of differentspecies or biovariants of the genus or speciesthat were identified were: Acinetobacter, 1;Arthrobacter, 3; Bacillus, 5; Flavobacterium, 2;Kurthia, 1; Lactobacillus, 11; Micrococcus, 1;Micromonospora, 1; and Pseudomonas, 7. Or-ganisms appear to be common soil and waterinhabitants (1, 4). Although many of the fungi inthese genera are known to be cellulolytic, partic-ularly species of Trichoderma (1, 28, 20), cellu-lose bacteria such as Cellulomonas, Cytophaga,or Sporocytophaga were not isolated in eitherdirect or enrichment cultures. Some species ofBacillus, Micrococcus, Micromonospora, and

APPL. ENVIRON. MICROBIOL.

Pseudomonas have been reported to be cellulo-lytic (1, 28), but subsequent testing of theseisolates proved negative.The densities of bacteria as determined by

plate counts in Dow and Hydranautics modulesare given in Table 2. The frequencies of occur-rence of fungi as determined by direct isolationand enrichment techniques for the same mod-ules are given in Table 3.The ability to degrade cellulose was indicated

by clearing of suspensions of acid-swollen cellu-lose and growth on filter paper as a sole carbonsource. Cellulolytic ability was widespreadamong the fungi isolated from the Roswell TestFacility (Table 4). None of the fungi cleared CA398-3, phosphoric acid-treated blend membrane,or CTA hollow fibers after an incubation inexcess of 4 months (data not shown). Fungalgrowth was sparse on blend membrane strips inBM-FW medium (Table 4) and was only slightlybetter growth on gas-sterilized blend membranein HUT-FW medium (data not shown). Growthon blend membrane in BSY medium (Table 4)was good, but this growth was undoubtedlysupported by yeast extract. None of the mem-brane strips exhibited any visual signs of degra-dation or loss of structural integrity after anincubation in excess of four months. Fungi grewvigorously on deacetylated blend membrane inBM-FW, and many strips were degraded to thepoint of fragmentation when the tubes wereagitated (Table 4). The data suggest that al-though many of the fungi were strongly cellulo-lytic, they were unable to degrade intact CAreverse-osmosis membranes or their constituentCA. The vigorous growth on deacetylated mem-brane suggested that deacetylation was prereq-uisite for degradation of CA.

Bacteria were more limited than the fungi inthe capacity to degrade cellulose. None of the 33bacterial groups isolated from Roswell Test Fa-cility was able to degrade acid-swollen cellulose,carboxymethyl cellulose, or filter paper (with orwithout yeast extract amendments) after 30 daysof incubation. Cellulomonas cultures exhibited apulping or macerative effect on filter paper after10 days. However, none of the bacteria pro-duced visible effects on strips of blend mem-brane in HUT-FW (with or without yeast extractamendment) after an incubation in excess of 2months (data not shown).Because none of the qualitative tests indicated

that the bacterial and fungal isolates from theRoswell Test Facility or acquired cultures coulddegrade CA membranes or suspensions of CA,more sensitive quantitative tests were per-formed to insure that visually undetected degra-dation was not occurring. If CA degradationoccurred, either the acetates or the cellulosepolymer (or both) would be cleaved, resulting in

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CELLULOSE ACETATE DEGRADATION 423

TABLE 4. Qualitative tests for cellulose and CA degradative ability of fungal isolates

Cellulose Growtha on:Culture Fungus depth Filter Blend membrane Deacetylated

(mm)depth aFiler BM-Fblend(mm) paper BM-FW BSY membrane

AcremoniumAlternariaAspergillusAspergillusAspergillusAureobasidiumAureobasidiumAureobasidiumCladosporiumCleistothecial ascomyceteFusariumFusariumFusariumFusariumFusariumFusariumFusariumFusariumGeotrichumPenicilliumPenicilliumPenicilliumPenicilliumPhialophoraPhialophoraPhialophoraPhialophoraPhialophoraPhialophoraPhialophoraPhialophoraTrichodermaTrichodermaTrichodermaTrichodermaTrichodermaTrichodermaYeastYeastYeast

1201215160019121223121511161619ND0152421202221171619201611ND27262118261200

2 1 ND2 1 ND2 1 22 1 22 1 20 0 ND0 1 ND2 1 22 1 22 1 ND2 0 ND2 1 ND1 1 ND2 0 ND2 1 22 1 22 1 22 1 ND0 0 ND2 0 ND2 1 22 1 NDND 1 ND2 0 22 1 ND2 1 22 1 22 1 ND2 1 ND2 1 22 1 ND2 ND ND2 1 22 0 22 1 ND2 1 22 0 22 1 ND0 0 ND0 0 0

a 0, No growth; 1, slight or questionable growth; 2, significant growth; ND, not determined.b Breakage of membrane.

a lower acetyl content of the CA or a decrease inthe viscosity of the cellulose polymer (or both).In addition, the reducing sugar content of themedia would increase with the cleaving of thecellulose polymer.Eastman CA 400-25 was not degraded by any

of the fungi tested (Table 5). Acetyl contentswere not significantly reduced in any of thesamples. The range of mean acetyl contents ofthe inoculated samples was narrow (0.5%), andthe mean acetyl content of the control (39.47%)compared favorably with determinations byTennessee Eastman (39.45%). Although the in-trinsic viscosity analysis of variance was statisti-

cally significant, only one value (1.58 dl/g, forFusarium RS2) was significantly different fromthe mean. Because degradation of CA wouldlower, rather than raise, the viscosity, depoly-merization of the cellulose polymer did notoccur. The amounts of reducing sugars releasedinto the medium from inoculated samples tendedto be greater than control values, but only threevalues (32.0, 29.7, and 28.5 dl/g) were outsidethe ranges of non-significantly different meansthat included the control. The fungi producingthese values (Fusarium RS 31, CladosporiumRS 42, and Penicillium RS 30, respectively)were all cellulolytic (Table 4); therefore, the

RS 18RS 26RS 7RS 9RS 10RS 1RS 11RS 33RS 42RS 21RS 22RS 27RS 23RS 2RS 35RS 12RS 31RS 41RS 4RS 25RS 30RS 39RS 40RS 3RS 13RS 5RS 24RS 32RS 36RS 16RS 17RS 14RS 15RS 28RS 29RS 37RS 38RS 6RS 19RS 20

222b220122b222222b2b2b212b22b2b2b22blb2122ND2b2b2b2b2b200

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TABLE 5. Assays for fungal degradation ofEastman CA 400 25a

Acetyl Intrinsic ReducingFungus content viscosity sugar

(%, wt/wt) (dUg) (nmonml)

Control 39.47a 1.51b 11.8cCladosporium RS 42 38.96a 1.53b 29.7abFusarium RS 22 39.00a 1.49b 15.1bcFusarium RS 2 39.10a 1.58a NDFusarium RS 12 39.21a 1.50b 17.1abcFusarium RS 31 39.30a 1.48b 32.OaPenicillium RS 30 38.96a 1.50b 28.5abPhialophora RS 13 39.47a 1.48b 7.6cPhialophora RS 24 39.21a 1.48b 14.ObcTrichoderma RS 15 39.27a 1.46b 18.7abcTrichoderma RS 28 39.98a 1.48b 22.7abcYeast RS 20 39.24a 1.51b 21.5abcAurobasidium RS 1 39.41 1.52 24.0

a Means within the same column followed by thesame letter are not significantly different at the 5%probability level by the Student-Newman-Keuls multi-ple comparison test. Aureobasidium was not includedin the analysis. ND, Not determined.

production of these relatively small amounts ofreducing sugars may have been the result ofcellulase activity on contaminating cellulose inthe sample (17). Other tests showed that thesefungi were not able to produce significant deace-tylation or depolymerization of the CA. Thus,we conclude that the production of smallamounts of reducing sugars was not regarded assufficient evidence for CA degradation.Eastman CA 400-25 was not degraded by any

of the 33 bacteria isolates tested from the Ros-well Test Facility (data not shown). No signifi-cant differences were found between the bacteri-al isolates and the control values for acetylcontent, intrinsic viscosity, and reducing sugarcontent. Again the reducing sugar content of thebacterial cultures was generally higher than thecontrol, but this was attributed to factors otherthan CA degradation.

Yeast extract was used in the CA 394-45medium to determine whether CA degradationrequired an alternate carbon source or a morenutritionally complete environment (Table 6).Growth of the microorganisms was vigorous,especially for the fungi, where the mycelia oftensequestered the bulk of the powdered CA. Theanalysis of variance of the acetyl contents andintrinsic viscosities was not significant. Theanalysis of variance for reducing sugar contentwas significant, but treatment values were lowerthan the control and were attributed to theuptake of reducing sugars during growth. Thehigh reducing sugar content in the control wasdue to the reducing sugar present in the yeastextract. The vigorous growth of the organisms

TABLE 6. Assays for fungal, bacterial, and mixedculture degradation of CA 394-45

Acetyl Intrinsic ReducingOrganism content viscosity sugar

(% wt/wt) (dUg) content

Control 38.05 1.38 146.6

FungiCladosporium RS 42 NDa 1.43 59.5Fusarium RS 35 37.82 1.38 85.5Fusarium RS 12 37.93 1.41 92.7Fusarium RS 31 38.24 1.40 90.5Penicillium RS 39 37.78 1.41 144.0Phialophora RS 24 37.63 1.39 117.1Phialophora RS 16 37.95 1.36 118.5Trichoderma RS 15 37.98 1.39 115.5Trichoderma RS 28 38.08 1.41 140.0

BacteriaSinclair 209 38.28 1.38 147.9Sinclair 226 38.17 1.36 127.3Sinclair C257 38.06 1.40 61.0ATCC 21681 38.07 1.41 140.8a ND, Not determined

was attributed to the yeast extract and not togrowth on CA since significant reductions in CAacetyl content and intrinsic viscosity did notoccur. Bacterial cultures from N. A. Sinclair,including a primary enrichment culture contain-ing a degraded CA strip and one Cellulomonasculture, did not demonstrate CA degrading abili-ty.

Primary enrichment cultures of CA mem-branes were analyzed for CA degradation toexplore the possibility that CA degradationmight require the concerted efforts of severalorganisms. The enrichment cultures includedflasks of BM medium containing 1% (wt/wt) CA398-3, inoculated with membrane pieces fromeach module and incubated for 100 days. Suffi-cient material from each sample was not avail-able to determine both acetyl content and intrin-sic viscosity or to perform analyses in replicate.However, analyses on control CA were done intriplicate, and 95% confidence intervals werecalculated for the control. No large declines ineither acetyl content or intrinsic viscosity werefound (Table 7). All values of acetyl content fellwithin the 95% confidence intervals of the con-trol. Values for intrinsic viscosity fell with the95% confidence intervals of the control, exceptfor two values that were higher and therefore notindicative of depolymerization. The amounts ofreducing sugars in the medium were small and inmost cases not detectable. Thus, no significantdeacetylation or depolymerization of CA oc-curred in these enrichment cultures.None of the bacteria or fungi inoculated onto

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CELLULOSE ACETATE DEGRADATION 425

TABLE 7. Assays for enrichment culturedegradation of CA 398-3

Intrinsic ReducingEnrichment Acetyl content Iscosic sugar

culture source (%, wt/wt) viscosity content(g) (nmoilml)Control 39.98 ± 1.21a 0.80 ± 0.21 NDbHydranautics 39.94 ND 0membrane

Fluid Sys- 39.58 ND NDtems mem-brane

Purtech mem-branes1 39.62 ND 5.42 ND 1.11 03 ND 0.76 04 ND 0.79 05 39.05 ND 06 39.77 ND 07 ND 0.84 08 39.63 ND ND9 ND 0.88 2.610 ND 0.85 0

Dow II mem- ND 0.90 3.9brane prod-uct water

Dow II mem- ND 1.02 8.2brane feedwatera Confidence interval, 95%; n = 3.b ND, Not determined.

CTA hollow fibers and incubated for up to 8weeks resulted in significant declines in mem-brane performance (Table 8). Three additionalcultures of Trichoderma, Candida, and inocu-lum from a primary enrichment culture of CA398-3 were also tested in BM and BM-FWmedia. However, declines in the performance ofthe CTA fibers were not detected after 8 weeksof incubation (Table 9).

DISCUSSIONReese (22) postulated that cellulose deriva-

tives became more resistant to enzymatic attackwith increasing degrees of substitution. Cowling(8) suggested that one substitution per anhydro-glucose unit (DS of 1) was sufficient to impartprotection from enzymatic attack. In the currentstudy, organisms capable of vigorously degrad-ing various forms of cellulose failed to degradein vitro CA membranes unless the membranehad been previously deacetylated. These resultsare consistent with the suggestions of Reese andCowling; deacetylation appears to be a prerequi-site for biological degradation of CA membranesand their constituent CAs. Although most of thefungi were strongly cellulolytic, there was noevidence that the fungal or the bacterial isolatesenzymatically deacetylated CA or depolymer-

ized the cellulose backbone of CA. Microorga-nisms did not appear to affect membrane per-formance when inoculated onto CTA hollowfibers; according to Cantor and Mechalas (6),the loss of salt rejection is the most sensitivemeasure of membrane damages.The occurrence of microorganisms and the

rate of decline of salt rejection appear to beinversely related. If the slopes of the regressionequations (Table 1) are compared with the densi-ties of fungi and bacteria (Table 2 and 3), it isclear that high rates of decline of salt rejectionare associated with low frequencies of microbialcontamination (with the exception of the FluidSystems membrane). In contrast, Dow Ilb hadthe highest levels of contamination, yet thelowest rate of decline in salt rejection. TheKendall's coefficients of rank correlation (24)between rate of decline and occurrence of fungiand bacteria in Dow I, Ilb, and III and inHydranautics are -0.67 and -0.91, respective-ly. This inverse relationship between occurrenceof microorganisms and rate of rejection declinedoes not support a microbial etiology for mem-brane failure.The levels of feedwater residual chlorine (Ta-

ble 1) do, however, appear to be directly relatedto the rate and magnitude of rejection declineexhibited by reverse-osmosis modules. TheKendall's coefficient of rank correlation calcu-lated for the seven pairs of slopes and values ofresidual chlorine for the Dow, Hydranautics,and Fluid Systems modules (Table 1) is 0.98.Within the Dow modules, rates of rejectiondecline were 3 and 49 times greater in chlorinat-ed modules (Dow I and III, respectively) than inthe average rates of decline of the unchlorinatedmodules (Dow Ila and Ilb). To verify this appar-ent relationship, chlorination was started onDow Ila, a module which had previously beenunchlorinated. After exhibiting extremely lowrates of rejection decline over a period of opera-tion in excess of 1 year while receiving unchlor-inated feedwater, the rate of decline in Dow Ilaincreased 1 order of magnitude in the 10-weekperiod during chlorination. Comparison of theslopes of regression equations of the 10-weekperiod directly preceding chlorination and the10-week period during chlorination shows a six-fold increase in rate of rejection decline; theslopes are different at P = 0.001. The ReverseOsmosis Technical Manual (23) recommends amaximum residual chlorine level of 1 mg/liter forCA and CTA membranes, and others (18) haveshown the performance ofCA membranes not tobe affected by short exposure (10 to 23 days) tohigh levels of chlorine (3 to 30 mg/liter). Never-theless, chlorination may affect membrane integ-rity. Hypochlorous acid may react directly withCA as in previously described nonspecific oxi-

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426 HO, MARTIN, AND LINDEMANN

TABLE 8. Dow Chemical Co. performance tests of CTA hollow fibers after incubation with variousmicrobial isolates

2-week incubation 4-week incubation 8-week incubation

Organism Water flux Salt Water flux Salt Water flux Salt(liters/m2/day) rejection (liters/m2/day) rejection (liters/m2/day) rejection

Control 297 96.5 322 97.4 232 97.5

FungiaAureobasidium 310 96.8 277 97.3 285 96.4Phialophora 269 96.3 314 96.4 297 96.9Trichoderma 310 95.4 293 97.6 281 97.0

BacteriabBacillus sp. 1 306 96.3 281 97.9 293 97.1Pseudomonas 318 95.4 293 95.5 253 97.7Bacillus sp. 2 285 96.7 289 97.2 277 98.3a Fungi were grown in BS-FW medium.b Bacteria were grown in nutrient broth-FW medium.

dations of cellulose by hypochlorite (19) or reactwith other chemical entities such as transitionalmetal ions, particularly Mn, as suggested byA. R. Marsh, Dow Chemical Co., and J. H.Hageman, New Mexico State University (per-sonal communications). In general, chlorinationreduced microbial contamination of those mem-branes receiving chlorinated feed water andmembranes cultured soon after shutdown.

Certain conditions might make CA mem-branes susceptible to microbial attack. Vos et al.(33) have shown that CA is deacetylated at pHconditions deviating from pH 5, with the reac-tion accelerating at higher temperatures. Pro-longed exposure of CA membranes to neutral orslightly alkaline conditions, or a short exposureto conditions of high pH, could cause sufficientdeacetylation to occur, allowing colonization bycellulolytic organisms. Rates of deacetylation ofoperational reverse-osmosis membranes may begreater than rates calculated by Vos et al.,because their measurements were made onmembranes in static as opposed to dynamicsolutions. Interactions with chlorine and othermetal ions may also increase rates of deacetyla-tion. The acetyl content of a CA membrane

TABLE 9. Dow Chemical Co. CTA hollow fibersafter 8 weeks of incubation

~~Water flux SaltOrganism (liter/m2/day) rejection

Control 313 95.4Trichodermaa 319 96.4Candidab 298 97.8Enrichment culturec 299 95.3

a Grown in BM plus yeast extract medium.b Grown in BM-FW medium.c Enrichment in BM-CA 398-3 medium.

determines the salt rejection properties in addi-tion to protecting the CA from enzymatic attack;thus, it is unlikely that deacetylation sufficient toallow microbial colonization would occur beforeserious impairment of membrane performancewould be apparent. If a deacetylation were tooccur, the decline in membrane performancewould be caused by chemical deacetylation. Theeffects of subsequent microbial colonizationwould be secondary.These data suggest that microbial degradation

of operational CA reverse-osmosis membranesat the Roswell Test Facility or other facilities isunlikely. Furthermore, it appears that as long asthe CA has a high degree of acetyl substitutionand the membrane is not exposed to conditionswhich favor deacetylation, microorganisms can-not readily degrade modern CA membranes.

ACKNOWLEDGMENTS

This research was funded by the Office of Water Researchand Technology, U.S. Department of the Interior grant 14-34-0001-9508.We thank Dow Chemical Co. for performance testing of the

CTA hollow fibers.

LITERATURE CITED

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2. Annual Book of ASTM Standards. 1978. Cellulose; leather;flexible barrier materials, part 21, D871 section 10, p. 55-57. American Society for Testing and Materials, Philadel-phia.

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12. Guy, D. B. 1980. Biodegradation of cellulosic membranesat the Roswell and Yuma desalination test facilities, p. 12and 15. In Pure water, vol. 2. International Desalinationand Environmental Association, Teaneck, N.J.

13. Hankin, L., and S. L. Anagnostatis. 1977. Solid mediacontaining carboxymethyl-cellulose to detect Cx celluloseactivity of microorganisms. J. Gen. Microbiol. 98:109-115.

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15. Kremen, S. S. 1980. An overview of the microbiologicalproblem and some potential causative agents. Workshopon microbiological membrane degradation. Eighth AnnualConference and International Trade Fair of the NationalWater Supply Improvement Association, Ipswich, Mass.

16. Leahy, T. M., III. 1980. Recent experience with CTAhollow fiber membranes at the Roswell Test Facility.Technical Proceedings Eighth Annual Conference andInternational Trade Fair of the National Water SupplyImprovement Association, Ipswich, Mass.

17. Lonsdale, H. K. 1966. Properties of cellulose acetatemembranes p. 93-160. In U. Merton, (ed.), Desalinationby reverse osmosis. MIT Press, Cambridge, Mass.

18. McCutchan, J. W., and J. Glater. 1980. The effect ofwater pretreatment chemicals on the performance and

durability of reverse osmosis membranes. Final report toMembrane Processes Division. Office of Water Researchand Technology, U.S. Department of the Interior, Wash-ington, D.C.

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22. Reese, E. T. 1957. Biological degradation of cellulosederivatives. Indust. Eng. Chem. 49:89-93.

23. Reverse Osmosis Technical Manual. 1979. Office of WaterResearch and Technology, U.S. Department of the Interi-or, Washington, D.C.

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27. Sinclair, N. A. 1981. Microbial degradation of reverseosmosis desalting membranes. A final report to the Bu-reau of Reclamation. U.S. Department of the Interior,Washington, D.C.

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31. Tanghe, L. J., L. B. Genung, and J. W. Mench. 1963b.Intrinsic viscosity, approximate degree of polymerizationand molecular weight of cellulose acetate, p. 210-212. InR. L. Whistler, (ed.), Methods in carbohydrate chemis-try, vol. 3, Cellulose. Academic Press, Inc., New York.

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33. Vos, K. D., F. 0. Burris, Jr., and R. L. Riley. 1966.Kinetic study of the hydrolysis of cellulose acetate in thepH range of 2-10. J. Appl. Polymer Sci. 10:825-832.

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