APPLIED MICROBIOLOGY, Sept. 1969, p. 376-386Copyright © 1969 American Society for Microbiology

Vol. 18, No. 3Printed in U.S.A.

Microbiological Study of Water-Softener ResinsJOHN M. STAMM,' WARREN E. ENGELHARD, AND JAMES E. PARSONS

Department of Microbiology, University of Nebraska, Lincoln, Nebraska 68508

Received for publication 23 June 1969

Microbial identification using effluents backflushed from exhausted urban andrural tank resins and cleaned resins containing the sulfonated copolymer of styreneand divinylbenzene (SDB) were completed, along with microbial assessment of theconcentrated stock salt brine. Forty-four different bacterial and fungal genera wereidentified. Extensive biochemical and animal virulence tests completed on one ofthe six bacterial salt brine isolates indicated a pathogenic staphylococcal strain. Theretention of Staphylococcus aureus, a Flavobacterium sp, and Escherichia coli B bac-teriophage was demonstrated both by using the nonexhausted sodium-regeneratedresin and by using the same resin exchanged with different mono-, di-, and trivalentcations. Effluent counts completed after bacterial seepage through the resins indi-cated the Pb4---exchanged resin removed 55% of the bacteria; Na+, Fe++, andAl++ removed 31 to 36% and Ca++ and Cu++ removed about 10 to 15%. Seventyper cent or more of the bacteriophage was removed by Fe++, Cu++, and Al+++,whereas the Ca++ and Na+ cations removed 25 to 31 %. Over a 77-day period, non-sterile tap water was passed through bacterial seeded and uninoculated SDB (Na)resin columns. Effluent and resin elution counts demonstrated the growth and sur-vival of 2 different bacteria per column. Increased bacterial retention, survival, andmultiplication occurred concomitantly with accumulation of organic and inor-ganic materials and the Ca++ and Mg++ cations from the tap water. Furthermore,microbial elution from resin particles taken from column depths of 1, 8, and 16 cmindicated a bacterial diminution with increasing depths.

Innumerable studies have described microbialsurvival and dissemination in public water supplysystems. We have contributed to this body ofknowledge by relating paralytic poliomyelitiscases to the water-borne virus (1), and by relatingsepticemia and deaths in prematures to a water-borne Achromobacter bacterium (13). Moreover,certain microorganisms indigenous to water,such as the Flavobacterium (6), Pseudomonas(26), Alcaligenes (10), and Paracolobactrum(23), have been associated with troublesomenursery infections caused by inhalation therapyequipment in hospitals or contaminated waterfrom other sources (21). Although such infec-tions have not been attributed to or associatedwith microorganisms cultured from cationicexchange resins used in water-softener units,they are directly involved in water microbiology.It seemed reasonable, therefore, to study theseresins. Consider, for example, (i) their micro-biological entrapment capabilities, (ii) theirfavorable environment for microbial main-tenance and multiplication, (iii) their longevity(7 to 9 years), and (iv) their reuse capability.

IPresent address: Captain, 6th U.S. Anny Medical Labora-tories, Fort Baker, Sausalito, Calif. 94965.

Moreover, their microbial dissemination poten-tial cannot be completely discounted when weconsider their ubiquitous distribution in water-softener units.

This report describes field and laboratorystudies completed on a synthetic cationic ex-change resin. Our objectives were simply toprovide: (i) a reasonable microbiological as-sessment of water-softener resins, a study whichentailed the generic identification of types andtotal numbers of the bacteria and fungi recover-able from the first backflush contents of theexhausted water-softener resin tanks servicingmainly urban and rural homes, but also hospitalsand dairies; (ii) a microbiological assessment ofthe 26% "stock salt brine" used to regenerate thecleaned resins; (iii) information regarding bac-terial survival time and filtration capabilitiesusing both the nonexhausted sodium-regeneratedresin (SDB) and the spent resin, determiningwhether these phenomena are the same whenthe resin is exchanged with different mono-, di-,and trivalent cations at the same pH, tempera-ture, and flow rate; (iv) information relative tothe influence of a progressively changing resin(resin in the process of exhaustion) upon bac-

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MICROORGANISMS IN WATER-SOFTENER RESINS

terial filtering capacity and survival time; (v) anevaluation of affluent and effluent pH, chlorine,hardness, and protein levels, finally, relatingthese factors to the maintenance and multipli-cation (or both) of the microorganisms in theresin, or conversely, to their inability to survive.

MATERIALS AND METHODSSample collection of field studies. Tank sources

included predominately urban and rural residences.A limited number were obtained from hospitals anddairies. The 9-inch diameter tanks contained 43 lb.of the SDB resin. Sterile plastic caps, placed on alltank pipe openings when the tanks were collected forcleaning, were removed. Sterile hose connectors wereinserted onto the backflush outlet and water inletpipes. Two gallons of the first backflush of eachexhausted tank was collected in sterile 3-gal plasticjars. Similar samples (one per month for 12 months)were obtained from the concentrated stock saltbrine. Effluents from 12 separate tanks containingthe backflushed, sanitized, regenerated, and washedresin were also cultured.

Enumeration and identification of microorganisms.All samples were processed the same day as collected,or were stored at 4 C and tested the following day.Samples (100-ml) of each shaken, undiluted, backflushsample were filtered using a 47-mm membrane filter(0.45 Mm, Millipore Corp., Bedford, Mass.). Thecontaminated filter pads were placed on plates ofTrypticase Soy Agar (TSA; BBL), TSA plates con-taining 5% defibrinated human blood (BA), EosinMethylene Blue Agar (EMB; Difco), SalmonellaShigella Agar (SS; Difco), and were incubated at37 C for 48 hr. Membrane filters were also placedon two plates of Sabouraud Dextrose Agar (SDA,Difco) and Mycophil Agar (MA, BBL). For 2 weeks,one set was incubated at 25 C and the other at 37 C.In addition, one Standard Plate Count Agar (Difco)plate was incubated for 5 days at 55 C and anotherwas incubated for 1 week at 4 C. Anaerobic micro-organisms were detected on Anaerobic Agar plates(BBL) incubated in Brewer anaerobic jars at 37 C for1 week.

Counts were recorded as colony-forming units(CFU) per 100 ml. The TSA plates were used for allcounts. If colonies were too numerous to count (morethan 300), the backflush samples were diluted 1:10and 1 :100, and the above procedures were repeated.

Generic identification of the different bacterialisolates was based upon colonial characteristics,staining procedures for identification of external andinternal structures (including the Gram and acid-fast stain), cellular morphology, and cultural andbiochemical characteristics as outlined in the Manualof Microbiological Methods (19).

Fungi were identified by their colonial character-istics on SDA, MA, BA, and by their morphologicaland spore characteristics in lactophenol cotton bluewet mounts and Shoemaker slides. Microbial identi-fication was based on guide lines outlined by Breedand Coake (5, 9).

Stock salt brine isolates. Salt brine dilutions of1:10 were similarly filtered by membrane filter (Milli-

pore Corp.) and cultured. In addition, 1:100 dilu-tions were filtered to isolate staphylococcal specieson BA. These isolates were cultured in Brain HeartInfusion broth (BHI, Difco). The sources of inoculumfor all tests were 24-hr, 37 C cultures. Extensive differ-ential tests were completed for the Staphylococcus.Colony characteristics were described after growthfor 48 hr at 37 C on Nutrient and Mannitol Salt(MS) Agar plates (Difco). Lactose, glucose, mannitol,xylose, sucrose, and maltose broth fermentation tests(nutrient broth, 0.5% carbohydrate, and phenol redindicator) were completed. Three to four drops of3% H202 were added to the growth on NutrientAgar plates to detect catalase production. Additionaltests included nitrate reduction (Trypticase NitrateBroth, BBL), gelatinase activity (Nutrient Gelatin,Difco), indole production (Tryptone Broth, BBL),Litmus Milk (Difco), and MR-VP medium (BBL).Dissolution of 5 ml of human plasma clotted withthrombin was used to demonstrate fibrinolysin.Deoxyribonuclease Test Agar (Difco) was used todetect deoxyribonuclease activity. An inoculum of0.05 ml was added to four separate 6-mm agar wells.Phosphatase activity was also determined (3). Theslide (7), tube (12), and the Coagulase Agar Base(Difco) methods were used to detect coagulase pro-duction. Phage typing was completed by loop spottingphage (National Communicable Disease Center,Atlanta, Ga.) diluted 1:10 on both TSA plates layeredwith the staphylococci and on soft agar overlay con-taining the isolates (4). The 24 phages used were: 3A,3B, 3C, 6, 7US, 29, 42D, 42E, 47, 47C, 52, 52A, 53,54, 55, 70, 71, 75, 77, 79, 80, 81, 83A, and 187.

Defibrinated (5%) rabbit, sheep and human bloodagar plates (Tryptose Blood Agar Base, Difco) werestreaked to determine the type of hemolysis. For thehemolysin tube titrations (16), the Staphylococcuswas grown in 100 ml of Tryptose Phosphate Broth(Difco) in an atmosphere of 20% CO2 at 37 C for48 hr. Unfiltered, centrifuged supernatant fluidswere diluted for hemolysin determinations againsthuman, rabbit, guinea pig, sheep, and ox red bloodcells (RBC). A 2% preparation of RBC (washedthree times with saline) was used, and an equivalentamount (0.6 ml) added to 0.85% saline dilutions ofthe supernatant fluid. Titers were recorded after 1hr incubation in a 37 C water bath and after over-night (4 C) refrigeration. The titer was the greatestdilution of supernatant fluid that produced 50%hemolysis. Disc antibiotic sensitivity tests (Sensi-disc, BBL) were completed on streaked TSA. Thefollowing were used: Streptomycin S/10, Chloro-mycetin C/5, Tetracycline Te/30, Aureomycin A/5,Neomycin N/30, Penicillin P/10, Terramycin T/30,Erythromycin E/15, Triple Sulfa SSS/1.0, Sulfa-methoxypyridazine Ky/1.0, Nitrofurantoin Fd/100,Colistin Cl/10, Novobiocin NB/30, SulfamethizoleTh/1.0. The sensitivity tests were positive when azone of inhibition, regardless of size, appeared aroundthe disc after 24 hr incubation at 37 C.

Animal virulence studies. All tests were completedwith an 18-hr staphylococcal BHI culture grown at37 C. For dermonecrosis studies, two rabbits wereinjected intradermally in four separate areas with

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seeded with P. fluorescens and S kWVW Tkd'lVtY5- A n & &itN Bacillus and a Corynebac-Columns no. !3 and i4 served as control co-lumns; terium were Irepeatedly cultured from the stocktap water only (no. 13) AdIA lPiw&ater only salt brine Or(no~+4)w~th uah v~'y uh~i ay.T, sat brne.On~ ~ Occasions we cultured a

ermoactinod fri efolte (lmostmia1~stmpiffe micro-cativi e cwoAhi'r; prqw~ed frokj efflL*~4,ITem1cq~ ne~ ohdlftff?6^abcolrsrlo. 9-14 at lhe same interv~fl~d o gaiilAs ere gram-tiegative diplococci whichmentioned ahove,7ao Wai slu ns6iiondays337-M b came- ptoumrphitc mpn-gucultureTheP64, and 77. Finally, onldays 80 and 81,jll resin beds d plocbcci fHiiled&to girow at .37.Ci,4 gSi%40were culturkd for microorganisms at depths df 1, ~, al 40, and gre~v op mally at room 5 xand 16 cm. A piece of sterile glass tubingidiamr8 mrs) tt Ire. h StaihyIcocc s Bacillus, anilwas inserted into the iesin column and a measured bicter m so. w iso ated from four o !amo~itwas dispEhsedi nto isterileJ9-m4water blanik s~mplqs of the ceaned (backfiushe&The tubes ere shaken vigorously for 3h sec to elufe -.the organis ns. Serial 1p-fold diluti ns were pnepar regenerated, an..1was.ed)controltank...s3from the spernatant fluid; tripljcate plate counr I...enti.icatioo.&.urban. tank n icrwere then inade0tor each dilution to determine tW rvealedseMjeral 5nteresting phenneni.IIurinnumber of colonies/g of resin. Similar meiasureil a 6 toF7 month oeriod the same.tanksoawk)samples of resin were dried and wei hed. Ifl .shed and cleafikd 4 to .5* times; lMitibMVN9

Inhuent Ind effluent water analrsis. bn a weekiN sessmdnts Were Wompleted (four't!qgEI))wgbasis, measurements or the pH, hlorine, and total t' e s4ne tank frvicil1gderent unI&" M_

eflet wtrwe bcterium ada Msu'hgdifern un.1 -~_fihardness oi' both influ nt and ef uen water wefmoas sp. Wpe~sii-determined tfor each kolurhn. Measurements weAe the foiar tim~s ths -ankwas returneu 1qemade usinglkits provid d by the Iach Chemical ca. Iijenes andge cterium

Furhemor, he montof proten in-both influefit .......adre.a.eiun ~and effluentiwaters was etermine in the niodifid rpeatedly isoatd oh three....itechnique of Lowry et al. (8), wih bline-album ii another tadk. Furthqrmore, siilatsi soAfraction V .s the standaird. V g nisnks were culturedj from. differen ba~kn se~

Of vicinglthe silme dkWellig, indicating -a periMEWtRl SULTS AIND DIScVSSION 8 scsurce of rmsin '-ontahination from thnfiffih'?

"' cS T.No 4ch studik with five tankff d# Xd%Microbi 1 content of water-softener unity. presence of Aspeillus sp and Padc& n

The back ushtq samples from 143 separate Firthermore, a flme1 waspepj i

exhausted water soflener tank resins were ex- fourth tank after thre vpamined, exclusbe of the contrOl tanks and salt the same unit inicate .an absence f auEor-brine s ;lesnisms-The total numbers of microorganisms varied

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STAMM, ENGELHARD, AND PARSONS

TABLE 1. Bacteria andfungi isolatedfrom the backflush contents of143 water softening units, 12 samples ofstock salt brine, and 12 control tank resins

No. of isolates from

Microorganism Tofaisolaeofisol

Farm Private Stock salt ControlMrgi.Farm |home Hospital Dairy brine tank resins

Achromobacter. .......... 12 1 7 4Aerobacter ............... 5 1 4Alcaligenes............... 8 1 6 1Aspergillus ............... 8 1 7Bacillus.................. 37 5 27 2 3 12 4Brevibacterium ........... 6 1 4 1Candida.................. 16 16Clostridium .............. 25 7 18Corynebacterium .......... 12 12 10Cryptococcus............. 5 3 2Escherichia............... 12 3 9Flavobacterium ........... 13 1 6 6 3Geotrichum............... 6 6Micrococcus.............. 8 8Mycobacterium ........... 10 8 1 1Paecilomyces............. 8 2 6Paracolobactrum ......... 8 1 5 2Penicillium............... 6 6Pseudomonas............. 9 7 1 1Rhodotorula.............. 8 1 7Sarcina .................. 11 1 10Serratia.................. 6 6Staphylococcus ........... 33 8 25 10 4Streptococcus ............ 11 1 10Streptomyces............. 5 5Gram-negative coccus 12 12Gram-negative diplo-COCCUS................. 12 12

staphylococcal salt brine isolate was also inter-esting. This organism appeared as a medium-size, white, glistening, convex, entirely edgedcolony on BHI agar. The optimal growth tem-perature was 37 C. Acid was produced from glu-cose, lactose, and sucrose; mannitol was notfermented. Growth was not observed on MSagar or nutrient agar. The catalase and MR testswere positive, acetoin was produced from glucose,nitrates were reduced, and litmus milk wasacidified and coagulated. Furthermore, phenyl-phosphate was hydrolyzed, and deoxyribonu-clease and fibrinolysin activity was demonstrated.Beta-type lysis occurred on rabbit and humanBA, but not on sheep BA. Sufficient free andbound coagulase was also demonstrated. Plaquedevelopment was absent for all 24 phages. Thetube hemolysin titrations indicated guinea pigRBC to be the most susceptible; the next mostsusceptible were those of the rabbit and human,and then the moderately susceptible sheep cells.Ox cells were not lysed. These titrations indicateda delta-type staphylococcal hemolysin. The iso-

late was resistant to tetracycline, neomycin,triple sulfa, sulfamethoxypyridazine, and sulfa-methizole.

Within 24 to 48 hr the ID injections producedinflammatory areas approximately 4.5 cm indiameter. This area receded within 6 days andproduced an area of necrosis. All signs of in-flammation were absent 8 days after injection.All rabbits survived the intravenous injectionswith no apparent sign of illness. All mice diedwithin 6 to 30 hr after intraperitoneal injections.The peritoneal cavities were covered with a whitemucous substance and staphylococci were iso-lated from both peritoneal fluid and heart blood.

Since the Staphylococcus was not susceptibleto the phages, the most definitive test to relatecleaned and exhausted softener resin and saltbrine staphylococcal isolates could not be used.

In spite of the absence of an alpha hemolysinand pigmentation, and in spite of the failure toferment mannitol aerobically and to liquefygelatin, the above cultural characteristics aresuggestive of the pathogenic staphylococci. For

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MICROORGANISMS IN WATER-SOFTENER RESINS

example, strains are generally considered patho-genic if they produce both free and bound co-agulase (11). Correlation has also been reportedbetween phosphatase synthesis and pathogenicity(2), and between deoxyribonuclease and coagu-lase activity (25).

Cation effect on bacterial filtration. Al ionexchanged resin columns removed bacteria(Table 2). Comparable percentages of both thegram-positive Staphylococcus and the gram-negative Flavobacterium were filtered from thesuspension. In addition to the massive bacterialdestruction by the Ag+-regenerated resin, Pb2+removed over one half of the total numbers oforganisms. The Na+, along with Fe2+- and A13+-regenerated resins, removed approximately athird of the organisms. The Cu2+ and Ca2+resins filtered a much smaller percentage of eachsuspension.

Cations, upon accumulation in the resin bed,can change the filtering characteristics. A smallermicrobial filtration capability would occur for a

TABLE 2. Capacity of the SDB resin column (Na+)and SDB resins, exchanged with mono-,

di-, and trivalent cations, to removemicroorganisms from a suspension

containing 106 CFU/mla

Per cent of organismsResin removedAtomic weights cations -______

Staphylococcus Flavobacterium

22.9 Na+ 36 33107.8 Ag+ 99 9955.85 Fe+ 35 31

207.21 Pb+ 56 6063.57 Cu++ 10 1540.08 Cal 14 1226.97 All 34 35

a Results are averages of triplicate-column runs.

TABLE 3. Capacity of the SDB resin column (Na+)and SDB resins, exchanged with mono-, di-,

and trivalent cations, to remove Esch-erichia coli B bacteriophage from asuspension containing 106 PFU/mla

Resincations ~~Per cent ofResin cations bacteriophage removed

Na............ 31Fe+ .......... 70Ag............ 99Cui .......... 74Ca' .......... 25All .......... 84

a Results are averages of triplicate column runs.

Ca2+_ or Cu2+-spent resin than for a Na+-,Fe2+-, Pb2+-, or Al3+-exhausted resin.

Except for the Ag+- regenerated resin, thesesame microorganisms were cultured from allresin effluent waters after 3 days. Dissolution ofthis metallic ion in water to the extent of 10-5g/liter is bactericidal. Bacterial counts de-creased exponentially in all columns thereafter,and, on day 13, all resin effluents, except Ca++,showed an absence of microorganisms. This isnoteworthy because the calcium ion protectsmicroorganisms against disinfecting agents (20).Furthermore, as the resin becomes exhausted(i.e., accumulates calcium), the maintenance ofentrapped microorganisms is prolonged. Never-theless, these results confirm the observations ofKlumb (17), namely, that siliceous resins in theabsence of accumulated, filtered organic matterare incapable of sustaining bacterial growth.Our experimental design with viable organisms

failed to reveal any significant role of valency inbacterial resin adsorption. It has been shown (14)that staphylococcal cell wall suspensions bindmetal ions and that divalent ions showed agreater affinity for cell walls than monovalentions. Furthermore, affinity increased with atomicweight. Others (27) observed that positivelycharged surfaces are more conducive to theattachment of a gram-negative bacteria such asEscherichia coli, than negatively charged ones.Puck and Sagik (22) confirmed the existence ofnegatively charged binding groups on the sur-face of Escherichia coli B cells at pH 7. Thesecells became rapidly attached to a positivelycharged sulfonic-acid polystyrene resin, but notto a negatively charged one. Obviously, bacterialsurface charges alone do not govern adsorption(24). Cell configuration, relative position of thecharges, age of the cells, and type of resin usedare equally important. Moreover, cell wall con-stituents, such as diaminopimelic acid, variousamino acids, teichoic acid, or hexosamine, mayprovide the necessary charges for adsorption tothe resin surface.

Cation effect on bacteriophage filtration. TheMn2+- and Pb2+-regenerated resins failed to ad-sorb the phage (Table 3). Moderate differences inthe affluent and effluent plaque counts were ob-served with the Na+- and Ca2+-regeneratedresins. Significant decreases, however, occurredwith Fe2+, Cu2+, Al3+, and the obvious destruc-tion of bacteriophage with Age.

In a somewhat similar study (22), using acationic resin of the sulfonic-acid type regeneratedwith 10% NaCl, it was demonstrated that atleast an 0.15 M NaCl concentration was requiredfor adsorption of Ti and T2 Escherichia coli

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Symbols: columns 1, (0); columns 2, (X); columns3, (0); columns 4, (A).shown IW &h"P ri$kYa'1l5)W %i 6limn 5(StaphW3n&1 AvWiPt rtii-d withvalues ot2 8"° e previous

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___ i~edth I~e| calcium-regenerated resin{columns 9, IO), i.e. %22% and |3c oFthePse4,omonas al St phyl4Cus, res evely.Onl4 8% and rct w re r qiedbythe (utriert-br¶t tregtediins (co 11,12).:jhe initiaL ltration sf1es (olum 1, 5,9, 1Q; 11, 12) ia4icatec theqaacity of4ht resinto rnove 'moriof the gpobitiv64h: thegram-negative orga isms However, when c-ornt~rh-Rk~b i~sE¢/fti dft29 thegram-positive organisms occurred within the

volufe,-ofsap aet pssdkhimagh eaM &obsmnevery -6thr~ays t1~1o~'r~anmXi~onf bac-teria re9l iaflh I After37 dayn\'!vf~xn\)l6i8'yf 40461mffi1 91 % ofthe Pseudomaos suspension, whereas approxi-matliynf'qgithe,-,taphylocoeRS ~ii§*moved.tUndoubted-'ly, the acainaUlationl oorgaMCr- -ma-terials and finorganic materials,-sudibhs the ex-changed mAnesium, calcium, and 6t er cationswhich accI ulate in the resin, Q9tibuted tothis retenti9. U+Both migoorganisms surviveda4 were re-

leased in fairly large numbers i e effluent.w~rrod~tl¢~e stu~ylF~ig.

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MIC.IMoinoA1N11It1 ER ESNLWUESINS

effluent ciunts of--Pseudomonas (col-o 054 iowedX a grii4al dodrease. TheIX fX106 tU/rtdl slow yedecreasedtaned -a pl4taqu affir aboi t :4 days of;s1ThM~iO4 teU/f! olu' 4 effluente~dgt de~la$t 2 paye ribd showedLet~argerxrnt.A-Althoug -the initial)ca Y unts 'rj ~slghtly lower, the

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384 STAMM, ENGELHARD, AND PARSONS

suits described above with the 13-day resincolumns and sterile deionized water.

Effluent counts of Pseudomonas and Staphy-lococcus from columns treated with calcium(columns 9, 10) and nutrient broth (columns 11,12) also declined and appeared to level off atapproximately 104 CFU/ml after 64 days (Fig.4). A 1-log increase for both microorganismsoccurred in the nutrient broth columns, i.e.,from an initial inoculum of 106 to approximately107 on day 13. However, both declined to popula-tion levels comparable to other columns. The in-fluence of the nutrient broth and the calcium didnot enhance the survival characteristics of thesetwo bacteria.

Effluent Flavobacterium counts increased withthe amount of tap water passage and leveled offat approximately 105 CFU/ml of effluent (Fig.5, 6). This undoubtedly reflects the maximalpopulation that our resin column will support.Flavobacterium counts from the effluents ofPseudomonas seeded columns 1-4 and tap watercontrol columns 13 and the staphylococcal per-colate, columns 5-8, are shown in Fig. 5 and 6,respectively.

Flavobacterium counts from columns 1 and 2,seeded with Pseudomonas on day 0 and 13, re-spectively, showed a steady 6-log increase,achieving the maximal stationary level of approxi-mately 106 CFU/ml on days 35 and 50, respec-tively (Fig. 5). Pseudomonas (columns 3 and 4),seeded on days 22 and 37, showed lower Flavo-bacterium counts (approximately 4.5-log in-crease) at days 37 and 50, respectively. Com-parable numbers were maintained over theremaining time period of 30 days. The unseededcolumns (no. 13, tap water passage only)showed a slow 4-log increase achieving a concen-tration of approximately 104 5 CFU/ml after60 days, with the major increase occurring over a37-day period. The final cell concentration wascomparable to that achieved in columns 3 and 4.

Several factors contribute to the enhancedFlavobacterium population in columns 1 and 2.For example, more Pseudomonas microorganismswere retained within the resin when columns 3and 4 were seeded on days 22 and 37 (Fig. 1).Competition for available substrates could causethe reduced Flavobacterium population. Further-more, we have established the direct relationshipbetween the accumulated, tap-water exchangedcalcium, magnesium, and other cations in theresin and the increased microbial retention (Fig.1).The staphylococcal columns (no. 5 and 6)

seeded on day 0 and 13, respectively, did notshow the same phenomenon (Fig. 6). TheFlavobacterium in column 5 showed an early3.5-log increase after 13 days and a 4.6-log in-crease at 22 days, before maintaining a relatively

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MICROORGANISMS IN WATER-SOFTENER RESINS

consistent population for the additional 55 days.Bacterial multiplication in column 6 was slower.A 3.5-log increase occurred after 22 days, withan additional gradual increase for approximately40 days before acheiving 50,000 CFU/ml. Thelarger numbers of Staphylococcus adsorbed by theresin seeded on day 13 contributed to this slowerincrease. However, column 7, Flavobacteriumnumbers (37 days) showed similarities withcolumn 5. Column 8 counts were highest on day51. A 5.5-log increase was observed which de-creased slightly over the 27-day period there-after. The fact that columns 7 contained moreStaphylococcus after day 22 than columns 5 didnot have the same depletion effect on the in-digenous Flavobacterium. The cocci added tocolumn 8 on day 37 caused a slight stimulatoryeffect. Columns 14, through which sterile tapwater passed for 77 days, showed no counts atany time.

Obviously, effluent counts alone are inadequateto disclose the extent of microbial growth. Themicroorganisms are not readily dislodged be-cause they are adsorbed to the resin and en-meshed within the resin matrix.

After 80 days, it was evident that considerablenumbers of Pseudomonas, Staphylococcus, andFlavobacterium were not washed from the resinwith the passage of 250 ml of tap water (Table 4).The average Pseudomonas counts from the top1-cm layer of the four paired resin columns(1-4) and the staphylococcal paired resin columns(5-8) contained 5.1 X 104 and 4.8 X 104 CFU/g of resin, respectively. At depths of 8 cm, theaverage counts of these same bacteria were 3.7X 104 and 3.8 X 104 CFU/g of resin; at 16 cm,the average counts were 3.1 X 104 and 3.5 X 104CFU/g of resin.

Bacterial diminution with increased resindepths was also observed with the Flavobacterium(Table 4) cultured from both the Pseudomonas-and Staphylococcus-seeded columns 1-4 and5-8, respectively. A similar reduction was appar-ent with columns 13, through which tap waterpassed only, although slightly larger numbers ofFlavobacterium were enmeshed within the resin.

Bacterial multiplication within these small resincolumns is best illustrated with paired columns 1,5, and 13; i.e., resin columns which had been ex-posed to Pseudomonas and Staphylococcus,respectively, prior to the addition of tap water(Fig. 1) and miroorganism indigenous to tapwater. Elution procedures completed on day 81on the entire 20 g in each paired resin columnrevealed average counts of 4 X 104 CFU/g ofresin, or 8 x 105 organisms/column no. 1. Sinceonly 28% of the 106 bacterial suspension was re-moved after the only initial bacterial passage onday 0, the above results represent more than athreefold increase, i.e., from 2.8 x 105 CFU/ml

(day 0) to 8 X 105 of the total number of cells(day 81). A smaller twofold increase was ob-served with the Staphylococcus, i.e., 3.8 X 105 to8.5 X 105 total numbers of cells. Furthermore,effluent counts taken on the same day would in-crease the total number of cells in both in-stances 1.5 X 105 (104 CFU/ml X 15 ml).The total inoculum for the indigenous tap

water organism over the 80-day period (500CFU/250 ml every other day) was 1.9 X 104cells. A moderate increase was shown with theeffluent count of 4.8 X 104 CFU/ml. Elutiontests, however, showed 5.1 X 104 CFU/g ofresin, or slightly over 106 colonies/resin column.Under our experimental conditions it is apparentthat at room temperature this resin can supportthe simultaneous growth and multiplication ofseveral different types of microorganisms.When compared to columns 1-4 (Table 4)

fewer Pseudomonas were eluted from the 1, 8, and16 cm depths of the SDB resin column exposed tonutrient broth. The same reduced numbers wereapparent with the Staphylococcus. However, theFlavobacterium counts were slightly higher thancounts observed for column 13. With the excep-tion of the resin, eluate counts taken at 1-cmdepths, the Pseudomonas and Staphylococcuscounts from the calcium-regenerated resin werecomparable to counts obtained from pairedcolumns 1-4 and 5-8. Furthermore, calciumeither had a slight inhibitory effect on the growthof the Flavobacterium or reduced its adsorptiveabilities, since the lowest numbers of CFU/g ofresin were detected in this resin.No change occurred in the pH of the influent

and effluent waters. Consistent pH values of 6.8to 7.1 were obtained. Influent tap water contained1 ppm of chlorine. No chlorine was detected insamples of the effluent water, indicating removalby the filtered organic matter and the SDB resin.Chlorine in the resin matrix had no marked bac-tericidal effect upon the seeded microorganismsor the Flavobacterium.

Total hardness values indicated that the resincontinued to efficiently soften water throughoutthe study period. Protein levels of column effluentwaters, over the 77 day period, increased from3 ,ug to approximately 25 ,ug of protein/ml.Adsorbed or filtered organic matter would be

more abundant in the upper layers of the resin,resulting in the establishment of a larger microbialpopulation. Cation exchangers of the sulfonictype have been shown to take up all cations, bothorganic and inorganic (8). Ampholytic chemicals,such as amino acids, are also removed (24).Furthermore, it is reasonable to conclude thatsolid surfaces, such as resin particles, enablebacteria to develop in substrates otherwise toodilute for growth, by adsorbing and concentratingorganic matter (15). Once bacterial growth is

VOL. 18, 1969 385

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