APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1986, p. 434-438 0099-2240/86/090434-05$02.00/0 Copyright 1986, Americn Society for Microbiology Vol. 52, No. 3 Bcteri Associted with Grnulr Activted Crbon Prticles in Drinking Wter ANNE K. CAMPER, MARK W. LECHEVALLIER,t SUSAN C. BROADAWAY, AND GORDON A. McFETERS* Deprtment of Microbiology, Montn Stte University, Bozemn, Montn 59717 Received 6 Jnury 1986/Accepted 28 My 1986 A smpling protocol ws developed to exmine prticles relesed from grnulr ctivted crbon filter beds. A guze filter/swinnex procedure ws used to collect crbon fines from 201 grnulr ctivted crbon-treted drinking wter smples over 12 months. Appliction of homogeniztion procedure (developed previously) indicted tht 41.4% of the wter smples hd heterotrophic plte count bcteri ttched to crbon prticles. With the enumertion procedures described, heterotrophic plte count bcteri were recovered t n verge rte of 8.6 times higher thn by conventionl nlyses. Over 17% of the smples contined crbon prticles colonized with coliform bcteri s enumerted with modified most-probble-number nd membrne filter techniques. In some instnces coliform recoveries were 122 to 1,194 times higher thn by stndrd procedures. Nerly 28% of the coliforms ttched to these prticles in drinking wter exhibited the fecl biotype. Scnning electron microgrphs of crbon fines from treted drinking wter showed microcolonies of bcteri on prticle surfces. These dt indicte tht bcteri ttched to crbon fines my be n importnt mechnism by which microorgnisms penetrte tretment brries nd enter potble wter supplies. Activted crbon hs been widely used in the tretment of drinking wter to remove unplesnt tstes nd odors s well s to control the formtion of trihlomethnes (11, 18, 21, 28). Grnulr ctivted crbon (GAC) is effective in dsorbing wide rnge of orgnic compounds, some of which serve s nutrients for bcteril growth (2, 4, 5, 9, 24, 28). Vrious reserchers hve shown tht GAC used to tret drinking wter supplies cn be hevily colonized by heterotrophic microorgnisms (2, 4, 5, 18, 28). Potentil problems could rise if these bcterium-coted prticles penetrte tretment brriers (23) or if orgnisms re sloughed or shered from colonized filter beds nd enter finished drinking wter supplies (26). Recent studies (14) hve shown tht bcteri ttched to GAC re resistnt to chlorintion. Previous investigtions into the effects of ctivted crbon on the microbiology of finished drinking wter hve indicted tht GAC hd little impct on the bcteril qulity of potble wter supplies (2, 5, 18, 24). It ws thought tht ny bcteri emnting from GAC filters were effectively treted by post-chlorintion. In ddition, it ws shown tht bcteril levels were generlly low nd of little helth significnce. All of these prior investigtions exmined crbon-treted wter by collecting stndrd grb smples nd enumerting microorgnisms by stndrd bcteriologicl procedures. However, these techniques give indequte results since single prticle coted with thousnds of bcteri would yield only one colony. Recently, we hve developed homogeniztion technique to quntittively desorb microorgnisms from GAC (6). This procedure ws shown to enumerte ttched microorgnisms t efficiencies of 80 to 90%. This report describes novel smple collection method cpble of trpping prticles from treted drinking wter. Appliction of the homogeniztion technique on prticles collected by this method llowed us to evlute the impct of crbon fines on drinking wter qulity. The results show tht bcteri ttched to crbon prticles pssing through GAC filters cn * Corresponding uthor. t Present ddress: Americn Wter Works Service Co., Inc., Belleville Lbortory, Belleville, IL 62220. 434 be n importnt source of contmintion in treted drinking wter supplies. MATERIALS AND METHODS Smpling device. A 47-mm Swinnex filter holder (Millipore Corp., Bedford, Mss.) ws modified by drilling ech end to 0.25-in. (0.64-cm) dimeter. A filter ws constructed by sewing 16 lyers of guze mteril together into 47-mmdimeter circle. The Swinnex unit nd filter were sterilized by utoclving (121 C, 15 min) prior to use. The Swinnex contining the guze filter ws ttched to smpling ports of drinking wter filters. Smple sites nd collection methods. A totl of nine drinking wter tretment fcilities (ech using full tretment, i.e., floccultion, sedimenttion, filtrtion, pre- nd postchlorintion) provided smples of filtered effluents for this study by instlling Swinnex units to smple ports immeditely following filtrtion, prior to finl chlorintion. Seprte guze filters were used to trp prticles during three time periods of norml drinking wter tretment. One ws the entire filter run until 1 h prior to bckwsh, the second guze filter ws used for the 1-h period before bckwsh, nd third filter ws used for the 1 h fter bckwsh. Ech guze filter ws septiclly removed from the Swinnex unit, plced in sterile Whirl-pk bg (Nsco) contining few drops of wter, nd shipped on ice vi overnight ir service to the lbortory. Informtion on the flow rte through the guze filter nd the time period of filtrtion were lso recorded. All filters were processed within 30 h of collection. Microbiologicl exmintions. Upon receipt, ech filter ws exmined nd rted for the mount of crbon fines observed. The filter rtings were s follows: 1, <10 crbon (blck) prticles per filter visible with dissecting microscope t x 10 mgnifiction; 2, >10 crbon (blck) prticles per filter visible with dissecting microscope t x 10 mgnifiction; 3, <10 crbon (blck) prticles per filter visible per filter by the nked eye; 4, >10 crbon (blck) prticles per filter visible by the nked eye. Filters were then septiclly cut in hlf nd plced in fleker contining 300 ml of cold, sterile, regent-grde wter (Milli-Q system; Millipore
VOL. 52, 1986 TABLE 1. Results of nlyses of prticles collected from GAC-treted effluents Typeof Totl No. (%) Fold increse Type of no. of showing nlysis no. >2-fold Men Mximum increse HPC 198 82 (41.4) 8.6 50.0 Coliform MF 201 14 (7.0) 124.3 1,194.0 MPN 191 33 (17.2) 24.5 122.2 Homogenized versus hnd-shken nlyses. Corp.). Ech fleker ws vigorously hnd shken to dislodge prticles from the filter. The guze ws removed nd the suspended prticles were chlorinted with 2.0 mg of sodium hypochlorite per liter for 30 min t 4.0 C (ph 6.5 to 7.0) in the drk. This chlorintion procedure effectively eliminted bcteri not ttched to prticulte mteril (14). Smples were dechlorinted with sodium thiosulfte (1). Chlorinted smples were divided into two prts; one hlf ws evluted fter hnd shking nd the other ws ssyed following homogeniztion (6). The homogeniztion procedure consisted of blending the smple t 16,000 rpm in solution of 10-6 M Zwittergent 3-12 (Clbiochem-Behring, L Joll, Clif.), 10-3 M ethylene glycol-bis(p-minoethyl ether)- N,N'-tetrcetic cid (EGTA), 0.1% peptone (Difco Lbortories, Detroit, Mich.), nd 1 mm Tris buffer, ph 7.0 (Sigm Chemicl Co., St. Louis, Mo.) for 3 min t 4 C. Coliform bcteri were enumerted by the membrne filter technique, using 0.45-,um HA filters (Millipore Corp.) nd m-t7 gr (12). A totl of 90 ml ws filtered, usully in three 30-ml quntities. Pltes were incubted t 35 C for 24 h. Yellow colonies were counted with the id of disecting microscope (x15) nd confirmed in luryl tryptose broth for gs formtion t 35 C for 48 h. Coliforms were lso enumerted by using modified three-tube, three-dilution, multipletube fermenttion technique (most probble number [MPN]). The method ws modified by dding sodium luryl sulfte (Sigm) to the luryl tryptose broth medium (prepred initilly without luryl sulfte) 4 h fter inocultion. Gs-positive tubes were confirmed by inocultion into brillint green bile broth nd isoltion on m-endo gr (Difco). Identifictions were performed with the API-20E system (Anlytb Products, Plinview, N.Y.). Coliforms were screened for fecl biotype by inocultion into EC broth (Difco) nd incubted for 24 h t 44.5 C. Heterotrophic plte count (HPC) bcteri were enumerted, in triplicte, by the spred plte technique, using R2A medium incubted for 7 dys t 25 C (21). HPC bcteri were identified by the procedure of LeChevllier et l. (15). An increse of greter thn twofold in bcteril counts of homogenized over hnd-shken smple ws selected s n indiction of ttched popultions for ll enumertion procedures. Prticle exmintion. Crbon smples were prepred for scnning electron microscopy by mounting criticlly pointdried prticles on copper support, using colloidl grphite (Ted Pell, Inc., Tustin, Clif.). Smples were shdow csted with pltinum nd exmined with JEOL model JEM-100 CX scnning trnsmission electron microscope. Prticles were lso observed by using the cridine ornge direct count procedure of Hobbie et l. (11). Additionl prticle nlyses were done with the id of n imge nlyzer system composed of n Olympus model H-2 microscope nd Quntimet Qlo stndrd progrm. Prticles BACTERIA ASSOCIATED WITH GAC 435 were prepred for viewing by concentrtion on 0.45-,um HA Millipore filters nd exmined under epi-white light illumintion, using Nomrski filters t x15 mgnifiction. All mesurements were mde on 10 fields per smple. Some smples were split nd hlf were treted with 2 N HCl to remove orgnics nd mngnese oxide mteril tht might be mistken s crbon. RESULTS A totl of 201 guze filters were collected from nine drinking wter tretment fcilities during the 1-yer smpling period. Anlysis of the prticles entrpped by the filters showed tht 41.4% of the smples hd greter thn twofold increse of HPC bcteri when enumerted by the homogeniztion technique compred with conventionl hnd-shken methods (Tble 1). An increse of this mgnitude ws presumed to be due to relese of ttched microorgnisms. The men increse of HPC bcteri fter homogeniztion ws 8.6-fold, while the mximum increse observed ws 50-fold. Coliform bcteri were enumerted by two techniques (Tble 1). Over 17% of the smples nlyzed by the modified MPN procedure showed greter thn twofold increse fter homogeniztion. The verge increse in coliform densities fter homogeniztion ws 24.5 times higher thn by the conventionl nlyses (mximum increse, 122.2 times higher). Coliform enumertions by the membrne filter (MF) technique showed only 7.0% of the smples with greter thn twofold increse, but the men increse ws 124.3 nd the mximum increse ws 1,194 times higher thn the unblended smples. An understnding of when crbon prticles penetrte tretment brriers is importnt in the mngement of drinking-wter filters. Experiments were designed to collect prticles during the full filter run s well s immeditely before nd fter the bckwsh cycle. Dt presented in Tble 2 indicte tht more crbon fines were found with the full filter run smples. The time periods before nd fter bckwsh were not ssocited with n especilly gret mount of prticle brek-through. The men filter rtings for smples tht yielded greter thn twofold increses in coliform nd HPC bcteri fter homogeniztion were lso slightly higher thn the overll men filter rting. Informtion presented in Tble 3 suggests n ssocition between the volume of wter pssed through the guze filters nd the increses observed in coliform densities fter homogeniztion. The results show tht one-qurter of the smples collected from the full filter run (18,600 liters) hd greter thn twofold increses of coliform bcteri fter homogeniztion. The men increse of coliform orgnisms ws 111 times higher thn the hnd-shken count. Wter TABLE 2. Summry of guze filter rtings for GAC-treted drinking wter NofMen Description of smple smples No. of filter rting All smples 201 1.8 Full filter run smples 72 2.6 Smples 1 h before bckwsh 63 1.5 Smples 1 h fter bckwsh 66 1.5 Smples with >2-fold incresed coliform 32 2.4 bcteri' Smples with >2-fold incresed HPC bcterib 82 2.2 Arithmetic men of filter rtings for ll smples from ech ctegory. b Homogenized versus hnd-shken nlyses.
436 CAMPER ET AL. TABLE 3. Summry of dt collected t different times during filter cycle Avg o% >2-fold increse Men increse Time of Totl no. of in: (fold) collection no.*l liters HPC HPC smples smpled bcteri Coliforms bcteri Coliforms Full cycle 72 18,600 43.1 25.0 11.1 111.0 1 h before 63 260 41.3 12.7 12.5 13.8 bckwsh 1 h fter 66 270 37.9 9.1 8.3 17.7 bckwsh Homogenized versus hnd-shken nlyses. collected 1 h before or fter bckwsh (260 to 270 liters filtered) hd coliform increses fter homogeniztion of 12.7 nd 9.1%, respectively. The men increses of coliforms in these smples were 13.8- nd 17.7-fold, respectively. The results for HPC bcteri were more uniform, for both the percent showing greter thn twofold increses (43.1 to 37.9% of the smples) nd the mgnitude of the increse (12.5 to 8.3 times higher thn hnd-shken nlyses). These results indicte tht populted GAC fines were relesed throughout the entire filter cycle with no peks in brekthrough occurring t the very end or beginning of the cycle. A distinct sesonl trend in the level of ttched coliform bcteri ws observed. Dt presented in Fig. 1 indicte mrked peks in both coliform isoltion rtes nd men number of ttched cells during the spring (Mrch-My) nd fll (October-December). HPC bcteri recovered from prticles did not show pprecible sesonlity. 100 200 90 180 80 160 wlj 7F0 Wt140 0 ol b60 120 Z 5-40 80 Z SJ O CI,FMAM MONTH~~~~~C 30 60 20~ ~ ~ 440 1 0 20 0 0 SO0N D J F M AM J J A MONTH FIG. 1. Smples showing greter thn twofold increse in coliform bcteri fter homogeniztion. Symbols: (40) percentge of smples with greter thn twofold increse; (0) men fold increse. 0 TABLE 4. Orgnism Identifiction of coliform bcteri isolted from prticles in finished drinking wter No. of coliforms isolted MPN h-mpn MF h-mf Kluyver spp. 0 6 0 0 Klebsiell oxytoc 4 24 2 40 Klebsiell pneumonie 1 (0)b 11 (5) 9 (3) 25 (10) Escherichi coli 10 (10) 23 (23) 3 (3) 13 (13) Enterobcter gglomerns 0 3 0 9 Enterobcter cloce 7 (0) 11 (2) 5 (5) 2 (0) Enterobcter erogenes 2 1 0 1 Enterobcter skzkii 1 6 0 0 Aeromons hydrophil 0 5 0 3 Citrobcterfreundii 0 1 0 1 Serrti spp. 2 5 0 6 Hfni spp. 2 3 0 0 h-mpn nd h-mf denote tht smples were homogenized prior to nlysis. b Numbers in prentheses give number expressing fecl biotype. Identifiction of bcteri from hnd-shken nd homogenized smples indicted tht, while recoveries of HPC bcteri incresed fter homogeniztion, the diversity of HPC microorgnisms did not become greter. Six gener of HPC bcteri (Arthrobcter, Alcligenes, Acinetobcter, Micrococcus, Corynebcter, nd Pseudomons) were recovered from five smples nlyzed by both hnd-shken nd homogenized methods from three distribution systems. An increse in species diversity ws observed for coliform bcteri fter homogeniztion for both the MPN nd MF procedures (8 versus 12 nd 4 versus 10, respectively) (Tble 4). Five species were detected only in homogenized smples (Kluyver spp., Enterobcter gglomerns, Aeromons hydrophili, Citrobcterfreundii, Serrti spp.). There were 2.5 more coliforms exhibiting fecl biotypes fter homogeniztion thn by hnd-shken nlyses (Tble 4). Blck prticles collected by the guze filters from GACtreted drinking wter were exmined with the id of n imge nlyzer (Tble 5). Results showed tht the prticles rnged from 1.0 to 3.5 x 103,um in length nd were nerly sphericl. Three smples were split nd hlf were treted with 2 N HCl to determine if mngnese dioxide nd drk orgnic mterils were influencing the filter rtings. Results showed no difference in the size distribution of treted nd untreted prticles, suggesting tht the mteril ws crbon nd not debris. Crbon mteril collected from GAC-treted distribution wter nd exmined by scnning electron microscopy showed microcolonies of bcteri on the prticle surfces. Bcteri coted with extrcellulr mteril were observed in the crcks nd crevices on the crbon surfces. Anlysis of TABLE 5. APPL. ENVIRON. MICROBIOL. Summry of dt collected by imge nlysis No. of Men Size Prmeter roundness Distribution, prticles vlue length (pum) All smplesb 2,333 1.36 x 103 1.0-3.5 x 103 Smples before cid 857 1.59 x 103 1.0-3.5 x 103 tretmentc Smples fter cid 1,617 1.48 x 103 1.0-5.7 x 102 tretmentc Perfectly round = 1.00 x 103; vlues cn rech 3.00 x 103. b Totl of eight smples, not cid treted. ctotl of three smples.
VOL. 52, 1986 crbon prticles by using cridine ornge nd Hoechst stin lso reveled spordic ptches of microcolonies (dt not shown). DISCUSSION The results of this study hve shown tht crbon prticles cn penetrte tretment brriers nd enter finished drinking wter. The development nd use of guze filter/swinnex smpling technique permitted the exmintion of lrge volumes of filtered drinking wter for crbon fines. Crbon prticles were observed routinely in the guze filters received from tretment fcilities. These findings support those of Robeck et l. (23), who in 1964 demonstrted tht powdered ctivted crbon frequently penetrted tretment filters nd cused problems in distribution wter, prticulrly when floccultion ws wek. Syrotynski (25) showed tht microorgnisms, turbidity, nd luminum ions could be ssocited with filtered drinking wter effluents t ll rnges of flows but especilly t high filtrtion rtes (1.44 to 2.52 liters/min per m2). In this study, experiments were designed to determine when crbon prticles entered finished wter supplies. It ws nticipted tht the time before or fter filter bckwsh would be the period of highest prticle penetrtion (3). However, results showed tht the ccumultion of crbon fines ws dependent only on the mount of wter filtered nd not on the time period before or fter filter bckwsh (Tble 2). These results suggest tht the relese of crbon fines ws rndom event not relted to filter opertion. Dt presented in this report indicted tht >40% of the smples contined crbon prticles colonized with HPC bcteri (Tble 1). HPC bcteri were recovered t rte 8.6 times higher thn with conventionl nlyses. Over 17% of the smples contined crbon prticles colonized with coliform bcteri (Tble 1). Mximum coliform recoveries rnged from 122 to 1,194 times higher thn when stndrd procedures were used. Exmintion of scnning electron microgrphs of crbon fines from treted drinking wter showed microcolonies of bcteri on prticle surfces. It is likely tht desorption of these bcteri by the homogeniztion procedure would result in lrge increses in recoveries of coliform nd HPC bcteri. The observtion tht incresed coliform recoveries were usully of single species lso supports the microcolony hypothesis. Consistent with this ide is the effect of sesonl chnges. The sesonl input of nutrients nd wrm-wter tempertures could stimulte growth of the microcolonies, resulting in higher bcteril recoveries. Indictor orgnisms ttched to crbon prticles in drinking wter could be responsible for spordic recovery of coliform bcteri in routine smples. Further, ccumultion of crbon prticles in distribution mins could be involved in the phenomenon known s coliform regrowth. It should lso be noted tht this study ws performed with the coopertion of tretment plnts tht were properly operted nd hd no history of coliform problems in the distribution wter. The impct of GAC prticles on wter qulity my be more severe in instnces where tretment is not diligently monitored. Previous studies hve shown tht bcteri ttched to GAC were unffected by disinfection with 2 mg of free residul chlorine per liter for 1 h (14). The experiments performed in this study support these results. All of the microorgnisms recovered in this study survived disinfection with 2 mg of free residul chlorine per liter for 30 min of contct. Some of the bcteri relesed from crbon fines, BACTERIA ASSOCIATED WITH GAC 437 including Acinetobcter spp., Pseudomons spp., Klebsiell pneumonie, Aeromons hydrophil, nd Serrti spp. my be opportunistic pthogens (9, 16, 19). Nerly 28% of the coliforms ttched to crbon prticles, including the 49 Escherichi coli isoltes from finished drinking wter, exhibited the fecl biotype (Tble 5). In ddition, Slmonell spp. were isolted once from GAC filter bed (A. K. Cmper, D. G. Dvies, S. C. Brodwy, M. W. LeChevllier, nd G. A. McFeters, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, N38, p. 223). While the survivl of pthogens on GAC hs been shown to depend on the interction between utochthonous wter orgnisms nd the pthogenic bcteri (7), the results of this study indicte tht relese of crbon fines from tretment filters my be mechnism by which virulent microorgnisms penetrte disinfection brriers nd enter potble wter supplies. However, further reserch is needed to determine the helth significnce of low levels of bcteri entering wter supplies by this route. The results of this study lso demonstrte the impct of suspended prticultes on coliform detection. Coliform enumertions by the MF technique showed only 7.0% of the smples with greter thn twofold increse, while over 17% of the smples nlyzed by the modified MPN procedure showed such n increse fter homogeniztion. Previous studies hve shown the deleterious effect of turbidity on the detection of coliforms by the MF technique (13). It hs been speculted tht prticles lter the surfce morphology of the membrne filter so s to inhibit coliform growth. This study hs indicted tht investigtors exmining turbid wter smples should consider (i) desorption technique to dislodge microorgnisms from prticle surfces nd (ii) the type of coliform enumertion technique used. As cumbersome s the MPN method my be, we recommend it for nlysis of turbid wter smples. In summry, the results of this report hve importnt implictions for the microbiologicl determintion of wter qulity. We hve shown tht bcteri ttched to crbon fines my be n importnt mechnism by which microorgnisms penetrte tretment brriers nd enter finished drinking-wter supplies. Some of the orgnisms ttched to crbon prticles hve been identified s possible pthogens or opportunistic pthogenic bcteri. In ddition, we hve developed techniques to detect bcteri ttched to crbon prticles. Appliction of these methods in tretment plnts experiencing coliform problems my provide importnt insights into the mechnisms of bcteril pssge, survivl, nd growth in finished drinking-wter supplies. ACKNOWLEDGMENTS We re grteful to the mngement of the Americn Wter Works Service Co. nd the stff of the prticipting drinking wter tretment plnts for providing smples. The ssistnce of Andy Escher with the imge nlysis is pprecited. We lso thnk Ajib Singh, Rich Robisco, Dine Mthson, Bruce Lpke, Jerrie Beyrodt, nd Nncy Burns for their excellent technicl ssistnce. This project ws supported by funds from the Microbiologicl Tretment Brnch of the Drinking Wter Reserch Division, U.S. Environmentl Protection Agency, Cincinnti, Ohio (grnt CR810015). LITERATURE CITED 1. Americn Public Helth Assocition. 1985. Stndrd methods for the exmintion of wter nd wstewter, 16th ed. Americn Public Helth Assocition, Wshington, D.C. 2. Americn Wter Works Assocition Reserch nd Technicl Prctice Committee on Orgnic Contminnts. 1981. An ssessment of microbil ctivity on GAC. J. Am. Wter Works Assoc.
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