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Environ. Sci. Technol. 2010, 44, 3514–3519

Removal of Sediment and Bacteria from Water Using Green Chemistry AUDREY L. BUTTICE,† JOYCE M. STROOT,‡ DANIEL V. LIM,‡ PETER G. STROOT,§ AND N O R M A A . A L C A N T A R * ,† Department of Chemical and Biomedical Engineering, Department of Cell Biology, Microbiology, and Molecular Biology, and Department of Civil and Environmental Engineering, University of South Florida, Tampa, Florida 33620

Received October 8, 2009. Revised manuscript received March 9, 2010. Accepted March 13, 2010.

Although nearly all newly derived water purification methods have improved the water quality in developing countries, few havebeenacceptedandmaintainedforlong-termuse.Fieldstudies indicate that the most beneficial methods use indigenous resources, as they are both accessible and accepted by communities they help. In an effort to implement a material that will meet community needs, two fractions of mucilage gum were extracted from the Opuntia ficus-indica cactus and tested as flocculation agents against sediment and bacterial contamination. As diatomic ions are known to affect both mucilage and promote cell aggregation, CaCl2 was studied in conjunction and compared with mucilage as a bacteria removal method. To evaluate performance, ion-rich waters that mimic natural water bodies were prepared. Column tests containing suspensions of the sediment kaolin exhibited particle flocculation and settling rates up to 13.2 cm/min with mucilage versus control settling rates of 0.5 cm/min. Bacillus cereus tests displayed flocculation and improved settling times with mucilage concentrations lower than 5 ppm and removal rates between 97 and 98% were observed for high bacteria concentration tests (>108 cells/ml). This natural material not only displays water purification abilities, but it is also affordable, renewable and readily available.

Introduction The United Nations has estimated that 1.1 billion people lack access to potable water (1, 2). With so many people living on the brink of illness, a great deal of attention has been drawn to designing and implementing new and innovative methods of water purification, particularly in developing countries. Gradually, the goal of bringing safe water to the world has developed into a series of goals from educating to finding a method of purification that will be culturally accepted and sustainable (2, 3). In an attempt to circumvent problems associated with implementing purification methods based solely on technology, the Opuntia ficus-indica cactus (also known as the Nopal or Prickly Pear) indigenous to Mexico is being tested as a flocculating agent for waterborne contaminant removal. * Corresponding author phone: (813) 974-8009; fax: (813) 9743651; e-mail: [email protected]. † Department of Chemical and Biomedical Engineering. ‡ Department of Cell Biology, Microbiology, and Molecular Biology. § Department of Civil and Environmental Engineering. 3514

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Through simple extraction processes, two fractions of mucilage gum can be obtained from fresh cut Opuntia ficusindica pads including a Gelling Extract (GE) and Non-Gelling Extract (NE) (4, 5). Mucilage consist of up to 55 sugars, mainly arabinose, galactose, rhamnose, xylose, glucose, and uronic acids, the percentage of which varies with mucilage type (4, 6, 7). Literature has indicated that these extracts, particularly the GE, undergo property alterations including viscosity changes in the presence of diatomic ions such as Ca2+ (4, 7). Young et al. demonstrated that cactus mucilage is an effective tool for separating sediments (clay and mud particles) represented by kaolin, from deionized (DI) water suspensions. In previous studies, kaolin settling rates observed in columns treated with mucilage and aluminum sulfate (Al2(SO4)3), a common commercially used flocculant, were compared and mucilage was concluded to induce a greater increase in settling rate (5, 8, 9). Related tests also indicate that mucilage extracted from the Opuntia spp. acts as an efficient coagulant in surrogate turbid water (10). In this work, the efficiency of mucilage and Al2(SO4)3 to remove kaolin suspended in ion-rich environments was evaluated. Hard water (HW) and soft water (SW) were prepared and are two dominant water types found in drinking water sources. The mineral concentration and type found in the water dictates whether it is classified as hard or soft and depends mainly on the mineral content of the soil around the well (11, 12). Aside from sediment intrusion, another common problem associated with drinking water has been bacterial contamination. Even in more developed countries where purification and distribution systems are technologically advanced and water is closely monitored, cases of waterborne illnesses caused by bacteria are occasionally observed (13). In developing countries, issues with bacterial contamination are more severe. Bacillus cereus, a Gram-positive, sporeforming, non-pathogenic, soil-dwelling rod (∼1 by 3 µm), was chosen to study bacterial aggregation with mucilage for its ease of use and potential as a possible surrogate for waterborne bacteria with similar structural characteristics (14). Calcium chloride (CaCl2) solutions were evaluated alone and in conjunction with mucilage treatments to determine removal efficiency, as recent studies have suggested that diatomic ions promote cell aggregation (15, 16).

Materials and Methodology Pads were obtained from an Opuntia ficus-indica cactus, originally purchased from Living Stones Nursery in Tucson, AZ then replanted and cultivated in Tampa, FL. The pads were processed according to the flowchart protocol outlined by Goycoolea and Ca´rdenas with the following specifications and alterations (4). The pads were steamed then liquidized with a 10 speed Osterizer blender. The pH was adjusted to 7 using approximately 4 mL of 1 M NaOH solution and the solids separated using an accuspin 400 centrifuge (Fisher Scientific) and a 4-place swinging bucket Rotor (Fisher Scientific) with a centripetal force of 2522 ×g for 10 min. The precipitate was removed and used in the extraction of GE and the supernatant was reserved for the acquisition of NE. A 50 mM NaOH with 0.75% (w/w) sodium hexametaphosphate solution was added until the precipitate was covered and then stirred for 30 min. The pH was adjusted to 2 using ∼4 mL 0.12 M HCl solution, the mixture centrifuged, and the supernatant discarded. The precipitate was resuspended in DI water, and the pH was increased to 8 using ∼5 mL of 1 10.1021/es9030744

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M NaOH solution. This suspension was filtered via vacuum filtration with a filter cut from a 100% continuous filament, double knit polyester cloth (Berkshire, Co.). The portion of the original suspension that was reserved for the extraction of NE was mixed with 400 mL of 1 M NaCl solution and filtered using a #41 Whatman filter and a vacuum filtration system. The filtrates of both the NE and GE were separately mixed with acetone (1:1 v/v) and left overnight allowing water to evaporate. The recovered mucilage was removed and washed in isopropanol (1:1 v/v). The mucilage was spread upon sterile Petri dishs, dried, ground using a mortar and pestle, and stored on the benchtop in sealed containers. Prior to experimentation, the mucilage powder was dispersed in DI water using a Pyrex Tenbroeck tissue grinder (Fisher Scientific) with final stock concentrations of 500 ppm which were diluted accordingly during column inoculation. Unused mucilage suspension was stored in the refrigerator for future use. The two distinct fractions of mucilage gum were studied for their different characteristics and removal abilities. If produced and utilized in developing countries, combined mucilage, containing both NE and GE studied here in a control manner, can be obtained by boiling the cactus pads and using the mucilage released in the water. Simple column tests were used to evaluate the flocculation effects of the mucilage. The column contents (suspended “contaminant”, mucilage and desired water type) were mixed to 10 mL in 15 mL centrifuge tubes, vortexed and poured into column arrays which were observed over a period of time. Fisherbrand serological 10 mL pipettes with the tops snapped off and tips wrapped in parafilm, were used for all column arrays. Experiments were performed at least three times for reproducibility and statistical information was calculated using OriginLab Data Analysis and Graphing Software. Kaolin (hydrated aluminum silicate; Fisher Scientific, S71954) suspensions with final concentrations of 50 g/L were set up in the appropriate water type at least 24 h prior to the run of the experiment to allow kaolin particles thorough hydration time (5, 8, 9). A 1-mL portion of 0.01 M phosphate buffered saline (PBS) (Sigma) with a pH of 7.4 was added to the kaolin suspension in order to maintain the same solution background used in tests with B. cereus, which were suspended in PBS. In column tests of specific concentrations, kaolin has been observed to form a clear interface which was read every minute for up to 60 min, and the volume marker at the interface was recorded for later plotting. These plots were truncated where compression in the column began and the settling rate in cm/min was obtained. Bacillus cereus (ATCC 10876) was cultured overnight in Luria-Bertani (LB) media in an Orbital Incubator Shaker, model Gyromax 727 (Amerex Instruments Inc.), operated at 35 °C and 200 rpm. Initial bacteria cell counts were performed prior to column inoculation using a cellometer cell counting chamber (Nexcelom Bioscience). The bacteria were washed once in PBS using a mini vortexer and a cell pellet collected using a centripetal force of 2522 ×g for 5 min. Cells were then resuspended in 15 mL of PBS to a final stock solution of 109 cells/mL, resulting in a final column cell count of 108 cells/mL. Unlike kaolin, B. cereus did not form a visible interface while settling. The time when flocs began to appear as small white flecks in the otherwise turbid water to the time that the flocs ceased to fall was recorded. Box plots were used to represent settling times, where the bottom of the box indicates the start time of the floc formation, while the top indicates the time when flocs were no longer falling in the column. The dotted lines represent the start (lower line) and completion time (upper line) of the control columns containing only CaCl2, as the addition of Ca2+ alone does result in flocculation. Anhydrous CaCl2 (MP Biomedicals) was suspended in the synthetic water of choice and filtered

using a 0.22 µm cellulose acetate tube top filter (Corning) to remove any bacterial contaminants. Once the flocs had completed their descent, a 1 mL sample was taken from the top of the column and final cell counts were evaluated using standard microbiology plate counting techniques on LB media (17-19). The resulting bacteria concentration was compared to the initial concentration and a removal rate was obtained. Synthetic HW and SW were prepared as outlined by Smith, Davison and Hamilton-Taylor (12). Three (SW) to Four (HW) stock solutions consisting of MgCl2 · 6H2O, CaCl2 · 6H2O, Ca(NO3)2 · 4H2O, Na2SO4, KHCO3, NaHCO3, K3PO4, MgSO4 (HW only), and CaO were prepared separately to avoid supersaturating the water and were then mixed accordingly. Chemicals used were of reagent grade from Fisher Scientific and Arcos. Gases used in the production process (N2, CO2 and Air) for pH changes and oxygen removal were high pressure, ultrapure gases from Airgas, Inc. Equilibrium with the atmosphere was achieved by bubbling the water with compressed air for 5 h and the final pH was determined to be 7.43 for the SW and 8.34 for the HW. Both synthetic waters were filtered using a 0.20 µm Cellulose Nitrate membrane filter (Fisher Scientific) to remove any unwanted bacterial contamination. Dynamic Light Scattering (DLS) outputs were obtained from a DLS manufactured by Malvern Instruments and were used to evaluate kaolin particle sizes using standard cuvettes. Transition Electron Microscope (TEM) images were obtained using a Morgagni 268D TEM with Formvar/Carbon 150 Mesh Copper grids from Electron Microscopy Sciences (FCF150-Cu-50) and were used to study mucilage structure, kaolin particle size, and flocs observed in kaolin columns. A 20-µL sample of the mucilage stock solutions (500 ppm) was deposited on the grid and soaked for 5 min. Remaining liquid was removed using the tip of a KimWipe and the grid was dried overnight. Samples obtained from the top 1 mL of kaolin columns were prepared for imaging following the same technique. Atomic Force Microscope (AFM) scans of GE and NE were obtained using a XE-100 AFM purchased from PSIA 100 and were scanned on mica (ASTM V-1 quality) purchased from Axim Mica using aluminum TAP300Al cantilevers (average force constant of 40 N/m) purchased from Budget Sensors using non-contact mode. Light microscope images of kaolin and B. cereus were produced using 10 µL of sample and a coverslip. Untreated and treated kaolin samples were obtained from the kaolin supernatant interface and B. cereus samples were obtained using small valves attached to the bottom of the columns.

Results and Discussion Mucilage Structure. Mucilage structure (500 ppm stock solution) was studied using both AFM (scanned areas of 2 × 2-µm x-y and 0.5 × 0.5 µm x-y) and TEM (Figure 1). Scans of GE displayed an orderly chain-like structure with similar orientation angles, whereas those of NE showed a denser net-like structure with a uniform distribution. Maximum heights of 2.011 nm (GE scans) and 1.42 nm (NE scans) were recorded by the AFM. The structural differences were similar to those observed with the TEM (provided as inserts in Figure 1A, B) and potentially contribute to the different removal abilities observed between the two fractions of mucilage. Kaolin Removal with Cactus Mucilage. Kaolin particle size was evaluated using DLS and TEM. A sharp peak indicated that there was a narrow size distribution and an average particle size of 518 ( 30 nm was determined. Kaolin suspensions in HW, SW, and DI water were treated with 0 to 100 ppm GE and NE, which demonstrated three characteristics of the mucilage-induced settling (Figure 2). First, it was observed that as mucilage concentration VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. AFM scans (2 × 2-µm x-y and 0.5 × 0.5-µm x-y areas) and TEM images as inserts (500 nm scale bar) of the 500 ppm GE (A) and the NE (B) stock solutions suspended in DI water.

FIGURE 2. The effects of NE (A), GE (B), and Al2(SO4)3 (C, Hard Water only) on the settling rate of kaolin (50 g/L) suspended in Hard (2), Soft (b), and DI water (0). increased, for both NE and GE, so did the settling rate of the kaolin regardless of water type. Initially, the relationship between concentration and settling rate appeared to be linear. However, as the concentration of mucilage increased, a point 3516

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was observed where the kaolin’s settling rate began to level off, indicating that an optimal concentration of mucilage exists and an equilibrium value had been reached. Second, from these plots the influence of ion-rich water on the mucilage was discerned. It was observed that with no mucilage treatment, all kaolin suspensions settled at a rate close to 0.5 cm/min, indicating that any differences observed among the treated suspensions were primarily due to ion interaction with the mucilage. With NE concentrations of 20-75 ppm, settling rates differed heavily depending on water type (Figure 2A). By analyzing the linear portions of the plots (Figure 2A,B) and comparing the settling rates with respect to mucilage concentration (Table 1), the influence of ionrich water is demonstrated. Treatment with NE produced average increases of 39% between DI water and SW suspensions, 38% between SW and HW suspensions, and 62% increases among suspensions in HW compared to DI water. In kaolin suspensions treated with GE, the settling rate/ mucilage concentration differences among water types were not observed to be as severe. An average increase amid suspensions of DI water and SW was 13% while the increase between SW and HW was determined to be 15%. Average increases of 27% were observed between DI water and HW suspensions. In HW columns treated with 100 ppm NE, an average settling rate of 13.2 cm/min was observed, while treatment with 100 ppm GE resulted in an average settling rate of 11 cm/min. Suspensions of SW and DI water displayed similar differences. Average differences in settling rate/ mucilage concentration between GE and NE treated kaolin were determined to be 6% in DI water, 33% in SW and 51% in HW suspensions. Efficiency variation between the mucilage types is likely attributed to the previously mentioned structural distinctions observed in the AFM and TEM scans (Figure 1). Due to the small size of the kaolin particles, the tighter packed mucilage structure exhibited by the NE could potentially be more efficient at entrapping and aggregating kaolin particles than the less dense more spread out structure of the GE. Also shown in Figure 2 is the settling rate of mucilage columns treated with Al2(SO4)3. Kaolin suspended in HW was used for comparison. Experiments with Al2(SO4)3 concentrations ranging from 0-500 ppm show little to no increase in settling rate when compared to the control indicating that, at low concentrations of treatment, mucilage is a more effective flocculating agent for sediment contaminated waters. At higher mucilage concentrations (∼15-100 ppm), changes in the kaolin particle size were visually observed from the resulting flocculation (Figure 3A). While Figure 3A depicts kaolin suspensions in DI water; the same changes were observed in columns of SW and HW. TEM images of samples obtained from kaolin suspensions with no treatment (Figure 3B), 50 ppm GE (Figure 3C), and 50 ppm NE (Figure 3D) provide microscopic confirmation of the flocculation observed in Figure 3A. All samples imaged from the control columns displayed similar results to those seen in Figure 3B. Single kaolin particles were found spread out in the sample, indicating that no flocculation had taken place in the absence of mucilage. In samples taken from columns containing mucilage (Figure 3C,D), flocs of kaolin particles were observed, verifying the presence of large particle aggregates with GE and NE treatment. Similar flocs were observed in columns of HW and SW. TEM images of kaolin treated with Al2(SO4)3 showed some flocs of kaolin, although not to the degree observed in Figure 3C,D. Light microscope images (included as inserts in Figure 3B-D) also demonstrate no flocculation in the control column and heavy particle aggregation in GE (50 ppm) and NE (50 ppm) treated columns. Bacillus cereus Removal with Cactus Mucilage. The changes in flocculation times with respect to CaCl2 con-

TABLE 1. Settling Rates with Respect to Mucilage Concentration (cm/min/ppm) for GE and NE Treatment of 50g/L Kaolin Suspensions in DI water, SW, and HW mucilage type, water type

change in settling rate/mucilage concentration (cm/min/ppm)

mucilage type, water type

change in settling rate/mucilage concentration (cm/min/ppm)

GE, DI water GE, SW GE, HW

0.11 ( 0.00 0.13 ( 0.01 0.15 ( 0.00

NE, DI Water NE, SW NE, HW

0.12 ( 0.01 0.19 ( 0.02 0.31 ( 0.02

centration [10 to 35 mM] were evaluated in HW columns treated with 2, 3, 4, and 5 ppm NE (Figure 4). As CaCl2 concentration increased, the settling time of the control and the experimental column also increased. As NE concentration increased by 1 ppm, columns with low CaCl2 concentrations

(10, 15, and 20 mM) were observed to progressively decrease in efficiency, while columns with higher CaCl2 concentrations (30 and 35 mM) maintained similar settling times. With the addition of 5 ppm NE, columns containing 10 mM and 15 mM CaCl2 did not form flocs at all. A range of mucilage concentrations were tested to evaluate the effects of mucilage on the settling time. The settling rates of B. cereus with 0.5-50 ppm NE and GE were obtained from columns containing HW and 20 mM CaCl2 (Figure 5). NE concentrations of 0.5 to 3 ppm demonstrated flocculation that both started and finished faster than the control (Figure 5B). With 4 ppm NE the settling time increased although, the mucilage still displayed faster setting than the control column. With NE concentrations of 5 ppm, the experimental column took longer than the control to complete its settling though the flocs began to form slightly faster. At NE concentrations of 10, 25, and 50 ppm, signs of flocculation occurred after the control column had finished settling, if at all. This indicates that there is an optimal concentration at which the mucilage no longer produced flocs faster than the control columns; in this case, concentrations above 4 ppm. By comparing these results to the settling times of GE (Figure

FIGURE 3. Digital image of Kaolin (50 g/L) in DI water (A) with no treatment (1), 25 ppm GE (2), and 50 ppm GE (3). TEM and light microscope (inserts) images of kaolin (50 g/L) in DI water with no treatment (B), 50 ppm GE (C), and 50 ppm NE (D). TEM and light microscope scale bars indicative of 500 nm and 20 µm, respectively.

FIGURE 4. Effects of CaCl2 concentration on settling times in Hard Water suspensions of Bacillus cereus (108 cells/mL) treated with 2 ppm NE (A), 3 ppm NE (B), 4 ppm NE (C), and 5 ppm NE (D). Boxes indicate the flocculation start (bottom line) and end (top line) times of suspensions treated with the addition of NE. Dashed lines represent the start (bottom line) and end (top line) time of flocculation in control columns.

FIGURE 5. Box plots of Bacillus cereus (108 cells/mL) settling rates in Hard Water with 20 mM CaCl2, with respect to the mucilage concentration (ppm) in the column. The boxes indicate the flocculation start (bottom line) and end (top line) time in columns treated with GE (A) and NE (B). Dashed lines indicate the flocculation start (bottom) and end (top) times of columns containing only 20 mM CaCl2. VOL. 44, NO. 9, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Light microscope images of Bacillus cereus (108 cells/mL) in Hard Water with no treatment (A), 20 mM CaCl2 (B), 20 mM CaCl2 and 2 ppm GE (D), and 20 mM CaCl2 and 2 ppm NE (F). Images also shown of Bacillus cereus suspensions in Soft Water with 40 mM CaCl2 (C), 40 mM CaCl2 and 2 ppm GE (E), and 40 mM CaCl2 and 2 ppm NE (G). Scale bars indicative of 10 µm. 5A), slight differences the mucilage fractions were observed. Contrary to the observations described above, suspensions treated with 5 ppm GE caused flocculation that started and completed well within the time that the control had completed. In addition, at a GE concentration of 10 ppm flocculation started before the control had completely settled, whereas the NE columns at this concentration were not observed to form flocs in this time range. Like the columns treated with NE, GE concentrations of 25 and 50 ppm showed no signs of flocculation even after the control column had completed. An important difference between the mucilage’s ability to aggregate kaolin particles compared to bacteria was that there existed a concentration where the bacteria no longer reacted to the mucilage in a positive manner. In columns containing kaolin, increases in mucilage concentration resulted in higher settling rates, but no concentrations were observed to restrict settling as seen in suspensions of B. cereus. Also, in columns containing kaolin, the NE appeared to cause 3518

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larger increases in settling rates while the GE seems to work slightly better as a treatment for B. cereus suspensions. This is most likely attributed to the structural differences of the mucilage types discussed earlier. Experiments concerning the removal of B. cereus suspended in SW have been observed to required higher concentrations of CaCl2 to achieve flocculation rates similar to those observed in HW most likely due to the ion differences in the waters. Microscopic B. cereus floc characteristics were studied in the presence of CaCl2 alone and in conjunction with mucilage (Figure 6). Control images from HW control columns indicated that without treatment, aggregation did not occur, and the bacteria were freely floating in the solution (Figure 6A). Comparisons of flocs obtained from HW (with 20 mM CaCl2) and SW columns (with 40 mM CaCl2) alone were performed (Figure 6B,C). The addition of GE (2 ppm) or NE (2 ppm) to suspensions containing the above CaCl2 concentrations resulted in the flocs portrayed in Figure 6D-G. From these images, it was observed that, although both concentrations of CaCl2 did cause flocculation, the flocs formed do not appear as organized, stable, or large as those formed in the presence of mucilage. The size difference observed here is most likely responsible for the increased settling rate in the columns containing mucilage, as the density will naturally cause higher settling rates. Bacteria removal rates associated with 0-5 ppm NE and GE were determined to be 97.71% ( 1.37% and 98.32% ( 0.27% respectively, in SW with 40 mM CaCl2. The control columns containing only CaCl2 produced similar removal rates of 98.21 ( 0.13% but were found to take longer to form flocs and settle than suspensions treated with mucilage. Although these removal rates are high, the level of bacteria remaining in the columns renders the water still unsafe to drink. This is due to the initial cell concentration (108 cell/mL) which would not be typically observed in the real world, but was used to obtain a visual indicator of flocculation and removal. Future experimentation will involve optimizing parameters for bacterial removal at lower levels of contamination. The results discussed in this work demonstrate the potential of mucilage extracted from the Opuntia ficus-indica as a flocculation agent for sediment and bacterial contamination in ion rich waters. The cactus’ prevalence, affordability, and cultural acceptance make it an attractive natural material for water purification technologies that could be beneficial in Mexico and around the world. The different mucilage fractions have been observed to provide diversity both in their structural features as well as in their reaction to the natural ion concentration in the water they are treating.

Acknowledgments The authors thank the National Science Foundation award (CBET 0808053) and the Florida Center of Excellence for Biomolecular Identification and Targeted Therapeutics (FCoEBITT) Seed Grant for partial student support. We thank the State of Florida-USF partnership-Sustainable Communities: Water award, and The Integrating global capabilities into STEM education: Critical technologies and strategies for meeting the United Nation’s Millennium Development Goals on water and sanitation Grant for their financial support. The authors thank Dr. Vinay Gupta for the use of the DLS apparatus under the NER award (CTS-0508309) from National Science Foundation. In addition, the authors thank Betty Lorramm and the Integrative Biology Microscopy Core Laboratory at USF for assistance and discussion regarding the TEM, as well as Samuel DuPont, Jr., Dr. Jeffy Jimenez, and Leigh West (FCoE-BITT) for instrumental assistance and support.

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(9) Young, K. A., Alcantar, N. A., Cunningham, A. Cactus goo purifies water. Science News 2005. (10) Miller, S. H.; Fugate, E. J.; Craver, V. O.; Smith, J. A.; Zimmerman, J. B. Toward understanding the efficacy and mechanism of Opuntia spp. as a natural coagulant for potential application in water treatment. Environ. Sci. Technol. 2008, 42, 4274–4279. (11) Alth, M., Alth, C. Wells and Septic Systems, 2nd ed.; TAB Books: Blue Ridge Summit, PA, 1992. (12) Smith, E. J.; Davison, W.; Hamilton-Taylor, J. Methods for preparing synthetic freshwaters. Water Res. 2002, 36, 1286–1296. (13) Hrudey, S. E.; Hrudey, E. J. Published case studies of waterborne disease outbreaks - Evidence of a recurrent threat. Water Environ. Res. 2007, 79, 233–245. (14) Hardwood, C. Bacillus; Plenum Press: New York, 1989. (15) Larsen, N.; Nissen, P.; Willats, W. G. T. The effect of calcium ions on adhesion and competitive exclusion of Lactobacillus ssp and E. coli O138. Int. J. Food Microbiol. 2007, 114, 113–119. (16) Zita, A.; Hermansson, M. Effects of ionic-strength on bacterial adhesion and stability of flocs in a waste-water activated-sludge system. Appl. Environ. Microbiol. 1994, 60, 3041–3048. (17) Bauman, R. W. Microbiology; Pearson Education, Inc.: San Francisco, CA, 2004. (18) Ingraham, J. L.; Ingraham, C. A. Introduction to Microbiology, 2nd ed.; Brooks/Cole: Pacific Grove, CA: 2000. (19) Lim, D., Microbiology, 3rd ed.; Kendall/Hunt: Dubuque, IA, 2003.

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