pubs.acs.org/Langmuir © 2010 American Chemical Society
SWNT-MWNT Hybrid Filter Attains High Viral Removal and Bacterial Inactivation Anna S. Brady-Estevez, Mary H. Schnoor, Seoktae Kang,† and Menachem Elimelech* Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06520-8286, United States. † Current address: Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta, Canada T6G 2W2 Received September 20, 2010. Revised Manuscript Received November 3, 2010 We describe the concept and demonstrate the efficacy of a novel SWNT-MWNT hybrid filter for the removal and inactivation of microbial pathogens from water. The filter is composed of a thin SWNT layer (0.05 mg cm-2) on top of a thicker MWNT layer (0.27 mg cm-2) supported by a microporous support membrane. The SWNT-MWNT filter exhibits high log removal of several model viruses (MS2, PRD1, and T4 bacteriophages) by depth filtration, which predominantly takes place in the thicker and more uniform MWNT layer. The filter removes all bacteria by a sieving mechanism, with the top SWNT layer providing high levels of inactivation of model bacteria (Escherichia coli K12 and Staphylococcus epidermidis), as well as microbes from river water and treated wastewater effluent. The dual-layer SWNT-MWNT filter lays the framework for new possibilities in point-of-use water filtration.
Introduction The lack of safe drinking water is one of the most serious challenges of the twenty-first century.1 At present, over one billion people lack access to clean water, nearly all of them in developing countries. Waterborne viral and bacterial pathogens are the primary cause for the vast majority of diarrheal diseases, accounting for over two million child deaths per year.2 Waterborne diseases also inflict significant economic burden through the loss of productivity in the workforce and increasing national health care costs.3 Thus, there is a need to develop new point-of-use technologies for the effective removal and inactivation of waterborne microbial pathogens. Nanotechnology has the potential to provide solutions to such problems afflicting developing countries.4,5 Our previous development of carbon nanotube-based filters has focused on micrometer-size deposit layers of either singlewalled carbon nanotubes (SWNTs)6 or multiwalled carbon nanotubes (MWNTs)7 that serve as a filter matrix supported by a microporous membrane. Comparison between the performance of the MWNT and SWNT filters has revealed differences in surface morphology and viral removal, with the MWNT filter exhibiting significantly higher viral removal on a per unit mass basis.7 Although the permeability was slightly lower for the MWNT *Corresponding author. E-mail:
[email protected]. Phone: þ1 (203) 432-2789. (1) Shannon, M. A.; Bohn, P. W.; Elimelech, M.; Georgiadis, J. G.; Marinas, B. J.; Mayes, A. M. Science and technology for water purification in the coming decades. Nature 2008, 452(7185), 301–310. (2) Elimelech, M. The global challenge for adequate and safe water. J. Water Supply: Res. Technol.;AQUA 2006, 55(1), 3–10. (3) Montgomery, M. A.; Elimelech, M. Water and sanitation in developing countries: Including health in the equation. Environ. Sci. Technol. 2007, 41(1), 17–24. (4) Diallo, M.; Savage, N. Nanoparticles and water quality. J. Nanopart. Res. 2005, 7, 325–330. (5) Hammond, P. T. Solutions for the Developing World. ACS Nano 2009, 3(9), 2431–2432. (6) Brady-Estevez, A. S.; Kang, S.; Elimelech, M. A single-walled-carbonnanotube filter for removal of viral and bacterial pathogens. Small 2008, 4(4), 481–484. (7) Brady-Estevez, A. S.; Schnoor, M. H.; Vecitis, C. D.; Saleh, N. B.; Elimelech, M. Multiwalled Carbon Nanotube Filter: Improving Viral Removal at Low Pressure. Langmuir 2010, 26(18), 14975–14982.
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filter, high removal of viruses was still achieved while operating in the microfiltration pressure range, in contrast with microfiltration systems that would be ineffective for viral removal. The removal and inactivation of microbes have previously been demonstrated on the SWNT filter.6 Numerous studies have addressed the cytotoxicity of a range of carbon-based nanomaterials.8-16 Cell membrane lysis has also been imaged for the case of E. coli on SWNT filters6 and MWNTs.8 Studies have shown that SWNTs are more effective than MWNTs in cell inactivation.8,16 Although cell inactivation is not yet fully understood, several mechanisms have been proposed, including physical membrane damage,10 physical piercing,17 disruption of metabolic (8) Kang, S.; Herzberg, M.; Rodrigues, D. F.; Elimelech, M. Antibacterial effects of carbon nanotubes: Size does matter. Langmuir 2008, 24(13), 6409–6413. (9) Kang, S.; Mauter, M. S.; Elimelech, M. Microbial Cytotoxicity of CarbonBased Nanomaterials: Implications for River Water and Wastewater Effluent. Environ. Sci. Technol. 2009, 43(7), 2648–2653. (10) Kang, S.; Pinault, M.; Pfefferle, L. D.; Elimelech, M. Single-walled carbon nanotubes exhibit strong antimicrobial activity. Langmuir 2007, 23(17), 8670– 8673. (11) Pulskamp, K.; Diabete, S.; Krug, H. F. Carbon nanotubes show no sign of acute toxicity but induce intracellular reactive oxygen species in dependence on contaminants. Toxicol. Lett. 2007, 168(1), 58–74. (12) Zhu, Y.; Ran, T. C.; Li, Y. G.; Guo, J. X.; Li, W. X. Dependence of the cytotoxicity of multi-walled carbon nanotubes on the culture medium. Nanotechnology 2006, 17(18), 4668–4674. (13) Magrez, A.; Kasas, S.; Salicio, V.; Pasquier, N.; Seo, J. W.; Celio, M.; Catsicas, S.; Forro, L. Cellular toxicity of carbon-based nanomaterials. Nano Lett. 2006, 6(6), 1121–1125. (14) Arias, L. R.; Yang, L. J. Inactivation of Bacterial Pathogens by Carbon Nanotubes in Suspensions. Langmuir 2009, 25(5), 3003–3012. (15) Yang, C.; Mamouni, J.; Tang, Y.; Yang, L. Antimicrobial Activity of Single-Walled Carbon Nanotubes: Length Effect. Langmuir 2010, 26(20), 16013– 16019. (16) Jia, G.; Wang, H. F.; Yan, L.; Wang, X.; Pei, R. J.; Zhao, Y. L.; Guo, X. B. Cytotoxicity of carbon nanomaterials: Single-wall nanotube, multi-wall nanotube, and fullerene. Environ. Sci. Technol. 2005, 39(5), 1378–1383. (17) Narayan, R. J.; Barry, C. J.; Brigmon, R. L. Structural and biological properties of carbon nanotube composite films. Mater. Sci. Eng., B 2005, 123(2), 123–129. (18) Nel, A. N.; Xia, T.; Madler, L.; Li, N. Toxic Potential of Materials at the Nanolevel. Science 2006, 311(5761), 622–627. (19) Manna, S. K.; Sarkar, S.; Barr, J.; Wise, K.; Barrera, E. V.; Jejelowo, O.; Rice-Ficht, A. C.; Ramesh, G. T. Single-Walled Carbon Nanotube Induces Oxidative Stress and Activates Nuclear Transcription Factor-κB in Human Keratinocytes. Nano Lett. 2005, 5(9), 1676–1684.
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pathways,18 and oxidative stress.11,18-20 Recent DNA microarray work has shown up-regulation of genes that supports inactivation by cell membrane damage and oxidative stress.8 An understanding of the advantages and limitations of MWNT and SWNT filters in terms of cost, permeability, viral removal, and microbial removal and inactivation informed the design of the SWNT-MWNT hybrid filter. In this paper we demonstrate the efficacy of the SWNT-MWNT filter in the removal and inactivation of a wide range of viruses and bacteria. In designing this hybrid filter, we attempted to leverage the strengths of each nanotube type. MWNTs were selected to compose the majority of the depth of the filter matrix due to the MWNT matrix’s enhanced adsorption of viruses, low cost, and reasonably high permeability. A thin upper layer of SWNTs was then deposited onto the MWNT matrix to take advantage of the antibacterial properties of SWNTs.
Materials and Methods SWNT-MWNT Filter Preparation. The lower layer of the SWNT-MWNT filter comprised commercially available multiwalled carbon nanotubes (MWNTs) (NanoTechLabs, Inc., Yadkinville, NC). These MWNTs were used in prior work and had been previously analyzed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), Raman spectroscopy, energy dispersive X-ray (EDX), and thermogravimetric analysis (TGA).21 We deposited MWNTs on a 5-μm pore size PTFE membrane (Omnipore filters, Millipore) by a sonication and filtration procedure similar to the preparation of the SWNT6 and MWNT7 filters. MWNTs were added at a concentration of 0.5 mg/mL to dimethyl sulfoxide (DMSO). The suspension was then sonicated for 15 min at a power output of 50 W with the Sonifier 450 probe sonicator (Branson model 102) to debundle MWNTs and achieve a more uniform dispersion. All MWNT suspensions were allowed time to cool but were used within a few hours of preparation to ensure uniform dispersion and consistent filter performance. Bath sonication of the MWNT suspension was also performed for 10 s immediately prior to filter deposition to disrupt any aggregates. Deposition of MWNTs from a 5-mL solution was achieved by vacuum filtration through the PTFE membrane to attain a loading of 0.27 mg/cm2 MWNTs on the base filter. The thin upper layer of the SWNT-MWNT hybrid filter was composed of commercially available, unfunctionalized singlewalled carbon nanotubes (SWNTs) with a purity of greater than 95% (w/w) SWNTs (Stanford Materials, SWNT-90, lot #082106). The manufacturer reports that as-received SWNTs had lengths of 10-20 μm, an average outer diameter of 1.2 nm, and a specific surface area of 407 m2 g-1. We analyzed the SWNTs in a previous study via TEM, Raman spectroscopy, EDX, and TGA.9 The as-received SWNTs were suspended in DMSO to form a dilute SWNT suspension (0.1 mg/mL) to ensure uniform coverage of the SWNT layer. The SWNT suspension was sonicated for 15 min with the Sonifier 450 probe sonicator (Branson model 102) at a power output of 50 W. We allowed the sonicated suspension to cool and then resonicated in a bath sonicator for 10 s prior to the layering procedure, which consisted of vacuum deposition of 5 mL of the 0.1 mg/mL SWNTs onto the MWNT layer that overlaid the 5-μm pore size PTFE membrane. After the SWNT layer of the nanotubes was laid, the dual filter was rinsed with 50 mL of ethanol followed by 50 mL of deionized water to remove residual DMSO. (20) Shvedova, A.; Castranova, V.; Kisin, E.; Schwegler-Berry, D.; Murray, A.; Gandelsman, V.; Maynard, A.; Baron, P. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity using Human Keratinocyte Cells. J. Toxicol. Environ. Health, Part A 2003, 66(20), 1909–1926. (21) Kang, S.; Mauter, M. S.; Elimelech, M. Physicochemical determinants of multiwalled carbon nanotube bacterial cytotoxicity. Environ. Sci. Technol. 2008, 42(19), 7528–7534.
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SWNT-MWNT Filter Characterization of Morphology and Permeability. The permeability of the prepared SWNTMWNT filters was evaluated by measuring permeate water flux as a function of transmembrane pressure drop as described previously.6 The surface morphology and cross-sectional views of the SWNT-MWNT filter were studied under various magnifications using field emission scanning electron microscopy (FESEM) (Hitachi S-4500, Hitachi). Additional FE-SEM images of MWNT and SWNT filters were also taken to visually compare with the dual layer filter. Viral Preparation. The present study aimed to demonstrate that viral removal on the filter is attained for several types of viruses. Three model bacteriophages;MS2, PRD1, and T4; were purchased from the American Tissue Culture Collection (ATCC), along with their host bacteria, E. coli 15597, E. coli 13706, and E. coli 11303, respectively. MS2 and PRD1 viral stocks were suspended at various dilutions in DI water and refrigerated at 4 °C until experiments were performed. A more highly concentrated stock of T4 was attained by injecting the virus into a suspension of E. coli 11303 host bacterium (suspended in tryptic soy broth) and allowing the phage to infect the bacteria overnight at 37 °C for viral replication. The lysed bacterial cells were then centrifuged to separate out the T4 viral supernatant, which was then filtered through a 0.22-μm pore size membrane to remove remaining cell debris. All viral stocks were diluted in 10 mM NaCl at pH 5.5 immediately prior to the filtration experiments. Viral Filtration. Viral filtration on the SWNT-MWNT filter was performed in the same manner as in our previous work.6 Briefly, the SWNT-MWNT filter was first preconditioned by flowing a 10 mM NaCl background solution at pH 5.5 (without virus) at constant flux using a peristaltic pump. Viral seed stock was then spiked into the 10 mM NaCl solution and pumped through the filter system. We performed all experiments at a water flux or filter approach velocity of 160 or 260 L m-2 h-1 as indicated. Filter permeate samples were collected in an autoclaved glass tube, and the viral concentration was determined by the plaque forming unit (PFU) method (U.S. Environmental Protection Agency Method 1601).22 For PFU measurement, E. coli (ATCC 15597, 13706, and 11303 for MS2, PRD1, and T4 bacteriophage, respectively) was used as the viral host bacterium and mixed with the various dilution tubes of the filter permeate with molten soft agar (0.7% TSA). The mixture was then poured onto presolidified tryptic soy agar (TSA) plates that, after overnight incubation, yielded plaque forming units of 20-200 PFU/plate to quantify virus presence. All experiments were, at a minimum, duplicated at each dilution and performed on two or more filters at each condition. Room temperature (23 °C) was maintained throughout the viral filtration experiments. Bacterial Preparation. Escherichia coli K12 and Staphylococcus epidermidis were selected to model Gram-negative and Gram-positive bacteria, respectively. The bacterial cultures had each been previously grown (separately) from a single colony of their plated culture. A sterile wire transfer loop was used to inoculate bacteria from discrete homogeneous colonies into individual tubes of LB. These LB tubes were then incubated overnight to the stationary growth phase (37 °C). Glycerol (25%, v/v) was then added to the bacterial suspension to allow freezing of the bacterial culture and storage at -80 °C. These frozen stocks were later used to seed LB for overnight incubation (37 °C) to the stationary phase, followed by a 50-fold dilution into LB to allow growth over ∼3.5 h incubation to the log growth phase. At this exponential growth phase, the E. coli and S. epidermidis were diluted to concentrations of 4 104 cells/mL into 50 mL of 0.9% (154 mM) or 10 mM NaCl in preparation for bacterial inactivation experiments. (22) Telliard, W. A. Method 1601: Male-specific (FR) and Somatic Coliphage in Water by Two-step Enrichment Procedure. In United States Environmental Protection Agency, 2001.
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Article Table 1. Properties of Carbon Nanotubes, Viruses, and Bacteria Used
CNT
diameter (nm)
length (μm)
residual metals (%)
Raman G/D ratio
MWNTa SWNTb
17 ( 9 1.2 ( 0.3
91 ( 21 17.8 ( 5.6
6.7 6.0
1.47 31.0
virus
diameter (nm)
nucleic acids
isoelectric point
MS2 PRD1 T4
25c 62g head 120 86k
ss-RNAd ds-DNAh ds-DNAl
3.9e 3-4,i 4.2j 4-5m
bacteria
diameter (μm)
Gram staining
structure icosahedralf sphericalj prolate capsid (head) and tail with contractile sheath, 6 long tail fibersl shape
class
0.5 2 D L Gram (-) rod-shaped gamma-Proteobacteria E. colin 1 Gram (þ) spherical Cocci S. epidermidiso a Kang et al.21 b Kang et al.9 c Hu et al.; Yuan et al.27,28 d Vi~ nuela et al.29 e Overby et al.; Pham et al.30,31 f Golmohammadi et al.32 g Pieper et al.33 h Olsen et al.34 i Harvey et al.35 j Dowd et al.36 k Leiman et al.37 l Fokine et al.38 m Gerba.39 n Trueba and Woldringh.40 o Parisi.41
Wastewater and River Water Samples. Microorganisms naturally present in the environment were modeled by wastewater effluent and river water samples. Wastewater was obtained from a local wastewater treatment plant (Wallingford, CT). The secondary wastewater effluent (from a rotating biological contactor) was collected prior to the disinfection stage at the treatment plant. Wastewater effluent was divided into two samples in the lab: (i) ascollected wastewater effluent that contained microorganisms and (ii) wastewater effluent that had been filtered through a 0.45-μm membrane for removal of suspended matter. The original wastewater effluent was then diluted 5-fold using the filtered wastewater effluent to avoid clogging of the CNT-hybrid filter and allow better imaging of individual microorganisms. The wastewater had a measured pH of 6.9 and an ionic conductivity of 279 μS (YSI Model 32). Total organic carbon (TOC) and dissolved organic carbon (DOC) (TOC analyzer, TOCVCSH, Shimadzu) were measured as 25.56 mg/L and 16.86 mg/L, respectively, while UV absorption at UV 254 nm (1 cm cell) gave a reading of 0.09 (UV/vis spectrophotometer model 8453, HP). Other typical solution chemistry characteristics of the wastewater effluent are given in our recent publication.9 River water samples were also obtained from the Mill River (New Haven, CT; coordinates 41.326046, -72.909019). The river water samples were transported back to the lab within 15 min of collection and maintained at room temperature (23 °C). The river water had a measured pH of 7.5 and an ionic conductivity of 147 μS. TOC and DOC levels of 15.41 mg/L and 10.39 mg/L, respectively, were measured, along with a UV absorption of 0.14 at 254 nm. It was possible to compare the amount of humic-like substances in the environmental samples by normalizing the UV reading at 254 nm by the DOC to obtain the specific UV absorbance (SUVA). The SUVA was 2.5 times higher for the river water than the wastewater, indicating higher concentrations of humic-like substances in the river sample. Other typical solution chemistry characteristics of the river water are given in our recent publication.9 Bacterial Inactivation Assays. Bacterial inactivation rates on the filters were measured by a standard fluorescent assay, which confirmed the membrane integrity of the cells. Bacterial suspensions were filtered through the SWNT-MWNT or MWNT filters. All bacterial cells were sieved and retained at the top layer of the filter. The filters were then incubated in the dark for the indicated times (0.5 and 2 h) in saline solution (154 or 10 mM NaCl) at 37 °C. After incubation, cells were shielded from light and stained with propidium iodide (PI, 50 μM) for 15 min and then counterstained with SYTO-9 for 5 min. The cells on the filter were imaged using an epifluorescence microscope (Olympus). For each filter, 10 representative images were taken, and percentage inactivation was determined by direct counting of PI-stained inactivated cells divided by the total number of cells that were stained with PI plus SYTO-9. More details on this technique are given elsewhere for Langmuir 2010, 26(24), 19153–19158
bacterial monocultures6,10 and microbes present in the river water and wastewater effluent samples.9
Results and Discussion SWNT-MWNT Filter Characteristics. Development of the SWNT-MWNT filter leveraged knowledge from previous studies involving MWNT and SWNT filters. Information on the properties of the SWNTs and MWNTs, as determined by SEM, TEM, EDX, TGA, and Raman spectroscopy, has been provided previously.9,21 Additional data on the electrophoretic mobilities of the SWNTs and MWNTs over a wide range of solution chemistries are also provided in our recent publications.7,23 Key properties of the SWNTs and MWNTs relevant to this work are summarized in Table 1. The permeability of the SWNT-MWNT filter indicated a highly porous matrix that operated in the microfiltration range of pressures. The SWNT-MWNT filter demonstrated a permeability of 9361 ( 550 L m-2 h-1 bar-1. This value was comparable to the permeabilities of the MWNT and SWNT filters, namely 11 900 ( 435 and 13 800 ( 320 L m-2 h-1 bar-1, respectively,6,7 which is in the range of microfiltration systems. One difference in the preparation of the SWNT-MWNT filter that may have influenced its permeability is that, in addition to the probe sonication of the nanotube suspensions, the mixture was also bath sonicated immediately prior to filter deposition. This likely contributed toward more evenly dispersed nanotubes, causing reduction of preferential flow paths in the filter and thereby reducing permeability slightly. It was expected that the SWNT layer laid on the MWNT matrix would give the surface of the SWNT-MWNT filter a morphology roughly equivalent to that of the SWNT filter. FESEM was performed to provide images of the morphology of all three filter types and allow for comparison (Figure 1). As expected, the surface of the SWNT-MWNT filter appeared similar to that of the SWNT filter. Both filters showed increased bundling of the SWNTs in comparison with the MWNTs on the MWNT filter. This bundling, which causes tighter blocking on some areas of the filter, may also allow flow to occur through gaps between bundles. In contrast, the MWNT filter images show much more consistent coverage of the nanotube matrix. Significant amounts of amorphous carbon were also imaged in the SWNT and SWNT-MWNT filters due to impurities in the asreceived SWNT sample used. (23) Brady-Estevez, A. S.; Nguyen, T. H.; Gutierrez, L.; Elimelech, M. Impact of solution chemistry on viral removal by a single-walled carbon nanotube filter. Water Res. 2010, 44(13), 3773–3780.
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Figure 2. Removal of the three model viruses by the SWNT-MWNT hybrid filter at the two indicated approach velocities (or water fluxes): 160 and 260 L m-2 h-1. The total CNT loading was 0.32 mg/cm2, composed of a layer of 0.27 mg/cm2 MWNTs covered by a layer of 0.05 mg/cm2 SWNTs. Other experimental conditions were 10 mM NaCl, pH 5.5, and 23 °C. At least two measurements were carried out at each dilution, and a minimum of two separate filters were tested for each experimental condition. Error bars indicate one standard deviation.
Figure 1. FE-SEM images of aerial and cross-section views of CNT filters. Top row: aerial view of MWNT-SWNT filter at 25000 magnification (left image); cross-section view of MWNT-SWNT filter at 4000 magnification (right image). Middle row: aerial view of MWNT filter at 25000 magnification (left image); cross-section view of MWNT filter at 4000 magnification (right image). Bottom row: aerial view of SWNT filter at 25000 magnification (left image); cross-section view of SWNT filter at 4000 magnification (right image).
Removal of MS2, PRD1, and T4 Viruses by the SWNTMWNT Filter. High levels of viral removal have been shown for the SWNT6 and MWNT7 filters. The design of the SWNTMWNT filter consists of a thin layer of SWNTs (0.05 mg/cm2) over a base matrix composed of 0.27 mg/cm2 MWNTs, for a total CNT loading of 0.32 mg/cm2. Since this total CNT loading was equivalent to that used for the MWNT and SWNT filters, we anticipated comparable levels of viral removal to those previously developed filters. Prior investigations for both the MWNT and SWNT filters showed the same trends for viral removal with regard to various parameters, such as filtration rate, ionic strength, pH, and the presence of natural organic matter.7,23 These studies demonstrated that viruses are removed by the CNT-filter matrix via a depth filtration mechanism.6,24 Having already established these trends, we instead tested the ability of the SWNT-MWNT filter to remove additional model viruses. The MS2, PRD1, and T4 viruses have different structures, ribonucleic acids, diameters, and isoelectric points (Table 1). We chose these viruses not to systematically determine how their individual characteristics may govern viral removal, but rather to demonstrate that the filter is effective at removing a wide range of viruses. The base solution chemistry selected for all viral removal experiments was (24) Elimelech, M. Effect of Particle-Size on the Kinetics of Particle Deposition Under Attractive Double-Layer Interactions. J. Colloid Interface Sci. 1994, 164(1), 190–199.
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10 mM NaCl at pH 5.5 to allow comparison with our previous studies on the MWNT and SWNT filters. Removal of MS2 was first examined to allow comparison of the SWNT-MWNT filter with our prior work (Figure 2). The log viral removal obtained was 4.10 ( 0.08 at the 260 L m-2 h-1 flux, which is slightly better than the 3.68 ( 0.2 log MS2 removal obtained on the MWNT filter at this approach velocity.7 Since this is a rather high flux, additional experiments were also performed at 160 L m-2 h-1, which resulted in complete removal (i.e., over 6.9 log removal from the initial starting concentration of 7.4 106 viral particles/mL). SWNT filter performance had been less effective in MS2 removal at this flux, attaining only 3.83 ( 0.41 log removal,6 while the MWNT filter attained 5.38 ( 0.80 log removal.7 It was expected that the SWNT-MWNT filter would more closely approximate the performance of the MWNT filter since we selected a nanotube loading of 83% MWNTs and only 17% SWNTs. We did not anticipate that the dual SWNT-MWNT filter would perform better than the 100% MWNT filter, since the SWNT filter had much lower viral removal. It is likely that this improvement is due to the procedural modification we made to follow probe sonication with bath sonication immediately prior to nanotube deposition. This method, which was only used in obtaining the SWNT-MWNT data, enabled the matrix to have more uniform surface coverage and higher contact opportunities for adsorption than filters that were prepared from more highly aggregated stocks. Additional experiments were made to provide an initial proofof-concept that the SWNT-MWNT filter retains various viruses. The filter attained 5.39 ( 0.76 log and complete viral removal of PRD1 (from an initial concentration of 1.9 106 viral particles/mL) at the flux rates of 260 and 160 L m-2 h-1, respectively. The T4 virus was also effectively removed, attaining complete viral removal at 160 L m-2 h-1 (from 2.6 104 viral particles/mL) and 3.84 ( 0.82 log viral removal at the higher flux of 260 L m-2 h-1. Inactivation of Gram-Negative Bacteria on the SWNTMWNT and MWNT Filters. E. coli, a rod-shaped Gramnegative bacteria, was selected for the inactivation experiments on the carbon nanotube filters. Previous studies have shown higher E. coli inactivation on SWNTs than MWNTs.8 Our prior work has also shown that the SWNT filter had relatively high inactivation of E. coli, even after only 15 min exposure to the nanotubes.6 The SWNT-MWNT filter essentially utilized a Langmuir 2010, 26(24), 19153–19158
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Figure 3. Summary of fluorescence-based toxicity assays of Gram-positive (S. epidermidis) and Gram-negative (E. coli) bacteria exposed to the MWNT filter or SWNT-MWNT hybrid filter at two exposure times (30 min or 2 h). Cell suspensions were passed through the filters and incubated in 10 mM NaCl (pH 5.5) at 37 °C for the time lengths indicated. Error bars indicate one standard deviation.
topmost layer of nanotubes that were equivalent to those used for the SWNT filter. The elevated inactivation of 91.4 ( 3.0% of the E. coli exposed to the SWNT-MWNT filter (Figure 3) was comparable to the slightly lower cytotoxicity levels for shorter exposure times on the SWNT filter.6 E. coli demonstrated greater resilience to exposure to the MWNT filter. The 30-min exposure time led to inactivation of only 58.5 ( 8.5% of this Gram-negative bacteria by the MWNT filter. This value, while far lower than that shown for exposure to the SWNT-MWNT filter, was still significantly higher than inactivation demonstrated in previous, direct-contact studies between E. coli and MWNTs.8,21 It is possible that the lower levels of salt in the current experiments (10 mM) in comparison to prior studies (154 mM NaCl) contributed to elevated stress for the bacterial cells. Differences in nanotube aggregation states between studies might also play a factor, as it has been suggested that increased dispersivity of the nanotubes can allow for greater cytotoxicity through enhanced contact with cells.21 Inactivation of Gram-Positive Bacteria on the SWNTMWNT and MWNT Filters. Staphylococcus epidermidis, a spherical cocci, was selected to model Gram-positive bacteria in the inactivation tests. S. epidermidis was demonstrated to have significantly lower inactivation rates than the Gram-negative E. coli. For the 10 mM NaCl solution at pH 5.5, S. epidermidis was 34.5 ( 3.4% inactivated on the MWNT filter, in comparison with the higher rates of 58.5 ( 8.5% for E. coli inactivation after 30 min of exposure (Figure 3). Although the inactivation of the Grampositive bacteria was much higher on the SWNT-MWNT filter (53.1 ( 3.6% at 30 min), the rates for S. epidermidis were still much lower than those for the Gram-negative E. coli (91.4 ( 3.0%) for the same exposure time. These results for the SWNTMWNT filter followed similar trends of previously demonstrated enhanced cytotoxicity toward E. coli in comparison with S. epidermidis on SWNT samples.9 While the mechanism of nanotube cytotoxicity toward bacteria is not well understood, it has been hypothesized that direct contact with the carbon nanotubes is necessary to attain inactivation.10 If direct contact with the bacterial surface does govern inactivation, then differences in cytotoxicity may be partially attributed to Langmuir 2010, 26(24), 19153–19158
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Figure 4. Inactivation of microorganisms in river water and secondary wastewater treatment plant (WWTP) effluent samples on the MWNT and SWNT-MWNT filters. Each 100-mL sample was filtered through the MWNT or SWNT-MWNT filters and incubated for 1 h in 0.9% NaCl solution at 37 °C. Error bars indicate one standard deviation.
surface characteristics of the bacteria. S. epidermidis, like other Gram-positive bacteria, has a thick peptidoglycan layer in its cell wall.25 This thick exterior may play a role in helping maintain cell structure under the stresses of exposure to carbon nanotubes. Impact of Exposure Time on Bacterial Inactivation. The inactivation of bacteria on the MWNT filter increased significantly over longer incubation times in 0.9% (154 mM) NaCl solution. The viability assay indicated that S. epidermidis cytotoxicity was enhanced to 48.1 ( 3.7% after 2 h, in comparison with 34.5 ( 3.4% after 30 min. E. coli showed heightened inactivation of 68.2 ( 4.0% after 2 h; this was a substantial increase compared with the 58.5 ( 8.5% inactivation at 30 min exposure to the MWNT surface. The level of microbial inactivation also increased on the SWNT-MWNT filter over time. This was consistent with previous studies that showed that E. coli inactivation increased with increasing exposure time to SWNTs.10,26 S. epidermidis underwent the greatest change, from 53.1 ( 3.6% inactivation after 30 min of exposure to 61.1 ( 3.3% inactivation when exposure time was increased to 2 h. Prior studies have shown time dependence for (25) Vollmer, W.; Blanot, D.; de Pedro, M. A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 2008, 32(2), 146–167. (26) Vecitis, C. D.; Zodrow, K. R.; Kang, S.; Elimelech, M. ElectronicStructure-Dependent Bacterial Cytotoxicity of Single-Walled Carbon Nanotubes. ACS Nano 2010, 4(9), 5471–5479. (27) Hu, J. Y.; Ong, S. L.; Song, L. F.; Feng, Y. Y.; Liu, W. T.; Tan, T. W.; Lee, L. Y.; Ng, W. J. Removal of MS2 bacteriophage using membane technologies. Water Sci. Technol. 2003, 47(12), 163–168. (28) Yuan, B. L.; Pham, M.; Nguyen, T. H. Deposition Kinetics of Bacteriophage MS2 on a Silica Surface Coated with Natural Organic Matter in a Radial Stagnation Point Flow Cell. Environ. Sci. Technol. 2008, 42(20), 7628–7633. (29) Vi~nuela, E.; Algranati, I. D.; Ochoa, S. Synthesis of virus-specific proteins in Escherichia coli infected with the RNA bacteriophage MS2. Eur. J. Biochem. 1967, 1(1), 3–11. (30) Overby, L. R.; Barlow, G. H.; Doi, R. H.; Jacob, M.; Spiegelman, S. Comparison of two serologically distinct ribonucleic acid bacteriophages. J. Bacteriol. 1966, 91(1), 442–448. (31) Pham, M.; Mintz, E. A.; Nguyen, T. H. Deposition kinetics of bacteriophage MS2 to natural organic matter: Role of divalent cations. J. Colloid Interface Sci. 2009, 338(1), 1–9. (32) Golmohammadi, R.; Valega˚rd, K.; Fridborg, K.; Liljas, L. The refined structure of bacteriophage MS2 at 2 3 8 A˚ resolution. J. Mol. Biol. 1993, 234(3), 620–639. (33) Pieper, A. P.; Ryan, J. N.; Harvey, R. W.; Amy, G. L.; Illangasekare, T. H.; Metge, D. W. Transport and recovery of bacteriophage PRD1 in a sand and gravel aquifer: Effect of sewage-derived organic matter. Environ. Sci. Technol. 1997, 31 (4), 1163–1170. (34) Olsen, R. H.; Siak, J.-S.; Gray, R. H. Characteristics of PRD1, a PlasmidDependent Broad Host Range DNA Bacteriophage. J. Virol. 1974, 14(3), 689–699. (35) Harvey, R. W.; Ryan, J. N. Use of PRD1 bacteriophage in groundwater viral transport, inactivation, and attachment studies. FEMS Microbiol. Ecol. 2004, 49(1), 3–16.
DOI: 10.1021/la103776y
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inactivation of the Gram-positive Bacillus subtilis by SWNTs.9 The inactivation of E. coli on the SWNT-MWNT filter was affected by time to a lesser degree. After 30 min exposure to the filter top surface, 91.4 ( 3.0% inactivation was obtained compared to a comparable value of 93.8 ( 1.9% after 2 h. Inactivation of Microbes Present in River Water and Wastewater. In addition to presenting the inactivation of specific monoculture bacteria, we also quantified the inactivation of microbes naturally present in the environment by the MWNT and SWNT-MWNT filters (Figure 4). The river water samples demonstrated less significant differences in cytotoxicity levels between the two filters than those found for the monoculture bacteria. The river water inactivation was 56.8 ( 3.4% for the MWNT filter, while 59.11 ( 2.96% was attained on the SWNT-MWNT filter at the 1 h exposure time. Prior work had hypothesized that it is the direct contact between cells and SWNTs that causes cell inactivation.10,26 It is possible that the high levels of natural organic matter (15.41 mg/L TOC) and colloidal materials in the river water modified the surfaces of the SWNT-MWNT and MWNT filters, thereby reducing the difference between the two filter surfaces for contact with the (36) Dowd, S. E.; Pillai, S. D.; Wang, S.; Corapcioglu, M. Y. Delineating the Specific Influence of Virus Isoelectric Point and Size on Virus Adsorption and Transport through Sandy Soils. Appl. Environ. Microbiol. 1998, 64(2), 405–410. (37) Leiman, P. G.; Chipman, P. R.; Kostyuchenko, V. A.; Mesyanzhinov, V. V.; Rossman, M. G. Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 2004, 118(4), 419–429. (38) Fokine, A.; Chipman, P. R.; Leiman, P. G.; Mesyanzhinov, V. V.; Rao, V. B.; Rossman, M. G. Molecular architecture of the prolate head of bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 2004, 101(16), 6003–6008. (39) Gerba, C. P. Applied and theoretical aspects of virus adsorption to surfaces. Adv. Appl. Microbiol. 1984, 30, 133–168. (40) Trueba, F. J.; Woldringh, C. L. Journal of Bacteriology. J. Bacteriol. 1980, 142(3), 869–878. (41) Parisi, J. Coagulase-Negative Staphylococci and the Epidemiological Typing of Staphylococcus epidermidis. Microbiol. Rev. 1985, 49, 129–139.
19158 DOI: 10.1021/la103776y
Brady-Est evez et al.
microbes. In contrast, the difference in cytotoxicity toward microbes present in the wastewater effluent was much more pronounced between the filters. The MWNT filter only attained 59.11 ( 3.0% inactivation, while the SWNT-MWNT filter achieved 71.49 ( 3.6%. This may also be attributed to differences in the microbial populations between the river water and wastewater effluent.
Conclusion Development of a dual-layer SWNT-MWNT filter lays the framework for new possibilities in water filtration. The filter shows significantly higher viral removal than both the previously developed SWNT and MWNT filters. Furthermore, we demonstrate effective removal of several bacteriophages with markedly different properties. The observed enhancement of viral adsorption was at the expense of only a modest reduction in filter permeability, which remained in the microfiltration pressure range. Bacterial inactivation was higher for the SWNT-MWNT filter than the filter composed of MWNTs alone. Our strategy for enhancing the filter bacterial inactivation was validated by the ability of the thin SWNT layer to inactivate more effectively across monocultured Gram-positive and Gram-negative bacteria, along with environmentally present microbes in wastewater and river water alike. These antimicrobial properties are useful for their role in inhibition of biofilm growth, which can reduce filter permeability and, thereby, increase energy and cleaning or filter regeneration needed for operation. Acknowledgment. Funding was provided by the National Science Foundation Graduate Research Fellowship Program for Anna S. Brady-Estevez and the National Science Foundation Research Grants CBET-0646247 and CBET- 0828795.
Langmuir 2010, 26(24), 19153–19158
