Experiments were performed using tap water and tap water amended with surface water to achieve target turbidity levels of <0.50 nephelometric turbidity units (NTU) (low-range turbidity), 1.0 NTU (midrange turbidity), and 4.0 NTU (high-range turbidity) relative to typical drinking-water turbidity levels. Tap water samples were collected from a faucet in the laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. Surface-water samples were collected from Murphey Candler Park (MCP) lake in Atlanta, GA. Investigators collected MCP lake water once a week by pumping the water with a peristaltic pump into plastic cubitainers and then transporting the samples back to the laboratory, where they were stored at 5°C when not being used for experiments. Tap water samples were collected by opening the cold-water faucet in the lab for at least 5 min prior to filling a 30-gallon high-density polyethylene tank that had been previously calibrated to 100 liters using 10-liter gradations. At the beginning of each experiment, the 30-gallon tank was filled with water to the 100-liter mark, which was mixed with a metal stir rod, and a 100-ml sample was collected for water quality testing. Water quality testing consisted of analyses for turbidity, temperature, and total organic carbon (TOC). The temperature of the in-tank bulk water samples was measured using an infrared/type K noncontact thermometer (catalog no. 15-077-57; Fisher Scientific). Turbidity was measured with a model 2100N laboratory turbidimeter (Hach Company). TOC was measured using a low-range TOC reagent set and a DR/2400 portable spectrophotometer (Hach Company).
Lake water was volumetrically added to tap water based on the turbidity of the lake water sample to achieve target mid- and high-range turbidity levels. Final mid- and high-range-turbidity sample volumes were ~102 liters. The sample water was dechlorinated with 50 ml of 10% sodium thiosulfate and then tested using N,N-diethyl-p-phenylenediamine-free chlorine reagents (Hach Company) to ensure no residual chlorine was present.
Dead-end UF setup.
The DEUF setup is shown in Fig. . Each 30-gallon tank was sterilized before each experiment with a 1:10 dilution of bleach followed by a 3% solution of hydrogen peroxide and then rinsed three times with deionized (DI) water and sprayed with a 1% sodium thiosulfate solution. The tank was then rinsed two more times with DI water. Contact time with each disinfectant was at least 30 min. A Cole-Parmer Masterflex L/S peristaltic pump (model 7550-30) and pump heads were used with L/S 36 and L/S 24 silicone tubing. New tubing was used for each of the microbe-seeding experiments. The ultrafilters were set up with the input port on the top, connected with L/S 24 tubing to a 30-lb/in2 liquid-filled pressure gauge and clamped with plastic tubing clamps to prevent leakage. The pressure gauge was then fastened with L/S 36 tubing, which extended into the pump head. The L/S 36 tubing was used so that a maximum pump rate of 2,900 ml/min could be achieved, if desired. After extension out of the pump head, the L/S 36 tubing was connected to L/S 24 tubing using a polypropylene reducing connector and the L/S 24 tubing connected to the ultrafilter using an autoclaveable DIN adapter (a female fitting with threading for dialyzers on one end and a male barb on the other end) (special order from Molded Products, Inc., Harlan, IA; similar to the item under catalog no. MPC-855). Filtered water exited the ultrafilter through the permeate port (top-side port in Fig. ) and was collected in a second 30-gallon “permeate” tank. For these DEUF experiments, the bottom output port was attached to a small piece of L/S 24 tubing to avoid leakage from the manufacturer's cap, and the tubing was clamped shut with a tubing clamp. All tubing connectors and clamps were autoclaved, and the brass fitting of the pressure gauge was sanitized with a 3% solution of hydrogen peroxide and a 1:10 dilution of bleach and then washed thoroughly with DI water prior to use in the filtration setup.
Schematic of the dead-end UF setup for filtering water samples.
Hydraulic performance of DEUF method with different ultrafilters.
Using nonamended tap water, the DEUF method was investigated with four ultrafilter types to evaluate the hydraulic characteristics (permeate flow rate versus pressure) of each filter type. Pressure changes were monitored versus different flow rates at two water temperature levels (room/tap temperature and ~5°C). Permeate flow rates were measured using a 2-liter graduated cylinder and a stopwatch. The test ultrafilters included the following: Fresenius Optiflux F200NR (polysulfone, 2.0-m2 filter area, ~30-kDa pore size), Baxter Exeltra Plus 210 (cellulose triacetate, 2.1-m2 filter area, 70-kDa pore size), Asahi Kasei REXEED 21S (polysulfone, 2.1-m2 filter area, ~30-kDa pore size), and Asahi Kasei REXEED 25S (polysulfone, 2.5-m2 filter area, ~30-kDa pore size). The pump speed was changed (and corresponding permeate flow rate and pressure recorded) every 5 min. Pump speeds were set at nominal flow rates of 500, 700, 900, 1,100, 1,300, 1,500, 1,700, 1,900, and 2,100 ml/min. Three replicate experiments were performed for each ultrafilter.
Additional experiments were performed with REXEED 25S filters to investigate the relationship between hydraulic performance (system pressure and permeate flow rate) for this filter and turbidity. One-hundred-liter samples of tap water (0.26 NTU, low-range turbidity) and tap water amended with surface water (to achieve a midrange target turbidity level of 1.0 NTU) were used. The system pressure and the permeate flow rate were measured every 5 min. Three replicate experiments were performed for each turbidity range.
Microbe recovery with DEUF and backflushing.
The recovery efficiency for a suite of microbes was assessed using the REXEED 25S filter and low-range-, midrange-, and high-range-turbidity water samples. After the filtration apparatus was set up, the filter was flushed with ~1 liter of nonamended tap water to flush out the storage liquid that the vendor uses to fill REXEED 25S ultrafilters. After the sample of amended tap water was prepared for each experiment in the 30-gallon tank, the following samples were collected from the tank: a 100-ml water quality sample, a 2-liter “background” sample, and a 100-ml control sample. The background sample was tested to quantify study microbes that were present in the water sample prior to microbe seeding for an experiment. The control sample was subsequently seeded with microbes to quantify the number of microbes seeded into the 100-liter DEUF microbe recovery experiment.
The suite of microbes used for microbe recovery experiments consisted of C. perfringens spores (10,000 CFU BioBalls; BTF Pty. Ltd.), MS2 bacteriophage (ATCC 15597-B1), E. faecalis (ATCC 19433), and Iowa strain C. parvum oocysts (from Mike Arrowood, CDC). MS2 and E. faecalis were diluted in a diluent containing 0.01 M phosphate-buffered saline (Dulbecco's modification; pH 7.40), 0.01% (vol/vol) Tween 80 (Fisher), and 0.001% (vol/vol) Y-30 antifoam emulsion (Sigma). The C. perfringens spore Bioballs were added to 10 ml diluent (same as above) and placed in a Pall laboratory shaker for 30 min at 960 oscillations/min to suspend and disaggregate the spores. The C. parvum oocysts were diluted in DI water and heat inactivated (30 min in a heat block at 50°C). MS2 bacteriophage, E. faecalis, and C. perfringens spores were prefiltered using 0.1-μm, 5-μm, and 3-μm filters, respectively, to reduce the presence of microbe aggregates in the seed stocks added to water samples. The suite of microbes was then seeded into both the ~100-liter input water sample and the 100-ml control sample. MS2 bacteriophage was seeded at 3.9 × 104 to 1.0 × 105 PFU, E. faecalis was seeded at 900 to 1,000 CFU, C. perfringens spores were seeded at 1,500 to 3,760 CFU, and C. parvum oocysts were seeded at 9 × 105 to 1.4 × 106 oocysts. The nominal filtration pump speed (2,100 ml/min) was constant throughout each experiment, and the permeate flow rate, system pressure, and temperature were monitored. At the 40- and 80-liter cumulative sample filtration time points, a permeate sample was collected and tested to evaluate filter integrity (i.e., microbial breakthrough).
After the seeded sample was filtered, the filtration setup was adjusted for backflushing (Fig. ). The input tubing was replaced with a filter port cap. The permeate port tubing was replaced by a clean piece of L/S 36 tubing that was used as the new input tubing (threaded through the pump head), and the output port tubing was removed. While the output tubing was being carefully unscrewed, a beaker was placed under the port so that there was no liquid loss from the filter. Five hundred milliliters of backflush solution (0.5% Tween 80, 0.01% sodium polyphosphate [Sigma-Aldrich, catalog no. 305553], and 0.001% Y-30 antifoam emulsion) was pumped through the permeate port and collected in a beaker positioned under the output port (Fig. ). The pump rate was set at a constant 650 ml/min. The volume of the recovered backflush sample was measured with a graduated cylinder, and all of the samples were stored at 5°C until assaying or secondary processing took place. Final backflush sample volumes were an average of 533 ml.
Schematic of the setup for backflushing hollow-fiber ultrafilters.
Secondary processing of DEUF backflush samples was completed the same day as the DEUF procedure. The DEUF backflush sample and control were assayed by immunofluorescence assay microscopy per EPA method 1623 for C. parvum
). The DEUF backflush, control, background, and permeate samples were analyzed for C. perfringens
spores, E. faecalis
, and MS2. MS2 was assayed by single-agar-layer plaque assay using EPA method 1602 with an E. coli
Famp host (ATCC C-3000) (17
) using 1 ml of control sample, 2 ml of UF concentrate, and 100 ml of permeate. E. faecalis
was assayed by membrane filtration and mEI agar culture using EPA method 1600 (16
) using 5 ml of control sample, 50 ml of UF concentrate, and 1.5 liters of permeate. C. perfringens
was assayed by membrane filtration and mCP agar culture (2
) using 10 ml of control sample, 50 ml of UF concentrate, and 1.5 liters of permeate.
Data analysis and statistical testing.
Percent recovery efficiencies were calculated by dividing the total number of each microbe measured in a backflush sample by the total number of each microbe measured in the input sample (microbes seeded plus microbes present as “background”) for the experiment and multiplying the fraction by 100. Tukey's studentized range (HSD) test was used to test for significant differences between mean recovery efficiencies for each microbe at each turbidity level (i.e., low-range, midrange, and high-range). An alpha value of 0.05 was used for all statistical tests.