Report no Test of bacterial retention of membrane filters. LiqTech International A/S Industriparken 22C DK-2750 Ballerup ATT Johnny Marcher

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1 LiqTech International A/S Industriparken 22C DK-2750 Ballerup ATT Johnny Marcher Teknologiparken Kongsvang Allé 29 DK-8000 Århus C Telefon Telefax info@teknologisk.dk Report no Test of bacterial retention of membrane filters Task: Danish Technological Institute has tested the bacterial retention of membrane filters to be used for liquid filtration according to the standard F Trial date: December 11 th 2012 Analysis periode: Staring from December 11 th 2012 and finalizing at December 19 th 2012 Note: The results of the analysis, sample labeling and explanation of the methods used in the report, relate only to the tested subjects and analyzed samples. The analysis is performed according to DTI general conditions for commissioned work. The analysis report may only be used in full length unless else has been accepted by DTI. Michael VW Kofoed Microbiologist, Ph.D. Direct no.: mvk@dti.dk Caroline Kragelund Rickers Microbiologist, Ph.D. Direct no.: cakr@dti.dk Summary of conclusions: The test determining the membrane filters ability to retain bacteria has been evaluated based on the ASTM standard F for liquid filtration. The Liqtech membrane has been challenged with a Pseudomonas diminuta ( recently renamed Brevundimonas diminuta) suspension corresponding to the concentration of 10 7 organisms per cm 2 of effective filtration area. The Liqtech membrane has the ability to retain bacteria corresponding to a log 7 reduction according to ASTM F Report no.: page 1 af 6

2 Filter identification. Two membranes, (OD25mm, 305mm length) were taken from the regular production. The tested and approved membranes were mounted in standard housing, flushed with clean water, with back-pulse every 30 second for 15 minutes. Water was exchanged and the membranes were back flushed for 15 minutes. Figure 1. Overview of Liqtech membrane filter device collected in the steel housing. To reduce the volume of the test, each membrane was cut in two, using a diamond saw so a final length was 150 mm as depicted in Figure 1. The membranes were placed in clean water in a Hilsonic ultrasonic bath, and were exposed to 60 minutes ultrasound at 80 C. The membranes were dried in a Binder drying oven at 120 C for 24 hours. Both ends of the four membrane samples were sealed with Epoxy resins (Resin 180 from LKR), air-dried for four hours and hardened at 75 C for 24 hours. Membrane samples were mounted into standard housing, with modified inlet/outlet. The descriptions of the two Liqtech membranes used for the experiments were as follows: 150 mm in length with 31 channels each with a diametre of ø3mm. The area of each membrane is calculated as cm 2 per membrane. Experimental setup The entire apparatus has been assembled as described in Figure 2 : Experimental setup trial 1 Pressure sensor Membrane housing Valve Filtrate outlet Valve Analytical filter (poresize 0,45µm) Pressure pot Sterile Saline solution or Bacterial suspension Air Inlet Figure 2: Experimental setup according to ASTM F Report no.: page 2 af 6

3 Equipment preparation The Liqtech membranes were placed in stainless steel housing and were flushed in sterile-filtered deionized water prior to assembly. Teflon (PTFE) tubing was used in the setup. Tubing, fittings and valves were initially flushed in sterile-filtered deionized water followed by flushing in 70 % ethanol, and hereafter in sterile-filtered deionized water. The entire experimental setup was as depicted in Figure 2. Everything was assembled (autoclavable hoses, membrane filter housing, analyses filters with 0.45 µm filter) and autoclaved at 121 C for 45 minutes. Except for the pressure vessel, and the non-autoclavable connection to air pressure, which were thoroughly cleaned by using 70 % ethanol and rinsed thoroughly with sterile-filtered deionized water prior to use. Test procedure The test procedure was performed according to the descriptions of ASTM F In Figure 3 is shown the experimental setup for determining the Liqtech Membrane ability to retain bacteria. Figure 3. Experimental setup. Liqtech membrane filter directed connected to exchangeable filter house containing a 0.45 µm analytical filter. Operating conditions The membrane was initially characterized with regards to pressure and flow-rate using sterile saline solution (0.9%). The pressure and flow-rate was automatically logged using a digital scale with pressure-sensors placed at in- and out-let of the membrane. Data is presented in Figure 4 and Report no.: page 3 af 6

4 Flow rate (ml/min) Flow rate (ml/min) Pressure - Flow rate Pressure Out Pressure In Absolute Pressure (Bar) Figure 4. Overview of flow rate and absolute pressure used for control suspension. The trans-membrane pressure (ΔP) was calculated from at pressure at in- and outlet, see Figure 5. Trial 1 ΔP - Flow rate Δ Pressure (Bar) Serie1 Figure 5. Overview of trans-membrane pressure and flow rate. Data suggests a minor decrease in pressure across the Liqtech membrane, whereas the analytical 0.45µm membrane showed high resistance. Challenge test During operation of the challenge-test, the inlet pressure was adjusted to 0.5 Bar on the pressure pot. The actual inlet pressure was logged using a small pressure-sensor at the inlet. For the challenge-test, the inlet pressure had to be increased during the experiment to above the sensors detection limit (> 2.8 bar). The operating conditions during the experiments were as depicted in Table 1 with respect to temperature and flow rates of control and challenge suspension..during tests, the weight increase in Report no.: page 4 af 6

5 grams was also logged to ensure that the required volume of control or challenge suspension had passed through the membrane filter setup. Table 1. Overview of operating conditions during challenge test. Operating conditions Temperature 24 C Flow rate (ml/min) - Control - Challenge test Inlet Pressure (bar) - Control - Challenge test 1.37 ± to above upper limit of pressure sensor (2.8 bar) Number of bacteria in challenge suspension. The total number of bacteria in the challenge suspensions was determined by filtration of suspension on a polycarbonate filter, followed by a DAPI stain. The concentration in the suspension was determined as 1.48*10 7 cell/ml, SE 1.33*10 6. The viable bacterial concentration for the suspension was determined to be 5*10 6 cfu/ml corresponding to 33 % viable cells. To challenge the filter according to the description of ASTM F with 10 7 cells/cm 2 and using the Liqtech membranes for experimental work with a total area of cm 2 per membrane, a total of 320 ml bacterial challenge suspension or autoclaved 0.9% Sodium Chloride solution was used for the experiments. Number of bacteria in filtrate The number of bacteria in the filtrate was determined on an analytical membrane filter with a 0.45 µm pore size (diameter 47mm, HAWP04700 from Millipore). Following the experimental approach, the filters were collected from the filter house, placed on PCA plates, and incubated at 30 C for 48 hours, 72 hours and 7 days. One sterile filter house was used for the control experiment; one sterile for the challenge test, and results can be seen in Table 2. Table 2. Overview of bacterial numbers collected on 0.45 µm filters after the control and challenge experiments 48 hours (cell/ml) 72 hours (cell/ml) 7 days (cell/ml) Control X 0 0 Challenge suspension 102 X Report no.: page 5 af 6

6 Filter performance The Liqtech membrane filter performance is calculated as the difference between bacteria in the challenge solution corresponding to a load of more than 10 7 cells/cm 2 membrane filtration area. In total more than 10 9 viable cells (cfu) were used for the challenge test and using guidelines by ASTM F838-05, this corresponded to a reduction of log7. Conclusions. The Liqtech membrane filter device is capable of retaining P. diminuta (identical to B. diminuta) and reduces the concentration of cells by a log 7 according to ASTM F This fulfills the requirements of liquid sterilization Report no.: page 6 af 6