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Effect of operating conditions on biofouling in reverse osmosis membrane processes: Bacterial adhesion, biofilm formation, and permeate flux decrease
Desalination 378 (2016) 74–79
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Desalination journal homepage: www.elsevier.com/locate/desal
Effect of operating conditions on biofouling in reverse osmosis membrane processes: Bacterial adhesion, biofilm formation, and permeate flux decrease Daisuke Saeki, Hamed Karkhanechi 1, Hirotaka Matsuura 1, Hideto Matsuyama ⁎ Center for Membrane and Film Technology, Department of Chemical Science and Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
H I G H L I G H T S • • • •
Biofouling behavior in reverse osmosis membrane processes was evaluated. A crossflow bacterial filtration system was designed for biofouling evaluation. Trans-membrane pressure and stirring rate changed with operating conditions. Low trans-membrane pressure and high stirring rates prevented biofilm formation.
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Article history: Received 15 June 2015 Received in revised form 18 September 2015 Accepted 19 September 2015 Available online xxxx Keywords: Biofouling Reverse osmosis membranes Operating conditions Concentration polarization
a b s t r a c t We systematically evaluated the effects of operating conditions on biofouling behaviors in reverse osmosis (RO) membrane processes. The biofouling experiment was performed by filtrating nutrient-containing feed water with a cross-flow cylindrical membrane cell through a circular polyamide RO membrane with pre-adhered Pseudomonas putida. The trans-membrane pressure (TMP) and stirring rates in the cell were controlled as operating conditions. The stirring rates brought about shear force and thus corresponded to cross-flow velocities on the membrane surfaces in commercial operations. Permeate flux was monitored during the filtration, and bacterial adhesion to the membrane surfaces was observed using a confocal laser scanning microscope. An increase in TMP increased both the reduction rate of permeate flux and the volume of adhered bacteria, thus facilitating biofouling. Osmolarity calculation on the membrane surface suggested that TMP affected bacterial growth by concentration polarization (CP) of nutrients. Higher stirring rate prevented reduction of permeate flux and bacterial growth on membrane surfaces. The hydrodynamic shear force generated by stirring effectively detached the adhered bacteria, while CP of nutrients was not remarkably affected by the range of stirring rates used in this study. These results suggest that lower TMP and higher stirring rates prevent biofilm formation by decreasing CP and promoting bacterial detachment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Global water shortage has become a critical problem owing to excessive usage and increasing contamination of natural water sources. Therefore, desalination techniques such as distillation and membrane filtration are attracting much attention for the use of clean and safe water from various water sources such as wastewater, brackish water, and seawater. It is reported that about half of the industrial plants for water desalination around the world use reverse osmosis (RO) membranes owing to their high efficiency of space and energy [1].
⁎ Corresponding author. E-mail addresses: [email protected] (D. Saeki), [email protected] (H. Matsuyama). 1 Equal contribution.
http://dx.doi.org/10.1016/j.desal.2015.09.020 0011-9164/© 2015 Elsevier B.V. All rights reserved.
However, RO membranes are vulnerable to biofouling, which reduces the water permeability of membranes due to biofilm formation [2]. Feed water for RO operation contains various organic foulants such as bacteria and their nutrients, which cause biofouling [3,4]. Although pretreatments such as pre-filtration and chemical sterilization are applied [5–7], total removal of biofilms from RO membranes is practically difficult [3]. On the other hand, surface properties of RO membranes affect bacterial adhesion and biofilm formation [8]. Therefore, modification of membrane surfaces with functional materials such as biocides [9–11] and hydrophilic polymers [12,13] has been attempted to prevent bacterial adhesion and biofilm formation. However, practical application of surface modification to RO membrane processes is difficult since these modification processes complicate membrane fabrication and are not warranted for their durability in long-term operation. Operating conditions such as applied trans-membrane pressure (TMP) and cross-flow velocity on membrane surfaces play an important
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role in the control of biofouling progress through effects on cell proliferation and biofilm maturation [14]. The effects of the applied TMP on the organic fouling [15,16] and biofouling [14,17] have been investigated. Higher TMP results in an increased deposition rate of foulants and formation of compact fouling layers. Moreover, the permeate flux generated by applying TMP affects concentration polarization (CP) which concentrates solutes on the membrane surface [18], resulting in bacterial growth and biofilm formation [17]. Cross-flow velocity on the membrane surface is a significant parameter which affects the shear force and CP on the membrane surface [19]. It has been reported that high cross-flow velocity is effective in controlling membrane biofouling [14]. Since these studies evaluated the effect of operating conditions on biofouling of RO membranes through filtration of bacterial suspensions, they did not consider the hydrodynamic accumulation and bacterial adhesion on the membrane surface separately. In this study, we investigated the effect of operating conditions such as TMP and stirring rates in the cylindrical RO membrane cell on the biofouling with respect to the bacterial growth pre-adhered on membrane surfaces and the permeate flux. Pseudomonas putida (P. putida), used as model bacterial species, was adhered on the surface of a circular polyamide RO membrane set on a cylindrical stainless steel membrane cell. The cell was then fed continuously with feed water containing only nutrients for the bacteria, and biofouling experiments were carried out by controlling the TMP and stirring rate on the membrane surface. The stirring rate in the cell corresponded to cross-flow velocity on the membrane surface in commercial operations. The biofouling experiments evaluated changes in the permeate flux and the bacterial volume adhered on the membrane surface. The osmolarity on the membrane surface was calculated as a function of the nutrient concentration to estimate the degree of CP. In addition, the influence of drag force caused by the permeate flux and hydrodynamic shear force caused by the stirring rate was evaluated. 2. Materials and experimental methods 2.1. Materials A commercial polyamide RO membrane was obtained from Nitto Denko Corporation (ES20; Osaka, Japan). The mean surface roughness (Ra) of this RO membrane was 97.1 nm from atomic force microscope (AFM) observation (Fig. S1, details in Supplementary data), and was a typical value of RO membranes [8]. Fe (III)–EDTA was purchased from Dojindo Laboratories (Kumamoto, Japan). A LIVE/DEAD BacLight Bacterial Viability kit was purchased from Thermo Fisher Scientific (Waltham, MA) and used to stain bacteria. All chemicals, except those particularly described, were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification. Milli-Q water (18.2 MΩ·cm; Merck Millipore, Billerica, MA, USA) was used to prepare all aqueous solutions and for rinsing. 2.2. Bacterial species and culture conditions P. putida (NBRC100650) was used because Pseudomonas species have been observed in biofilms on water treatment membranes [20]. Modified FAB medium was used for bacterial cultivation because it was suitable for biofilm formation of P. putida [21,22]. The modified FAB medium contained the following components: glucose 2.0 g/L, (NH4)2SO4 2 g/L, Na2HPO4 4.7 g/L, KH2PO4 3.0 g/L, NaCl 3.0 g/L, CaCl2 11.0 mg/L, MgCl2 95.0 mg/L, and Fe(III)–EDTA 4.2 mg/L. P. putida was firstly precultured in the FAB medium overnight at 150 rpm at 30 °C. The precultured bacterial suspension was then diluted 5 times with the FAB medium and cultured at 150 rpm at 30 °C for 4 h. The cultured bacterial suspension was diluted to a final optical density of 0.05 at 450 nm (106–107 cfu/mL in bacterial concentration) and used for each experiment. The optical density was measured by a UV–VIS spectrophotometer (V-650, JASCO Corporation, Tokyo, Japan).
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Fig. 1. Schematic of the cross-flow membrane test unit.
2.3. Cross-flow membrane test unit A laboratory-scale cross-flow membrane test unit, depicted in Fig. 1, was used to measure the permeate flux [11]. A circular RO membrane was set in a cylindrical stainless steel membrane cell. The effect of operating conditions such as applied TMP and stirring rate in the cell was evaluated on biofouling which included the biofilm formation on the membrane surface and permeate flux decrease. Feed water was fed into the cell using a plunger pump (NPL-120; Nihon Seimitsu Kagaku Corporation, Tokyo, Japan) at 1 mL/min. The temperature of the feed reservoir and cell was kept at 30 °C using a water bath. The effective membrane area was 8.0 cm2. The feed water side in the cell was stirred by using a cylindrical magnetic bar of 3.0 cm length. The TMP was adjusted by two back-pressure valves on the permeate and condensate water sides. The permeate flux was calculated from the weight of the permeate water measured using an electric balance. 2.4. Biofouling experiments 2.4.1. Biofouling protocol The biofouling experiment was carried out by filtrating the feed water containing nutrients for bacteria through a polyamide RO membrane with pre-adhered bacteria. Before each experiment and prior to inserting the RO membrane into the cell, the membrane test units were autoclaved. The outline of the biofouling experiment is shown in Fig. 2. Firstly, Milli-Q water and 5-fold diluted FAB medium were continuously supplied into the cell for 15 min to stabilize the membrane conditions. The bacterial suspension was then supplied for 15 min and stopped for 45 min to allow bacterial adhesion to the membrane surface. This was followed by supply of 5-fold diluted FAB medium and permeation by applying TMP for biofouling. We investigated the effect of controlling the applied TMP and stirring rate in the cell on biofouling. The TMP and the stirring rate in the biofouling step shown in Fig. 2 were changed from 0 to 1.50 MPa with a constant stirring rate of 300 rpm and from 100 to 300 rpm (from 16.7 to 50.1 cm/s as maximum cross-flow velocity on the membrane surface) with a constant TMP of 0.75 MPa, respectively. 2.4.2. Biofilm characterization After completion of the biofouling experiments, the bacteria adhered to the membrane surface were characterized by confocal laser scanning
Fig. 2. Timeline of the biofouling experiment.
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microscopy (CLSM; FLUOVIEW FV1000, Olympus Corporation, Tokyo, Japan) and image analysis [11]. The membranes were detached from the cell and rinsed thoroughly with aqueous 0.85 wt.% NaCl solution. The adhered bacteria on the membrane surface were then stained using the LIVE/DEAD BacLight Bacterial Viability kit in 0.85 wt.% NaCl solution. The membranes were rinsed again with 0.85 wt.% NaCl solution and immersed in aqueous 2.5 vol.% glutaraldehyde solution for 1 h at 4 °C to fix the adhered bacteria. The membrane surface was observed using CLSM, and the CLSM images were analyzed using the COMSTAT software [23] to calculate the volume per unit area (μm3/μm2) of the adhered bacteria on the membrane surface. 2.4.3. Effect of drag force on bacterial adhesion To estimate the effect of the drag force on bacterial adhesion, the pre-formed biofilm on the membrane surface was exposed to different permeate flux, and the remaining biofilm was analyzed by CLSM. The biofilm was pre-formed by 24 h in the biofouling experiment using the same protocol described in Section 2.4.1 at 1.50 MPa of the TMP and 300 rpm of the stirring rate. Then, we continuously supplied 0.85 wt.% NaCl aqueous solution for 24 h into the cell at the various permeate flux from 0 to 0.4 m3/m2 day, to prevent further bacterial growth and to eliminate the effect of the CP of nutrients. The permeate flux was adjusted by the backpressure valve on the permeate water side. The TMP and stirring rate were maintained at 1.50 MPa and 300 rpm, respectively. 2.4.4. Effect of hydrodynamic shear force on bacterial adhesion To estimate the effect of the hydrodynamic shear force on the bacterial adhesion, the pre-formed biofilm on the membrane surface was exposed to the different stirring rates, and the detached bacteria were measured. After biofilm pre-formation using the same protocol as described in Section 2.4.1 at 1.50 MPa of the TMP and 100 rpm of the stirring rate, we continuously supplied 5-fold diluted FAB medium into the cell at 1.50 MPa of the TMP and at various stirring rates from 100 to 300 rpm. The bacterial concentration in the condensate water was measured by a pour plate method to evaluate the bacteria detached from the membrane surface due to the shear force caused by stirring. Briefly, 1 mL of condensate water was added to 27 mL of warm FAB medium containing 1.5 g/L agar. The mixed solution was poured onto plastic plates, cooled at room temperature, and solidified. The prepared plates were incubated for 24 h at 30 °C, and the bacterial colonies on the plates were then counted.
Fig. 3. Time course of the normalized permeate fluxes, J/J0, on the biofouling experiments operated at different TMPs: (●) 0.75 MPa, (○) 1.0 MPa, (■) 1.25 MPa, and (□) 1.5 MPa. The stirring rate was 300 rpm.
at just 58% of J0 after 24 h of the biofouling experiment, while that at 0.75 MPa was retained at about 77%. The increase of TMP enhanced the reduction rate in J. The value of J0 increased with increasing TMP (Fig. S2, details in Supplementary data). It is well known that a higher permeate flux results in increased severity of biofouling [14,17]. The increase in J would increase the drag force and CP on the membrane surface leading to transport of bacteria and nutrients, resulting in the faster biofilm development. Fig. 4A to D shows the analyzed CLSM images of stained bacteria on membrane surfaces after 24 h of the biofouling experiments at different TMPs. At the TMP of 0.75 MPa, bacteria that covered the membrane
2.5. Evaluation of concentration polarization by osmolarity calculation The effect of CP caused by the permeate flux was evaluated by calculating the osmolarity on the membrane surface from the difference of the permeate flux with the feed solution and pure-water permeability. The osmotic pressure on the membrane surface, πm, can be calculated using the Morse equation: πm = CmRT, in which Cm, R, and T are the osmolarity on the membrane surface, gas constant, and temperature, respectively. The permeate flux with the feed solution, J, can be described as J = L(P − πm), in which L and P are the pure-water permeability and TMP respectively [19]. From these equations, the osmolarity on the membrane surface is calculated as Cm = (P − J / L) / RT. The values of J and L were measured by using the same apparatus as shown in Fig. 1. 3. Results and discussion 3.1. Effect of TMP on biofouling The time courses of normalized permeate flux, J/J0, on the biofouling experiment at different TMPs are shown in Fig. 3, in which J0 is the initial permeate flux. The value of J/J0 decreased with time at all experimental conditions as biofouling occurred. The value of J at 1.5 MPa was retained
Fig. 4. Effect of TMP on bacterial adhesion to the membrane surface after 24 h of biofouling experiments: (A to D) Analyzed CLSM images of the membrane surface at different TMPs of 0.75 (A), 1.0 (B), 1.25 (C), and 1.5 MPa (D). (E) Relation of the calculated volume of the bacteria adhered to the membrane surface (●) with J/J0 (○) after the biofouling experiments. The stirring rate was 300 rpm.
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surface were sparser than at the TMP of 1.5 MPa. Fig. 4E shows the calculated volume of the bacteria adhered on the membrane surface and J/J0 of the biofouling experiments at different TMPs. The bacterial volume increased with an increment in the applied TMP. Increased bacterial volume indicates bacterial growth and biofilm formation which results in the resistance to water permeation [15,24]. Increased TMP resulted in decreased permeate flux and increased volume of adhered bacteria in the above biofouling experiment (Fig. 4). The possible reasons for this may include, the effect of hydraulic pressure due to TMP on bacterial growth, adhesion of bacteria to the membrane surface due to drag force, and the CP of nutrients in medium on the membrane surface. It is reported that the bacterial growth on the membrane surface is inhibited by application of TMP in the range of several dozens of MPa [25,26]. We confirmed that the applied hydraulic pressure in the range in the above experiments hardly affects bacterial growth adhered to the membrane surface (Fig. S3, details in Supplementary data). Thus, the TMP in this study was not high enough to inhibit bacterial growth on the membrane surface. The impact of drag force caused by water permeation through the membrane on detachment of bacteria from the membrane surface was investigated. The bacterial volume of the pre-formed biofilm as a function of J is shown in Fig. 5. The bacterial volume of the biofilm on the membrane surface at high J was almost equal to those obtained just before the experiment (dashed line), while they decreased with decreasing J and reached values lower than those obtained before the experiment. When the drag force is weak, it is not enough to maintain bacterial adhesion and the bacteria detach from membrane surfaces due to shear force generated by cross-flow velocity [27]. On the other hand, a higher drag force presses and retains bacteria on the membrane surface and expedites bacterial growth on the membrane surface due to the presence of nutrients enriched by CP. Because NaCl solution was used to eliminate the effect of CP, the increase in bacterial volume could mostly be attributed to bacteria pressing on the membrane surface due to drag force. Fig. 6 shows the calculated osmolarity on the membrane surface under different applied TMPs. The osmolarity on the membrane surface increased with an increase in the TMP, and reached at least 2 times higher than that of the feed water. This increment in osmolarity is attributed to the CP phenomenon enhanced by the permeate flux. The enriched nutrients facilitate bacterial growth and biofilm formation on the membrane surface resulting in resistance to water permeation. The tendency of the decrease in J/J0 with an increase in the TMP (Fig. 3) is clearly attributed to the increase of osmolarity at the membrane surface. These results indicate that CP is the cause of biofouling of the RO membranes.
Fig. 5. Effect of permeate flux, J, on the bacterial volume of pre-formed biofilm on the membrane surface. The dashed line indicates the bacterial volume of pre-formed biofilm before supplying the NaCl aqueous solution. The TMP and stirring rate were maintained at 1.50 MPa and 300 rpm, respectively.
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Fig. 6. The calculated osmolarity on the membrane surface. The dashed line indicates the osmolarity of the feed water. The stirring rate was 300 rpm.
3.2. Effect of stirring rate on biofouling The time courses of J/J0 on the biofouling experiment at the different stirring rates are shown in Fig. 7. The value of J/J0 at 100 rpm decreased to 65% after 24 h of the biofouling experiment, while that at 300 rpm was retained at about 80%. These results clearly show that the decline rate of J/J0 increased with a decrease in the stirring rate. Fig. 8A to C shows the analyzed CLSM images of the stained bacteria on the membrane surfaces after 24 h of the biofouling experiments at different stirring rates. The thickness and coverage of the bacteria adhered to the membrane surface decreased with an increase in the stirring rate. The bacterial volume was also sufficiently decreased by the stirring rate (Fig. 8D). The stirring rate would mainly affect two factors, the hydrodynamic shear force and CP. Decreased stirring rates reduce the hydrodynamic shear force on the membrane surface, which would reduce bacterial detachment from the membrane surface. On the other hand, decreased stirring rates promote the CP and increase nutrient concentration on the membrane surface resulting in increased bacterial growth. Therefore, we focused on the effect of stirring rates on these two factors, hydrodynamic shear force and CP. Hydrodynamic shear force resulting from higher cross-flow velocity possibly detaches bacteria from the membrane surface and prevents biofilm formation [14,27]. We measured the bacterial concentration in the condensate water from the biofouling experiments at various stirring rates (Fig. 9). The bacterial concentration in the condensate water increased upon increasing the stirring rate. This result indicates that the shear force generated by stirring detaches bacteria from the membrane surface. Thus, one reason for the decrease of permeate flux with
Fig. 7. Time course of the J/J0 on the biofouling experiments operated at the different stirring rates: (■) 100 rpm, (○) 200 rpm, and (●) 300 rpm. The TMP was 0.75 MPa.
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Fig. 10. Effect of stirring rates on calculated osmolarity of the membrane surface. The dashed line indicates the osmolarity of the feed water. The TMP was 0.75 MPa.
detachment and not by CP reduction. Thus, higher stirring rates could reduce biofouling by both CP reduction and bacterial detachment. 4. Conclusion
Fig. 8. Effect of stirring rates on bacterial adhesion to the membrane surface after 24 h of biofouling experiments: (A to C) Analyzed CLSM images of the membrane surface at different stirring rates of 100 rpm (A), 200 rpm (B), and 300 rpm (C). (D) Relation of the calculated volume of the bacteria adhered to the membrane surface (●) with J/J0 (○) after the biofouling experiments. The TMP was 0.75 MPa.
increasing stirring rates was bacterial detachment from the biofilm formed on the membrane surface. Fig. 10 shows the calculated osmolarity on the membrane surface at different stirring rates. The osmolarity on the membrane surface was higher than that of the feed water and slightly decreased with increased stirring rates. The increased stirring rate prevents CP due to decreasing thickness of the boundary layer on the membrane surface [28]. In this study, the range of stirring rates analyzed was relatively small and therefore stirring rates did not affect the CP remarkably. The theoretical study by Qiu and Davies showed that cross-flow velocity slightly decreased the degree of CP [19]. In this study, inhibition of biofouling with increasing stirring rates was mainly caused by bacterial
The effects of operating conditions on biofouling in RO membrane processes were systematically investigated. The biofouling of the RO membranes was evaluated by filtrating bacterial medium through a circular polyamide RO membrane with pre-adhered bacteria. The TMP and stirring rate affecting the membrane surface in a cylindrical membrane cell were changed as the operating conditions. The application of TMP facilitated decreased rates of the permeate flux and the increased bacterial adhesion on the membrane surface. The increase in calculated osmolarity on the membrane surface suggested that the TMP caused CP of bacterial nutrients and accelerated the biofouling. An increase in the stirring rate prevented the decrease of permeate flux as well as bacterial growth on the membrane surface. This biofouling inhibition was mainly caused by reduction in bacterial volume due to hydrodynamic shear force and not the CP. These results show that lower TMP and higher stirring rates are favorable to prevent the biofouling by reducing CP reduction and promoting bacterial detachment. Supplementary data An AFM image of the unused polyamide RO membrane and methods of AFM observation (Fig. S1), effect of TMP on J0 (Fig. S2), and effect of the hydraulic pressure on the bacterial growth (Fig. S3). Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j. desal.2015.09.020. References
Fig. 9. Effect of stirring rates on bacterial detachment from the pre-adhered bacteria on the membrane surface. The bacterial concentration in the condensate water was counted by the pour plate method.
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Desalination journal homepage: www.elsevier.com/locate/desal
Effect of operating conditions on biofouling in reverse osmosis membrane processes: Bacterial adhesion, biofilm formation, and permeate flux decrease Daisuke Saeki, Hamed Karkhanechi 1, Hirotaka Matsuura 1, Hideto Matsuyama ⁎ Center for Membrane and Film Technology, Department of Chemical Science and Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan
H I G H L I G H T S • • • •
Biofouling behavior in reverse osmosis membrane processes was evaluated. A crossflow bacterial filtration system was designed for biofouling evaluation. Trans-membrane pressure and stirring rate changed with operating conditions. Low trans-membrane pressure and high stirring rates prevented biofilm formation.
a r t i c l e
i n f o
Article history: Received 15 June 2015 Received in revised form 18 September 2015 Accepted 19 September 2015 Available online xxxx Keywords: Biofouling Reverse osmosis membranes Operating conditions Concentration polarization
a b s t r a c t We systematically evaluated the effects of operating conditions on biofouling behaviors in reverse osmosis (RO) membrane processes. The biofouling experiment was performed by filtrating nutrient-containing feed water with a cross-flow cylindrical membrane cell through a circular polyamide RO membrane with pre-adhered Pseudomonas putida. The trans-membrane pressure (TMP) and stirring rates in the cell were controlled as operating conditions. The stirring rates brought about shear force and thus corresponded to cross-flow velocities on the membrane surfaces in commercial operations. Permeate flux was monitored during the filtration, and bacterial adhesion to the membrane surfaces was observed using a confocal laser scanning microscope. An increase in TMP increased both the reduction rate of permeate flux and the volume of adhered bacteria, thus facilitating biofouling. Osmolarity calculation on the membrane surface suggested that TMP affected bacterial growth by concentration polarization (CP) of nutrients. Higher stirring rate prevented reduction of permeate flux and bacterial growth on membrane surfaces. The hydrodynamic shear force generated by stirring effectively detached the adhered bacteria, while CP of nutrients was not remarkably affected by the range of stirring rates used in this study. These results suggest that lower TMP and higher stirring rates prevent biofilm formation by decreasing CP and promoting bacterial detachment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Global water shortage has become a critical problem owing to excessive usage and increasing contamination of natural water sources. Therefore, desalination techniques such as distillation and membrane filtration are attracting much attention for the use of clean and safe water from various water sources such as wastewater, brackish water, and seawater. It is reported that about half of the industrial plants for water desalination around the world use reverse osmosis (RO) membranes owing to their high efficiency of space and energy [1].
⁎ Corresponding author. E-mail addresses: [email protected] (D. Saeki), [email protected] (H. Matsuyama). 1 Equal contribution.
http://dx.doi.org/10.1016/j.desal.2015.09.020 0011-9164/© 2015 Elsevier B.V. All rights reserved.
However, RO membranes are vulnerable to biofouling, which reduces the water permeability of membranes due to biofilm formation [2]. Feed water for RO operation contains various organic foulants such as bacteria and their nutrients, which cause biofouling [3,4]. Although pretreatments such as pre-filtration and chemical sterilization are applied [5–7], total removal of biofilms from RO membranes is practically difficult [3]. On the other hand, surface properties of RO membranes affect bacterial adhesion and biofilm formation [8]. Therefore, modification of membrane surfaces with functional materials such as biocides [9–11] and hydrophilic polymers [12,13] has been attempted to prevent bacterial adhesion and biofilm formation. However, practical application of surface modification to RO membrane processes is difficult since these modification processes complicate membrane fabrication and are not warranted for their durability in long-term operation. Operating conditions such as applied trans-membrane pressure (TMP) and cross-flow velocity on membrane surfaces play an important
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role in the control of biofouling progress through effects on cell proliferation and biofilm maturation [14]. The effects of the applied TMP on the organic fouling [15,16] and biofouling [14,17] have been investigated. Higher TMP results in an increased deposition rate of foulants and formation of compact fouling layers. Moreover, the permeate flux generated by applying TMP affects concentration polarization (CP) which concentrates solutes on the membrane surface [18], resulting in bacterial growth and biofilm formation [17]. Cross-flow velocity on the membrane surface is a significant parameter which affects the shear force and CP on the membrane surface [19]. It has been reported that high cross-flow velocity is effective in controlling membrane biofouling [14]. Since these studies evaluated the effect of operating conditions on biofouling of RO membranes through filtration of bacterial suspensions, they did not consider the hydrodynamic accumulation and bacterial adhesion on the membrane surface separately. In this study, we investigated the effect of operating conditions such as TMP and stirring rates in the cylindrical RO membrane cell on the biofouling with respect to the bacterial growth pre-adhered on membrane surfaces and the permeate flux. Pseudomonas putida (P. putida), used as model bacterial species, was adhered on the surface of a circular polyamide RO membrane set on a cylindrical stainless steel membrane cell. The cell was then fed continuously with feed water containing only nutrients for the bacteria, and biofouling experiments were carried out by controlling the TMP and stirring rate on the membrane surface. The stirring rate in the cell corresponded to cross-flow velocity on the membrane surface in commercial operations. The biofouling experiments evaluated changes in the permeate flux and the bacterial volume adhered on the membrane surface. The osmolarity on the membrane surface was calculated as a function of the nutrient concentration to estimate the degree of CP. In addition, the influence of drag force caused by the permeate flux and hydrodynamic shear force caused by the stirring rate was evaluated. 2. Materials and experimental methods 2.1. Materials A commercial polyamide RO membrane was obtained from Nitto Denko Corporation (ES20; Osaka, Japan). The mean surface roughness (Ra) of this RO membrane was 97.1 nm from atomic force microscope (AFM) observation (Fig. S1, details in Supplementary data), and was a typical value of RO membranes [8]. Fe (III)–EDTA was purchased from Dojindo Laboratories (Kumamoto, Japan). A LIVE/DEAD BacLight Bacterial Viability kit was purchased from Thermo Fisher Scientific (Waltham, MA) and used to stain bacteria. All chemicals, except those particularly described, were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification. Milli-Q water (18.2 MΩ·cm; Merck Millipore, Billerica, MA, USA) was used to prepare all aqueous solutions and for rinsing. 2.2. Bacterial species and culture conditions P. putida (NBRC100650) was used because Pseudomonas species have been observed in biofilms on water treatment membranes [20]. Modified FAB medium was used for bacterial cultivation because it was suitable for biofilm formation of P. putida [21,22]. The modified FAB medium contained the following components: glucose 2.0 g/L, (NH4)2SO4 2 g/L, Na2HPO4 4.7 g/L, KH2PO4 3.0 g/L, NaCl 3.0 g/L, CaCl2 11.0 mg/L, MgCl2 95.0 mg/L, and Fe(III)–EDTA 4.2 mg/L. P. putida was firstly precultured in the FAB medium overnight at 150 rpm at 30 °C. The precultured bacterial suspension was then diluted 5 times with the FAB medium and cultured at 150 rpm at 30 °C for 4 h. The cultured bacterial suspension was diluted to a final optical density of 0.05 at 450 nm (106–107 cfu/mL in bacterial concentration) and used for each experiment. The optical density was measured by a UV–VIS spectrophotometer (V-650, JASCO Corporation, Tokyo, Japan).
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Fig. 1. Schematic of the cross-flow membrane test unit.
2.3. Cross-flow membrane test unit A laboratory-scale cross-flow membrane test unit, depicted in Fig. 1, was used to measure the permeate flux [11]. A circular RO membrane was set in a cylindrical stainless steel membrane cell. The effect of operating conditions such as applied TMP and stirring rate in the cell was evaluated on biofouling which included the biofilm formation on the membrane surface and permeate flux decrease. Feed water was fed into the cell using a plunger pump (NPL-120; Nihon Seimitsu Kagaku Corporation, Tokyo, Japan) at 1 mL/min. The temperature of the feed reservoir and cell was kept at 30 °C using a water bath. The effective membrane area was 8.0 cm2. The feed water side in the cell was stirred by using a cylindrical magnetic bar of 3.0 cm length. The TMP was adjusted by two back-pressure valves on the permeate and condensate water sides. The permeate flux was calculated from the weight of the permeate water measured using an electric balance. 2.4. Biofouling experiments 2.4.1. Biofouling protocol The biofouling experiment was carried out by filtrating the feed water containing nutrients for bacteria through a polyamide RO membrane with pre-adhered bacteria. Before each experiment and prior to inserting the RO membrane into the cell, the membrane test units were autoclaved. The outline of the biofouling experiment is shown in Fig. 2. Firstly, Milli-Q water and 5-fold diluted FAB medium were continuously supplied into the cell for 15 min to stabilize the membrane conditions. The bacterial suspension was then supplied for 15 min and stopped for 45 min to allow bacterial adhesion to the membrane surface. This was followed by supply of 5-fold diluted FAB medium and permeation by applying TMP for biofouling. We investigated the effect of controlling the applied TMP and stirring rate in the cell on biofouling. The TMP and the stirring rate in the biofouling step shown in Fig. 2 were changed from 0 to 1.50 MPa with a constant stirring rate of 300 rpm and from 100 to 300 rpm (from 16.7 to 50.1 cm/s as maximum cross-flow velocity on the membrane surface) with a constant TMP of 0.75 MPa, respectively. 2.4.2. Biofilm characterization After completion of the biofouling experiments, the bacteria adhered to the membrane surface were characterized by confocal laser scanning
Fig. 2. Timeline of the biofouling experiment.
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microscopy (CLSM; FLUOVIEW FV1000, Olympus Corporation, Tokyo, Japan) and image analysis [11]. The membranes were detached from the cell and rinsed thoroughly with aqueous 0.85 wt.% NaCl solution. The adhered bacteria on the membrane surface were then stained using the LIVE/DEAD BacLight Bacterial Viability kit in 0.85 wt.% NaCl solution. The membranes were rinsed again with 0.85 wt.% NaCl solution and immersed in aqueous 2.5 vol.% glutaraldehyde solution for 1 h at 4 °C to fix the adhered bacteria. The membrane surface was observed using CLSM, and the CLSM images were analyzed using the COMSTAT software [23] to calculate the volume per unit area (μm3/μm2) of the adhered bacteria on the membrane surface. 2.4.3. Effect of drag force on bacterial adhesion To estimate the effect of the drag force on bacterial adhesion, the pre-formed biofilm on the membrane surface was exposed to different permeate flux, and the remaining biofilm was analyzed by CLSM. The biofilm was pre-formed by 24 h in the biofouling experiment using the same protocol described in Section 2.4.1 at 1.50 MPa of the TMP and 300 rpm of the stirring rate. Then, we continuously supplied 0.85 wt.% NaCl aqueous solution for 24 h into the cell at the various permeate flux from 0 to 0.4 m3/m2 day, to prevent further bacterial growth and to eliminate the effect of the CP of nutrients. The permeate flux was adjusted by the backpressure valve on the permeate water side. The TMP and stirring rate were maintained at 1.50 MPa and 300 rpm, respectively. 2.4.4. Effect of hydrodynamic shear force on bacterial adhesion To estimate the effect of the hydrodynamic shear force on the bacterial adhesion, the pre-formed biofilm on the membrane surface was exposed to the different stirring rates, and the detached bacteria were measured. After biofilm pre-formation using the same protocol as described in Section 2.4.1 at 1.50 MPa of the TMP and 100 rpm of the stirring rate, we continuously supplied 5-fold diluted FAB medium into the cell at 1.50 MPa of the TMP and at various stirring rates from 100 to 300 rpm. The bacterial concentration in the condensate water was measured by a pour plate method to evaluate the bacteria detached from the membrane surface due to the shear force caused by stirring. Briefly, 1 mL of condensate water was added to 27 mL of warm FAB medium containing 1.5 g/L agar. The mixed solution was poured onto plastic plates, cooled at room temperature, and solidified. The prepared plates were incubated for 24 h at 30 °C, and the bacterial colonies on the plates were then counted.
Fig. 3. Time course of the normalized permeate fluxes, J/J0, on the biofouling experiments operated at different TMPs: (●) 0.75 MPa, (○) 1.0 MPa, (■) 1.25 MPa, and (□) 1.5 MPa. The stirring rate was 300 rpm.
at just 58% of J0 after 24 h of the biofouling experiment, while that at 0.75 MPa was retained at about 77%. The increase of TMP enhanced the reduction rate in J. The value of J0 increased with increasing TMP (Fig. S2, details in Supplementary data). It is well known that a higher permeate flux results in increased severity of biofouling [14,17]. The increase in J would increase the drag force and CP on the membrane surface leading to transport of bacteria and nutrients, resulting in the faster biofilm development. Fig. 4A to D shows the analyzed CLSM images of stained bacteria on membrane surfaces after 24 h of the biofouling experiments at different TMPs. At the TMP of 0.75 MPa, bacteria that covered the membrane
2.5. Evaluation of concentration polarization by osmolarity calculation The effect of CP caused by the permeate flux was evaluated by calculating the osmolarity on the membrane surface from the difference of the permeate flux with the feed solution and pure-water permeability. The osmotic pressure on the membrane surface, πm, can be calculated using the Morse equation: πm = CmRT, in which Cm, R, and T are the osmolarity on the membrane surface, gas constant, and temperature, respectively. The permeate flux with the feed solution, J, can be described as J = L(P − πm), in which L and P are the pure-water permeability and TMP respectively [19]. From these equations, the osmolarity on the membrane surface is calculated as Cm = (P − J / L) / RT. The values of J and L were measured by using the same apparatus as shown in Fig. 1. 3. Results and discussion 3.1. Effect of TMP on biofouling The time courses of normalized permeate flux, J/J0, on the biofouling experiment at different TMPs are shown in Fig. 3, in which J0 is the initial permeate flux. The value of J/J0 decreased with time at all experimental conditions as biofouling occurred. The value of J at 1.5 MPa was retained
Fig. 4. Effect of TMP on bacterial adhesion to the membrane surface after 24 h of biofouling experiments: (A to D) Analyzed CLSM images of the membrane surface at different TMPs of 0.75 (A), 1.0 (B), 1.25 (C), and 1.5 MPa (D). (E) Relation of the calculated volume of the bacteria adhered to the membrane surface (●) with J/J0 (○) after the biofouling experiments. The stirring rate was 300 rpm.
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surface were sparser than at the TMP of 1.5 MPa. Fig. 4E shows the calculated volume of the bacteria adhered on the membrane surface and J/J0 of the biofouling experiments at different TMPs. The bacterial volume increased with an increment in the applied TMP. Increased bacterial volume indicates bacterial growth and biofilm formation which results in the resistance to water permeation [15,24]. Increased TMP resulted in decreased permeate flux and increased volume of adhered bacteria in the above biofouling experiment (Fig. 4). The possible reasons for this may include, the effect of hydraulic pressure due to TMP on bacterial growth, adhesion of bacteria to the membrane surface due to drag force, and the CP of nutrients in medium on the membrane surface. It is reported that the bacterial growth on the membrane surface is inhibited by application of TMP in the range of several dozens of MPa [25,26]. We confirmed that the applied hydraulic pressure in the range in the above experiments hardly affects bacterial growth adhered to the membrane surface (Fig. S3, details in Supplementary data). Thus, the TMP in this study was not high enough to inhibit bacterial growth on the membrane surface. The impact of drag force caused by water permeation through the membrane on detachment of bacteria from the membrane surface was investigated. The bacterial volume of the pre-formed biofilm as a function of J is shown in Fig. 5. The bacterial volume of the biofilm on the membrane surface at high J was almost equal to those obtained just before the experiment (dashed line), while they decreased with decreasing J and reached values lower than those obtained before the experiment. When the drag force is weak, it is not enough to maintain bacterial adhesion and the bacteria detach from membrane surfaces due to shear force generated by cross-flow velocity [27]. On the other hand, a higher drag force presses and retains bacteria on the membrane surface and expedites bacterial growth on the membrane surface due to the presence of nutrients enriched by CP. Because NaCl solution was used to eliminate the effect of CP, the increase in bacterial volume could mostly be attributed to bacteria pressing on the membrane surface due to drag force. Fig. 6 shows the calculated osmolarity on the membrane surface under different applied TMPs. The osmolarity on the membrane surface increased with an increase in the TMP, and reached at least 2 times higher than that of the feed water. This increment in osmolarity is attributed to the CP phenomenon enhanced by the permeate flux. The enriched nutrients facilitate bacterial growth and biofilm formation on the membrane surface resulting in resistance to water permeation. The tendency of the decrease in J/J0 with an increase in the TMP (Fig. 3) is clearly attributed to the increase of osmolarity at the membrane surface. These results indicate that CP is the cause of biofouling of the RO membranes.
Fig. 5. Effect of permeate flux, J, on the bacterial volume of pre-formed biofilm on the membrane surface. The dashed line indicates the bacterial volume of pre-formed biofilm before supplying the NaCl aqueous solution. The TMP and stirring rate were maintained at 1.50 MPa and 300 rpm, respectively.
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Fig. 6. The calculated osmolarity on the membrane surface. The dashed line indicates the osmolarity of the feed water. The stirring rate was 300 rpm.
3.2. Effect of stirring rate on biofouling The time courses of J/J0 on the biofouling experiment at the different stirring rates are shown in Fig. 7. The value of J/J0 at 100 rpm decreased to 65% after 24 h of the biofouling experiment, while that at 300 rpm was retained at about 80%. These results clearly show that the decline rate of J/J0 increased with a decrease in the stirring rate. Fig. 8A to C shows the analyzed CLSM images of the stained bacteria on the membrane surfaces after 24 h of the biofouling experiments at different stirring rates. The thickness and coverage of the bacteria adhered to the membrane surface decreased with an increase in the stirring rate. The bacterial volume was also sufficiently decreased by the stirring rate (Fig. 8D). The stirring rate would mainly affect two factors, the hydrodynamic shear force and CP. Decreased stirring rates reduce the hydrodynamic shear force on the membrane surface, which would reduce bacterial detachment from the membrane surface. On the other hand, decreased stirring rates promote the CP and increase nutrient concentration on the membrane surface resulting in increased bacterial growth. Therefore, we focused on the effect of stirring rates on these two factors, hydrodynamic shear force and CP. Hydrodynamic shear force resulting from higher cross-flow velocity possibly detaches bacteria from the membrane surface and prevents biofilm formation [14,27]. We measured the bacterial concentration in the condensate water from the biofouling experiments at various stirring rates (Fig. 9). The bacterial concentration in the condensate water increased upon increasing the stirring rate. This result indicates that the shear force generated by stirring detaches bacteria from the membrane surface. Thus, one reason for the decrease of permeate flux with
Fig. 7. Time course of the J/J0 on the biofouling experiments operated at the different stirring rates: (■) 100 rpm, (○) 200 rpm, and (●) 300 rpm. The TMP was 0.75 MPa.
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Fig. 10. Effect of stirring rates on calculated osmolarity of the membrane surface. The dashed line indicates the osmolarity of the feed water. The TMP was 0.75 MPa.
detachment and not by CP reduction. Thus, higher stirring rates could reduce biofouling by both CP reduction and bacterial detachment. 4. Conclusion
Fig. 8. Effect of stirring rates on bacterial adhesion to the membrane surface after 24 h of biofouling experiments: (A to C) Analyzed CLSM images of the membrane surface at different stirring rates of 100 rpm (A), 200 rpm (B), and 300 rpm (C). (D) Relation of the calculated volume of the bacteria adhered to the membrane surface (●) with J/J0 (○) after the biofouling experiments. The TMP was 0.75 MPa.
increasing stirring rates was bacterial detachment from the biofilm formed on the membrane surface. Fig. 10 shows the calculated osmolarity on the membrane surface at different stirring rates. The osmolarity on the membrane surface was higher than that of the feed water and slightly decreased with increased stirring rates. The increased stirring rate prevents CP due to decreasing thickness of the boundary layer on the membrane surface [28]. In this study, the range of stirring rates analyzed was relatively small and therefore stirring rates did not affect the CP remarkably. The theoretical study by Qiu and Davies showed that cross-flow velocity slightly decreased the degree of CP [19]. In this study, inhibition of biofouling with increasing stirring rates was mainly caused by bacterial
The effects of operating conditions on biofouling in RO membrane processes were systematically investigated. The biofouling of the RO membranes was evaluated by filtrating bacterial medium through a circular polyamide RO membrane with pre-adhered bacteria. The TMP and stirring rate affecting the membrane surface in a cylindrical membrane cell were changed as the operating conditions. The application of TMP facilitated decreased rates of the permeate flux and the increased bacterial adhesion on the membrane surface. The increase in calculated osmolarity on the membrane surface suggested that the TMP caused CP of bacterial nutrients and accelerated the biofouling. An increase in the stirring rate prevented the decrease of permeate flux as well as bacterial growth on the membrane surface. This biofouling inhibition was mainly caused by reduction in bacterial volume due to hydrodynamic shear force and not the CP. These results show that lower TMP and higher stirring rates are favorable to prevent the biofouling by reducing CP reduction and promoting bacterial detachment. Supplementary data An AFM image of the unused polyamide RO membrane and methods of AFM observation (Fig. S1), effect of TMP on J0 (Fig. S2), and effect of the hydraulic pressure on the bacterial growth (Fig. S3). Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j. desal.2015.09.020. References
Fig. 9. Effect of stirring rates on bacterial detachment from the pre-adhered bacteria on the membrane surface. The bacterial concentration in the condensate water was counted by the pour plate method.
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