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The efficacy of different cleaners and sanitisers in cleaning biofilms on UF membranes used in the dairy industry
Journal of Membrane Science 352 (2010) 71–75
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Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
The efficacy of different cleaners and sanitisers in cleaning biofilms on UF membranes used in the dairy industry X. Tang a,∗ , S.H. Flint a , R.J. Bennett a , J.D. Brooks b a b
Institute of Food, Nutrition and Human Health, Massey University, Riddet Reception, Reddet Road, Palmerston North, New Zealand School of Applied Sciences, Auckland University of Technology, Auckland, New Zealand
a r t i c l e
i n f o
Article history: Received 15 September 2009 Received in revised form 18 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: Ultrafiltration PES membrane Biofilm Klebsiella oxytoca Electrolysed water
a b s t r a c t The efficacy of different cleaners and sanitisers for removing or killing Klebsiella oxytoca in biofilms on ultrafiltration (UF) membranes from the dairy industry was investigated. K. oxytoca B006 was grown individually and combined with another K. oxytoca strain, TR002, on polyethersulfone (PES) UF membranes in 5% whey medium in CBR 90 biofilm reactors. Both strains were previously isolated from New Zealand dairy plants. Three enzymatic cleaners were compared with sodium hypochlorite (pH 10.8–11) at 200 ppm free available chlorine (FAC) commonly used for cleaning-in-place (CIP) of UF membranes in the dairy industry. In addition, 4 sanitisers were used to treat the membranes after a CIP wash regime. The efficacy in reducing culturable bacteria in biofilms was measured using pour plate counting on standard plate count agar. QuatroZyme® , which is composed of mixed enzymes, performed slightly better than the other cleaners. The treatments with all the sanitisers improved cleaning. MIOX® EW anolyte (pH 6.8) (120 ppm) was the most effective sanitiser compared to the control CIP. However, PES UF membranes are known to be sensitive to oxidants at low pH and therefore the damage to these membranes by MIOX® EW anolyte needs to be determined. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ultrafiltration is frequently used in dairy product manufacture (i.e. for concentration of whey and milk), and spiral-wound, polyethersulfone (PES) UF membranes are most commonly used [1]. However, fouling is a serious problem in the application of membrane technology [2–4]. Dairy components, such as proteins, fats and minerals, are considered to be the key membrane fouling particles. To maintain the permeability and the selectivity of the membranes, regular chemical cleaning is required every 18–24 h. Many studies related to membrane cleaning have been done in the past 10 years, and most of them have focused on cleaning of protein fouling [1,4–6]. However, the cleaning of biofilm on the membranes has been rarely studied. Biofilms growing on the membranes have been reported to be a problem, resulting in membrane blockage, product contamination, and reduction of membrane life due to the microbial action on the membrane material [7,8]. A typical dairy clean-in-place (CIP) process consists of an alkaline or acid wash followed by a sodium hypochlorite wash at pH 11–12 (200 ppm). The alkaline treatment solubilises proteins, fats and carbohydrates, while the acid dissolves minerals. Sodium hypochlorite is widely used as a disinfectant. However, when it is
∗ Corresponding author. Tel.: +64 6 350 5600x7372; fax: +64 6 350 5757. E-mail address: [email protected] (X. Tang). 0376-7388/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2010.01.063
used at alkaline pH, it is not considered a true sanitiser, as this pH limits the amount of hypochlorous acid produced [9]. Treatment with hypochlorous acid reportedly damages polyamide reverse osmosis membranes [10]. PES membranes were found to be unstable in solutions containing chlorine [11]. However, in another study, Wienk et al. [12] stated that there was no reaction between PES and hypochlorite at pH 6.9–11.5 after analysing the molecular mass of PES. Enzymes (proteases and lipases) are often selected as complementary cleaning agents when simple chemicals (alkaline and acid) are not enough for cleaning and recovering the membrane capacity. However, most of the studies using enzyme cleaners focused on removing protein fouling, but did not evaluate the microbial component i.e. biofilms [6,13]. Although many cleaners have some ability to disinfect, control of biofilms always requires detergent cleaning followed by the use of a sanitiser [14]. There is a wide range of sanitisers available for use in food processing industries. Peroxyacetic acid (PAA) is a sanitiser with high oxidising potential sometimes used in dairy plants and it is effective against bacteria, fungi and spores [15]. It is not inactivated by catalase or peroxidase. Ozone has been used for many years in European countries. The main use is to disinfect drinking water [16]. Greene et al. [17] reported that both ozonated water and chlorine have equivalent decontamination efficacies. However, application of ozone does not
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Table 1 Standard CIP for dairy membrane processing plants (the sixth step of using sodium hypochlorite at high pH was considered as the control). Step 1 2 3 4 5 6 7
Water pre-flush Alkaline recirculation Water flush Acid recirculation Water flush Alkali + sodium hypochlorite recirculation Water flush
Chemicals Reflux® B615 Reflux® R410 Reflux® B615 + Reflux® S800
require heating and the necessary contact time is likely to be less than when using chlorine, as ozone is a more powerful oxidizer than chlorine [17]. Most recent studies have found that ozonated water was effective in inactivating biofilms of Pseudomonas fluorescens on glass slides [18]. Electrolysed water (EW) is an alternative treatment for decontamination. The advantages of using EW are that it is easy to produce, is stable if stored in a sealed container [19] and it does not require a high temperature for operation. Mahmoud [20] reviewed the production of EW and its antimicrobial activity on different foods. Other researchers also reported the efficiency of using EW as a sanitiser for food. Ongeng et al. [21] suggested using electrolysis for sanitising water used for final rinsing of vegetables. Cao et al. [22] found that slightly acidic electrolysed water was effective for inactivating Salmonella enteritidis. The performance of those cleaners and sanitisers described above in terms of removing and killing biofilms on membranes is unknown. Our previous study reported membrane contamination by biofilms of two Klebsiella strains isolated from dairy UF membrane plants [23]. The objective of this study was to investigate the efficacy of selected cleaners and sanitisers in removing and killing biofilms comprising single and dual Klebsiella strains on PES UF membranes. 2. Experimental 2.1. Sources of strains Klebsiella oxytoca B006 was isolated from a liquid sample taken from a UF membrane plant processing whey, and K. oxytoca TR002 was isolated from a biofilm sample scraped from a UF membrane plant processing whey at a second dairy manufacturing plant. These Klebsiella strains are known to attach [23] and form biofilms on membrane surfaces [24]. 2.2. Preparation of medium Five percent whey medium was prepared by mixing whey protein concentrate powder (Fonterra Co-operative Group Ltd., Auckland, New Zealand) with sterilized lactose (Fonterra Cooperative Group Ltd., Auckland, New Zealand) and artificial whey permeate, which was prepared by mixing the following minerals in deionised water to make 1 L (pH 6.0–6.1) (52.7 mL of 2 mol L−1 KOH (BDH, Poole, England), 24.29 g Na3 C3 H5 O(CO2 )3 ·2H2 O (Merck KGaA, Darmstadt, Germany), 4.99 g C6 H5 K3 O7 ·2H2 O (UNIVAR, Auckland, New Zealand), 3.67 g CaCl2 ·2H2 O (Biolab, Clayton, Australia), 5.85 g MgCl2 ·6H2 O (J.T. Baker, Phillipsburg, Mexico), 23.36 g KH2 PO4 (Merck KGaA, Darmstadt, Germany) and 17.1 mL of 3 mol L−1 H2 SO4 (Biolab, Clayton, Australia)). 2.3. Preparation of inocula Pure cultures of K. oxytoca B006 and TR002 were grown on skim milk agar (SMA) for 24 h at 30 ◦ C and then a colony was inoculated
Time (min)
Temperature (◦ C)
10 30 20 25 20 30 20
50 50 50 50 50 50 50
pH target 10.8–11.0 1.8–2.0 10.8–11.0 (200 ppm FAC)
into 10 mL whey and incubated for 24 h at 30 ◦ C. This was diluted in whey to reach a density of 106 –107 CFU mL−1 , confirmed by agar plate counting on standard plate count agar (SPCA) (Merck KGaA, Darmstadt, Germany). 2.4. Membranes Spiral-wound PES membranes that had been used in industrial production were provided by a New Zealand dairy manufacturing plant processing cheese whey by membrane filtration. Previous studies, using the same membranes, found that denser biofilms formed on those used membranes than on new membranes [24]. Membranes were cut into several small rolls using a band saw sterilized by 95% ethanol, and stored in a 4 ◦ C cold room. Before each experiment, a piece of membrane sheet was cut using sterile scalpel blades to provide a surface area of 1.27 cm2 , to fit a CBR 90 laboratory biofilm reactor (BioSurface Technologies, Bozeman, USA). The membranes were treated in the biofilm reactor with a typical CIP before being used for biofilm development [24]. The membranes were supported in the holders by standard polycarbonate coupons. This necessity introduced a limitation on the experiment, in that the membrane was positioned with one side against an impermeable surface. This configuration is not the same as found in the plant, where there is a constant flux of cleaning solutions through the membrane in addition to the cross flow. However, this approach allowed easier evaluation of different sanitisers under comparable conditions. 2.5. Biofilm development For developing biofilm of a single culture, 1 mL of the prepared inoculum was inoculated into the reactor. For preparing binary biofilms, 1 mL of each inoculum was inoculated. Biofilms on the membranes were generated after 24 h incubation at 25 ◦ C in the CBR 90 with stirring at 180 rpm and a continuous supply of 5% whey medium flowing through at 5.5 ± 0.5 mL/min. The operating volume of the reactor was 330 mL. The flow rate was set, based on the calculated planktonic growth data obtained from batch experiments, to ensure that the hydraulic retention time was less than the shorter cell doubling time of the two strains (B006 1.15 h, TR002 1.23 h), thus minimizing planktonic cell growth [25]. To measure the extent of biofilm growth before CIP, membrane samples were taken after 24 h incubation and rinsed for 1 min in a sterile glass bottle containing 15 mL sterilized reverse osmosed (RO) water. The samples were then transferred into 10 mL sterilized peptone water (Merck KGaA, Darmstadt, Germany) with 4 glass balls (d = 5 mm) and treated for 2 min in a sonicator water bath (Soniclean Pty Ltd., Thebarton, SA, Australia) to remove biofilm from the membrane surface and disrupt biofilm clumps. This method was shown in a previous study to effectively remove attached cells in a previous study [24]. The resulting peptone solution containing detached biofilm cells was then diluted in peptone in serial 10-fold dilutions and surface-plated (0.1 mL) onto SPCA.
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Table 2 The list of cleaners used to compare with the control (sodium hypochlorite at pH 10.8–11). Chemicals ®
Reflux E2001 (protease and lipase) Reflux® E1000 (protease) QuatroZyme® (lipase, protease, cellulase, amylase)
Dose (v/v)
Temperature (◦ C)
0.2% 0.2% 0.3%
48 48 48
pH
Exposure time (min)
8.5–9.5 9.0–10.0 7.0–8.0
45 45 30
Table 3 The list of sanitisers used following the CIP. Chemicals
Dose
Temperature (◦ C)
Exposure time (min)
Sodium hypochlorite (Reflux® S800) pH 6.5 Perform® (H2 O2 /PAA) MIOX® EW anolyte pH 6.8 (1 day old) Ozonated water pH 7.0
200 ppm FAC 2%, v/v 120 ppm FAC 0.5 ppm FAO
30 25 20 ± 1 20 ± 1
20 20 10 10
2.6. Cleaning and sanitising After generating the biofilm on the membrane surfaces, different CIP treatments using different cleaners were tested, followed by treatment with a selection of sanitisers (Tables 1–3). The standard CIP procedure (Table 1) was as used by the New Zealand dairy industry and was considered as a control. The free available chlorine (FAC) was determined using a standard sodium thiosulfate titration. Cleaning solution (500 mL) was recirculated through the reactor at a rate of 198 mL/min and flowed over the membranes. The cleaners listed in Table 2 were used to take the place of the “alkali + hypochlorite” step in the standard CIP (Table 1). Reflux® chemicals and enzymes including QuatroZyme® and Perform® were obtained from Orica, Auckland, New Zealand. Sanitisers (Table 3) were used as an additional step in the CIP. EW was generated by a laboratory scale mixed oxidants brine pump system (MIOX® BPS) (MIOX Corporation, New Mexico, USA) using 1% NaCl solution at 5 A and 12 V. The mixed oxidant solutions from the anolyte were stored in a sterile container at 4 ◦ C for 1 day before use. The recommended storage life of EW is 48 h (MIOX Corporation, New Mexico, USA). The pH was adjusted to 6.8 and the tested FAC was 120 ppm. Ozonated water containing 0.5 ppm free available ozone (FAO) as previously used by others [17] was generated using an ozone generator (Ozonator model VT-2A, EnvirOzone, Napier, New Zealand). FAO was calculated from ozone pumping speed and time. Its pH was adjusted to 7.0. 2.7. Sanitiser screening test After the CIP step (Table 1, steps 1–5), coupon holders with 3 membrane samples on each were removed from the CBR 90 reactor and placed into a 200 mL beaker containing 150 mL sanitiser and sanitised as described in Table 3 with stirring at 180 rpm. After sanitising, each membrane sample was inserted into a 25 mL glass bottle containing 10 mL Dey/Engley (D/E) neutralizing solution (DifcoTM , Sparks, MD) and incubated for 10 min to neutralize the sanitisers. Membrane samples with no sanitiser treatment were considered as the control. To estimate the culturable cells left on the membrane surfaces, the membrane samples were sonicated for
2 min in 10 mL sterile peptone water with glass beads and the liquid was then centrifuged for 10 min at 2500 × g. Eight millilitres of the supernatant was discarded and the pellet was resuspended to obtain a final 2 mL sample. The centrifugation method was tested to verify the recovery of cells and it was found that the recovery was 99% (p < 0.05). Serial 10-fold dilutions were prepared in sterile peptone water, and tempered SPCA was inoculated with 2 mL samples using pour plating and incubated at 30 ◦ C for 3 days before the colonies were counted. 2.8. Statistical analysis Plate counts for each membrane sample were converted to log10 values. Each mean and standard deviation was calculated from the counts of three identical tested membrane samples. All the data were analysed using the analysis of variance (ANOVA) test in Minitab software (Release 15; Minitab Inc., State College, PA, USA) at the 95% confidence level. 3. Results and discussion 3.1. The efficacy of standard CIP Both this and earlier studies [24] using the CBR 90 biofilm reactor generated consistent biofilms of K. oxytoca with a log density of approximately 7.42 ± 0.30 log CFU cm−2 on membrane surfaces. A typical CIP procedure involving alkali, acid and sodium hypochlorite at high pH was chosen as the cleaning control because this CIP procedure is widely used in the dairy industry. Unfortunately, the CIP control in the CBR 90 did not completely remove the biofilm, perhaps as a result of imperfect cleaning between the membrane sample and the holder in the reactor. The mounting of the membrane sample also affects the efficiency of cleaning, as has been pointed out previously (Section 2.4). In some ways, this experimental deficiency mimics the situation in a plant, where rubber seals become cracked and harbour biofilms. In our experiments, we used a small surface area (1.27 cm2 ) and the results showed that a standard CIP allowed a culturable count of 1.40–2.18 log CFU cm−2 to remain on the membrane surface. This number would be a concern when multiplied to reflect the total area of an industrial scale plant.
Table 4 The efficacy of different cleaners in reducing the viable cells on membrane surfaces (means and standard deviations were taken from triplicates). Cleaners
Reduction (log CFU cm−2 ) Single strain K. B006
Alkaline hypochlorite 200 ppm FAC (control) Reflux® E2001 Reflux® E1000 QuatroZyme®
6.01 6.02 5.17 6.15
± ± ± ±
0.54 0.31 0.14 0.22
Dual strains K. B006 and TR002 5.23 5.31 4.98 5.31
± ± ± ±
0.13 0.36 0.12 0.23
± ± ± ± 2.06 2.06 2.06 0.40 0.11a 0.11a 0.11a 0.23 ± ± ± ± 2.19 2.19 2.19 0.78 0.26 0.28 0.55 0.22 ± ± ± ± 0.64 0.95 1.87 0.46 0.16 0.14 0.47 0.04 ± ± ± ± a
0.55 0.62 0.51a 0.66 ± ± ± ± 0.61 0.51 1.21 0.59 Sodium hypochlorite 200 ppm FAC, pH 6.5 Perform® 2%, v/v MIOX EW anolyte 120 ppm FAC, pH 6.8 Ozonated water 0.5 ppm FAO, pH 7.0
Cleaner
Reduction (log CFU cm
The case that all the detectable viable cells remaining after cleaning were killed. The results were obtained from pour plate counting.
0.63 0.57 1.76 0.33 0.27 0.31 0.20a 0.25 ± ± ± ± 1.45 1.39 1.55 0.56 0.03a 0.03a 0.03a 0.07 ± ± ± ± 1.85 1.85 1.85 0.27 0.28 0.49 0.07a 0.24 ± ± ± ±
Reflux® E2001
)
Reflux® E2001
Reflux® E1000
QuatroZyme®
Alkali + sodium hypochlorite 200 ppm FAC (control)
Reduction (log CFU cm−2 )
Dual strains (K. B006 and TR002)
−2
The efficacy of different sanitisers (applied in beakers with stirring at 180 rpm) in reducing counts of culturable bacteria from residual biofilm following CIP with different cleaners is shown in Table 5. The effectiveness of sanitising was not significantly (p > 0.05) influenced by the presence of a second bacterial strain. MIOX® EW anolyte (120 ppm FAC, pH 6.8) gave the highest log reduction in all experiments and was the most effective sanitiser in reducing culturable cell numbers (Table 5). The lowest culturable counts were obtained from surfaces after treatment with the MIOX® EW anolyte. In most cases, counts were below the lower detection limit of −0.1 log CFU cm−2 . Ozonated water was the weakest sanitiser tested, with the lowest log reduction recorded around 0.27 log CFU cm−2 . Sodium hypochlorite and Perform® resulted in very similar log reductions to the MIOX® EW anolyte when used after treatment with Reflux E2001, Reflux E1000 and Quatrozyme cleaners for single species biofilms, and after treatment with Reflux E1000 and Quatrozyme cleaners for dual species biofilms (Table 5). The limitation of using plate counting for assessing cell numbers is that this method may not recover all the viable cells and only culturable cells are countable. Therefore, it is possible that viable but non-culturable cells may persist in the different treatments. The significance of such non-culturable cells in an industrial plant is not known. Our laboratory trials differed from the industrial scale in the amount of cleaning agent used per unit area of membrane surface, even though the concentrations used for our laboratory experiments were the same as the industrially applied values. The volume of cleaning solution used in dairy membrane plants is 4–5 L m−2 [29], while in our laboratory reactor systems the amount of cleaning solution was more than 82 L m−2 membrane. This was mainly due to the operating volume of the CBR 90 reactor at 330 mL and a membrane sample with a small surface area (total 30.48 cm2 ). Thus, the main differences between the laboratory- and industrial scale-cleaning regime were the volumes of water for flushing, and reagents for cleaning or sanitising. This suggests that using the same cleaners or sanitisers in an industrial scale membrane plant
Single strain (K. B006)
3.3. The efficacy of sanitisers
Sanitiser
The efficacy of different cleaners used in the CBR 90 in reducing counts of culturable bacteria on membrane surfaces is shown in Table 4. All the cleaners were more effective on biofilms of a single strain, than those composed of the dual strains, when no sanitiser was applied, as cleaning efficiency was significantly (p = 0.038) affected by the inoculation of single or dual strains. However, the effectiveness of different cleaners in reducing culturable bacterial numbers did not differ significantly (p > 0.05). The control clean removed 70–80% of culturable cells from the membrane surfaces. The effectiveness in reducing the number of culturable cells in biofilms using mixed enzymes has been reported [28]. In our studies, QuatroZyme® , containing a mixture of enzymes, performed slightly better than the other cleaners, but still left at least 1.2 log CFU cm−2 culturable cells on the membrane surfaces. Reflux® E1000 was less effective than the control in removing the bacteria from the membrane surface.
Table 5 The efficacy of different sanitisers in reducing viable cells on cleaned membrane surfaces (means and standard deviations were calculated from triplicates).
3.2. The efficacy of cleaners
1.86 1.74 1.92 0.41
Reflux® E1000
QuatroZyme®
There is evidence elsewhere that biofilms may protect bacterial cells against CIP chemicals and that culturable bacterial cells can remain attached to dairy manufacturing surfaces following a CIP [26,27]. The cells remaining on the surface will enable rapid regeneration of biofilm once suitable conditions are restored during the processing of dairy liquids.
0.18a 0.18a 0.18a 0.27
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Alkali + sodium hypochlorite 200 ppm FAC (control)
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might result in higher residual culturable bacterial counts than those which were achieved in our trials. Ozonated water appeared to be the weakest sanitiser among those used in this study. This may be because ozonated water application must be strictly controlled, such as being used freshly made in a completely closed unit; otherwise the ozone will transform into oxygen and lose the disinfectant activity [16]. Also, the effectiveness of ozone in terms of killing microorganisms is affected by ozone concentration, strains, temperature and pH [30]. 4. Conclusions Our study demonstrated that the use of sanitisers following a CIP procedure improved the reduction of culturable bacterial cells on the membrane surfaces. The most effective sanitiser from our studies was the MIOX® EW anolyte (120 ppm FAC, pH 6.8) when compared with the control CIP clean. Sodium hypochlorite and Perform® functioned equally well when combined with Reflux E1000 for single species biofilms, and with Reflux E1000 or Quatrozyme for dual species biofilms. This study would indicate that if a dairy processor is to use a standard CIP (like the control) on membrane systems, then a further flush with MIOX® EW anolyte would reduce residual attached microbial populations further. In addition, using enzyme cleaners, followed by a sanitiser, would be even more effective though in the latter case, one would have to use the enzyme cleaners with care, as residuals may impair dairy product being produced. The active disinfectant agent in the EW is believed to be hypochlorous acid [19]. However, our results showed that the effectiveness of the MIOX® EW anolyte with 120 ppm FAC (pH 6.8) in reducing culturable bacteria was greater than sodium hypochlorite with 200 ppm FAC (pH 6.5). This indicates that there might be something else (e.g. other oxidants), not only hypochlorous acid killing the culturable cells. For the application of anolyte of EW on membranes, the presence of chlorine and the low pH are the main concerns. This aspect of membrane sensitivity requires investigation to determine the effects of using the anolyte of EW at different pH values on the life of PES membranes. Acknowledgments We would like to thank the New Zealand dairy industry for providing used membrane materials and Davey Water Products, Auckland, New Zealand, for the MIOX BPS laboratory scale equipment for this study. The Department of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand, provided the laboratory and some assistance. This study was funded by Foundation of Research, Science and Technology, New Zealand. References [1] M. Rabiller-Baudry, L. Bégoin, D. Delaunay, L. Paugam, B. Chaufer, A dual approach of membrane cleaning based on physico-chemistry and hydrodynamics: application to PES membrane of dairy industry, Chem. Eng. Process. 47 (2008) 267–275. [2] J.L. Nilsson, Fouling of an ultrafiltration membrane by a dissolved whey protein concentrate and some whey proteins, J. Membr. Sci. 36 (1988) 147–160. [3] A.D. Marshall, P.A. Munro, G. Trägårdh, The effect of protein fouling in microfiltration and ultrafiltration on permeate flux, protein retention and selectivity: a literature review, Desalination 91 (1993) 65–108.
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Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
The efficacy of different cleaners and sanitisers in cleaning biofilms on UF membranes used in the dairy industry X. Tang a,∗ , S.H. Flint a , R.J. Bennett a , J.D. Brooks b a b
Institute of Food, Nutrition and Human Health, Massey University, Riddet Reception, Reddet Road, Palmerston North, New Zealand School of Applied Sciences, Auckland University of Technology, Auckland, New Zealand
a r t i c l e
i n f o
Article history: Received 15 September 2009 Received in revised form 18 January 2010 Accepted 28 January 2010 Available online 4 February 2010 Keywords: Ultrafiltration PES membrane Biofilm Klebsiella oxytoca Electrolysed water
a b s t r a c t The efficacy of different cleaners and sanitisers for removing or killing Klebsiella oxytoca in biofilms on ultrafiltration (UF) membranes from the dairy industry was investigated. K. oxytoca B006 was grown individually and combined with another K. oxytoca strain, TR002, on polyethersulfone (PES) UF membranes in 5% whey medium in CBR 90 biofilm reactors. Both strains were previously isolated from New Zealand dairy plants. Three enzymatic cleaners were compared with sodium hypochlorite (pH 10.8–11) at 200 ppm free available chlorine (FAC) commonly used for cleaning-in-place (CIP) of UF membranes in the dairy industry. In addition, 4 sanitisers were used to treat the membranes after a CIP wash regime. The efficacy in reducing culturable bacteria in biofilms was measured using pour plate counting on standard plate count agar. QuatroZyme® , which is composed of mixed enzymes, performed slightly better than the other cleaners. The treatments with all the sanitisers improved cleaning. MIOX® EW anolyte (pH 6.8) (120 ppm) was the most effective sanitiser compared to the control CIP. However, PES UF membranes are known to be sensitive to oxidants at low pH and therefore the damage to these membranes by MIOX® EW anolyte needs to be determined. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ultrafiltration is frequently used in dairy product manufacture (i.e. for concentration of whey and milk), and spiral-wound, polyethersulfone (PES) UF membranes are most commonly used [1]. However, fouling is a serious problem in the application of membrane technology [2–4]. Dairy components, such as proteins, fats and minerals, are considered to be the key membrane fouling particles. To maintain the permeability and the selectivity of the membranes, regular chemical cleaning is required every 18–24 h. Many studies related to membrane cleaning have been done in the past 10 years, and most of them have focused on cleaning of protein fouling [1,4–6]. However, the cleaning of biofilm on the membranes has been rarely studied. Biofilms growing on the membranes have been reported to be a problem, resulting in membrane blockage, product contamination, and reduction of membrane life due to the microbial action on the membrane material [7,8]. A typical dairy clean-in-place (CIP) process consists of an alkaline or acid wash followed by a sodium hypochlorite wash at pH 11–12 (200 ppm). The alkaline treatment solubilises proteins, fats and carbohydrates, while the acid dissolves minerals. Sodium hypochlorite is widely used as a disinfectant. However, when it is
∗ Corresponding author. Tel.: +64 6 350 5600x7372; fax: +64 6 350 5757. E-mail address: [email protected] (X. Tang). 0376-7388/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2010.01.063
used at alkaline pH, it is not considered a true sanitiser, as this pH limits the amount of hypochlorous acid produced [9]. Treatment with hypochlorous acid reportedly damages polyamide reverse osmosis membranes [10]. PES membranes were found to be unstable in solutions containing chlorine [11]. However, in another study, Wienk et al. [12] stated that there was no reaction between PES and hypochlorite at pH 6.9–11.5 after analysing the molecular mass of PES. Enzymes (proteases and lipases) are often selected as complementary cleaning agents when simple chemicals (alkaline and acid) are not enough for cleaning and recovering the membrane capacity. However, most of the studies using enzyme cleaners focused on removing protein fouling, but did not evaluate the microbial component i.e. biofilms [6,13]. Although many cleaners have some ability to disinfect, control of biofilms always requires detergent cleaning followed by the use of a sanitiser [14]. There is a wide range of sanitisers available for use in food processing industries. Peroxyacetic acid (PAA) is a sanitiser with high oxidising potential sometimes used in dairy plants and it is effective against bacteria, fungi and spores [15]. It is not inactivated by catalase or peroxidase. Ozone has been used for many years in European countries. The main use is to disinfect drinking water [16]. Greene et al. [17] reported that both ozonated water and chlorine have equivalent decontamination efficacies. However, application of ozone does not
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Table 1 Standard CIP for dairy membrane processing plants (the sixth step of using sodium hypochlorite at high pH was considered as the control). Step 1 2 3 4 5 6 7
Water pre-flush Alkaline recirculation Water flush Acid recirculation Water flush Alkali + sodium hypochlorite recirculation Water flush
Chemicals Reflux® B615 Reflux® R410 Reflux® B615 + Reflux® S800
require heating and the necessary contact time is likely to be less than when using chlorine, as ozone is a more powerful oxidizer than chlorine [17]. Most recent studies have found that ozonated water was effective in inactivating biofilms of Pseudomonas fluorescens on glass slides [18]. Electrolysed water (EW) is an alternative treatment for decontamination. The advantages of using EW are that it is easy to produce, is stable if stored in a sealed container [19] and it does not require a high temperature for operation. Mahmoud [20] reviewed the production of EW and its antimicrobial activity on different foods. Other researchers also reported the efficiency of using EW as a sanitiser for food. Ongeng et al. [21] suggested using electrolysis for sanitising water used for final rinsing of vegetables. Cao et al. [22] found that slightly acidic electrolysed water was effective for inactivating Salmonella enteritidis. The performance of those cleaners and sanitisers described above in terms of removing and killing biofilms on membranes is unknown. Our previous study reported membrane contamination by biofilms of two Klebsiella strains isolated from dairy UF membrane plants [23]. The objective of this study was to investigate the efficacy of selected cleaners and sanitisers in removing and killing biofilms comprising single and dual Klebsiella strains on PES UF membranes. 2. Experimental 2.1. Sources of strains Klebsiella oxytoca B006 was isolated from a liquid sample taken from a UF membrane plant processing whey, and K. oxytoca TR002 was isolated from a biofilm sample scraped from a UF membrane plant processing whey at a second dairy manufacturing plant. These Klebsiella strains are known to attach [23] and form biofilms on membrane surfaces [24]. 2.2. Preparation of medium Five percent whey medium was prepared by mixing whey protein concentrate powder (Fonterra Co-operative Group Ltd., Auckland, New Zealand) with sterilized lactose (Fonterra Cooperative Group Ltd., Auckland, New Zealand) and artificial whey permeate, which was prepared by mixing the following minerals in deionised water to make 1 L (pH 6.0–6.1) (52.7 mL of 2 mol L−1 KOH (BDH, Poole, England), 24.29 g Na3 C3 H5 O(CO2 )3 ·2H2 O (Merck KGaA, Darmstadt, Germany), 4.99 g C6 H5 K3 O7 ·2H2 O (UNIVAR, Auckland, New Zealand), 3.67 g CaCl2 ·2H2 O (Biolab, Clayton, Australia), 5.85 g MgCl2 ·6H2 O (J.T. Baker, Phillipsburg, Mexico), 23.36 g KH2 PO4 (Merck KGaA, Darmstadt, Germany) and 17.1 mL of 3 mol L−1 H2 SO4 (Biolab, Clayton, Australia)). 2.3. Preparation of inocula Pure cultures of K. oxytoca B006 and TR002 were grown on skim milk agar (SMA) for 24 h at 30 ◦ C and then a colony was inoculated
Time (min)
Temperature (◦ C)
10 30 20 25 20 30 20
50 50 50 50 50 50 50
pH target 10.8–11.0 1.8–2.0 10.8–11.0 (200 ppm FAC)
into 10 mL whey and incubated for 24 h at 30 ◦ C. This was diluted in whey to reach a density of 106 –107 CFU mL−1 , confirmed by agar plate counting on standard plate count agar (SPCA) (Merck KGaA, Darmstadt, Germany). 2.4. Membranes Spiral-wound PES membranes that had been used in industrial production were provided by a New Zealand dairy manufacturing plant processing cheese whey by membrane filtration. Previous studies, using the same membranes, found that denser biofilms formed on those used membranes than on new membranes [24]. Membranes were cut into several small rolls using a band saw sterilized by 95% ethanol, and stored in a 4 ◦ C cold room. Before each experiment, a piece of membrane sheet was cut using sterile scalpel blades to provide a surface area of 1.27 cm2 , to fit a CBR 90 laboratory biofilm reactor (BioSurface Technologies, Bozeman, USA). The membranes were treated in the biofilm reactor with a typical CIP before being used for biofilm development [24]. The membranes were supported in the holders by standard polycarbonate coupons. This necessity introduced a limitation on the experiment, in that the membrane was positioned with one side against an impermeable surface. This configuration is not the same as found in the plant, where there is a constant flux of cleaning solutions through the membrane in addition to the cross flow. However, this approach allowed easier evaluation of different sanitisers under comparable conditions. 2.5. Biofilm development For developing biofilm of a single culture, 1 mL of the prepared inoculum was inoculated into the reactor. For preparing binary biofilms, 1 mL of each inoculum was inoculated. Biofilms on the membranes were generated after 24 h incubation at 25 ◦ C in the CBR 90 with stirring at 180 rpm and a continuous supply of 5% whey medium flowing through at 5.5 ± 0.5 mL/min. The operating volume of the reactor was 330 mL. The flow rate was set, based on the calculated planktonic growth data obtained from batch experiments, to ensure that the hydraulic retention time was less than the shorter cell doubling time of the two strains (B006 1.15 h, TR002 1.23 h), thus minimizing planktonic cell growth [25]. To measure the extent of biofilm growth before CIP, membrane samples were taken after 24 h incubation and rinsed for 1 min in a sterile glass bottle containing 15 mL sterilized reverse osmosed (RO) water. The samples were then transferred into 10 mL sterilized peptone water (Merck KGaA, Darmstadt, Germany) with 4 glass balls (d = 5 mm) and treated for 2 min in a sonicator water bath (Soniclean Pty Ltd., Thebarton, SA, Australia) to remove biofilm from the membrane surface and disrupt biofilm clumps. This method was shown in a previous study to effectively remove attached cells in a previous study [24]. The resulting peptone solution containing detached biofilm cells was then diluted in peptone in serial 10-fold dilutions and surface-plated (0.1 mL) onto SPCA.
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Table 2 The list of cleaners used to compare with the control (sodium hypochlorite at pH 10.8–11). Chemicals ®
Reflux E2001 (protease and lipase) Reflux® E1000 (protease) QuatroZyme® (lipase, protease, cellulase, amylase)
Dose (v/v)
Temperature (◦ C)
0.2% 0.2% 0.3%
48 48 48
pH
Exposure time (min)
8.5–9.5 9.0–10.0 7.0–8.0
45 45 30
Table 3 The list of sanitisers used following the CIP. Chemicals
Dose
Temperature (◦ C)
Exposure time (min)
Sodium hypochlorite (Reflux® S800) pH 6.5 Perform® (H2 O2 /PAA) MIOX® EW anolyte pH 6.8 (1 day old) Ozonated water pH 7.0
200 ppm FAC 2%, v/v 120 ppm FAC 0.5 ppm FAO
30 25 20 ± 1 20 ± 1
20 20 10 10
2.6. Cleaning and sanitising After generating the biofilm on the membrane surfaces, different CIP treatments using different cleaners were tested, followed by treatment with a selection of sanitisers (Tables 1–3). The standard CIP procedure (Table 1) was as used by the New Zealand dairy industry and was considered as a control. The free available chlorine (FAC) was determined using a standard sodium thiosulfate titration. Cleaning solution (500 mL) was recirculated through the reactor at a rate of 198 mL/min and flowed over the membranes. The cleaners listed in Table 2 were used to take the place of the “alkali + hypochlorite” step in the standard CIP (Table 1). Reflux® chemicals and enzymes including QuatroZyme® and Perform® were obtained from Orica, Auckland, New Zealand. Sanitisers (Table 3) were used as an additional step in the CIP. EW was generated by a laboratory scale mixed oxidants brine pump system (MIOX® BPS) (MIOX Corporation, New Mexico, USA) using 1% NaCl solution at 5 A and 12 V. The mixed oxidant solutions from the anolyte were stored in a sterile container at 4 ◦ C for 1 day before use. The recommended storage life of EW is 48 h (MIOX Corporation, New Mexico, USA). The pH was adjusted to 6.8 and the tested FAC was 120 ppm. Ozonated water containing 0.5 ppm free available ozone (FAO) as previously used by others [17] was generated using an ozone generator (Ozonator model VT-2A, EnvirOzone, Napier, New Zealand). FAO was calculated from ozone pumping speed and time. Its pH was adjusted to 7.0. 2.7. Sanitiser screening test After the CIP step (Table 1, steps 1–5), coupon holders with 3 membrane samples on each were removed from the CBR 90 reactor and placed into a 200 mL beaker containing 150 mL sanitiser and sanitised as described in Table 3 with stirring at 180 rpm. After sanitising, each membrane sample was inserted into a 25 mL glass bottle containing 10 mL Dey/Engley (D/E) neutralizing solution (DifcoTM , Sparks, MD) and incubated for 10 min to neutralize the sanitisers. Membrane samples with no sanitiser treatment were considered as the control. To estimate the culturable cells left on the membrane surfaces, the membrane samples were sonicated for
2 min in 10 mL sterile peptone water with glass beads and the liquid was then centrifuged for 10 min at 2500 × g. Eight millilitres of the supernatant was discarded and the pellet was resuspended to obtain a final 2 mL sample. The centrifugation method was tested to verify the recovery of cells and it was found that the recovery was 99% (p < 0.05). Serial 10-fold dilutions were prepared in sterile peptone water, and tempered SPCA was inoculated with 2 mL samples using pour plating and incubated at 30 ◦ C for 3 days before the colonies were counted. 2.8. Statistical analysis Plate counts for each membrane sample were converted to log10 values. Each mean and standard deviation was calculated from the counts of three identical tested membrane samples. All the data were analysed using the analysis of variance (ANOVA) test in Minitab software (Release 15; Minitab Inc., State College, PA, USA) at the 95% confidence level. 3. Results and discussion 3.1. The efficacy of standard CIP Both this and earlier studies [24] using the CBR 90 biofilm reactor generated consistent biofilms of K. oxytoca with a log density of approximately 7.42 ± 0.30 log CFU cm−2 on membrane surfaces. A typical CIP procedure involving alkali, acid and sodium hypochlorite at high pH was chosen as the cleaning control because this CIP procedure is widely used in the dairy industry. Unfortunately, the CIP control in the CBR 90 did not completely remove the biofilm, perhaps as a result of imperfect cleaning between the membrane sample and the holder in the reactor. The mounting of the membrane sample also affects the efficiency of cleaning, as has been pointed out previously (Section 2.4). In some ways, this experimental deficiency mimics the situation in a plant, where rubber seals become cracked and harbour biofilms. In our experiments, we used a small surface area (1.27 cm2 ) and the results showed that a standard CIP allowed a culturable count of 1.40–2.18 log CFU cm−2 to remain on the membrane surface. This number would be a concern when multiplied to reflect the total area of an industrial scale plant.
Table 4 The efficacy of different cleaners in reducing the viable cells on membrane surfaces (means and standard deviations were taken from triplicates). Cleaners
Reduction (log CFU cm−2 ) Single strain K. B006
Alkaline hypochlorite 200 ppm FAC (control) Reflux® E2001 Reflux® E1000 QuatroZyme®
6.01 6.02 5.17 6.15
± ± ± ±
0.54 0.31 0.14 0.22
Dual strains K. B006 and TR002 5.23 5.31 4.98 5.31
± ± ± ±
0.13 0.36 0.12 0.23
± ± ± ± 2.06 2.06 2.06 0.40 0.11a 0.11a 0.11a 0.23 ± ± ± ± 2.19 2.19 2.19 0.78 0.26 0.28 0.55 0.22 ± ± ± ± 0.64 0.95 1.87 0.46 0.16 0.14 0.47 0.04 ± ± ± ± a
0.55 0.62 0.51a 0.66 ± ± ± ± 0.61 0.51 1.21 0.59 Sodium hypochlorite 200 ppm FAC, pH 6.5 Perform® 2%, v/v MIOX EW anolyte 120 ppm FAC, pH 6.8 Ozonated water 0.5 ppm FAO, pH 7.0
Cleaner
Reduction (log CFU cm
The case that all the detectable viable cells remaining after cleaning were killed. The results were obtained from pour plate counting.
0.63 0.57 1.76 0.33 0.27 0.31 0.20a 0.25 ± ± ± ± 1.45 1.39 1.55 0.56 0.03a 0.03a 0.03a 0.07 ± ± ± ± 1.85 1.85 1.85 0.27 0.28 0.49 0.07a 0.24 ± ± ± ±
Reflux® E2001
)
Reflux® E2001
Reflux® E1000
QuatroZyme®
Alkali + sodium hypochlorite 200 ppm FAC (control)
Reduction (log CFU cm−2 )
Dual strains (K. B006 and TR002)
−2
The efficacy of different sanitisers (applied in beakers with stirring at 180 rpm) in reducing counts of culturable bacteria from residual biofilm following CIP with different cleaners is shown in Table 5. The effectiveness of sanitising was not significantly (p > 0.05) influenced by the presence of a second bacterial strain. MIOX® EW anolyte (120 ppm FAC, pH 6.8) gave the highest log reduction in all experiments and was the most effective sanitiser in reducing culturable cell numbers (Table 5). The lowest culturable counts were obtained from surfaces after treatment with the MIOX® EW anolyte. In most cases, counts were below the lower detection limit of −0.1 log CFU cm−2 . Ozonated water was the weakest sanitiser tested, with the lowest log reduction recorded around 0.27 log CFU cm−2 . Sodium hypochlorite and Perform® resulted in very similar log reductions to the MIOX® EW anolyte when used after treatment with Reflux E2001, Reflux E1000 and Quatrozyme cleaners for single species biofilms, and after treatment with Reflux E1000 and Quatrozyme cleaners for dual species biofilms (Table 5). The limitation of using plate counting for assessing cell numbers is that this method may not recover all the viable cells and only culturable cells are countable. Therefore, it is possible that viable but non-culturable cells may persist in the different treatments. The significance of such non-culturable cells in an industrial plant is not known. Our laboratory trials differed from the industrial scale in the amount of cleaning agent used per unit area of membrane surface, even though the concentrations used for our laboratory experiments were the same as the industrially applied values. The volume of cleaning solution used in dairy membrane plants is 4–5 L m−2 [29], while in our laboratory reactor systems the amount of cleaning solution was more than 82 L m−2 membrane. This was mainly due to the operating volume of the CBR 90 reactor at 330 mL and a membrane sample with a small surface area (total 30.48 cm2 ). Thus, the main differences between the laboratory- and industrial scale-cleaning regime were the volumes of water for flushing, and reagents for cleaning or sanitising. This suggests that using the same cleaners or sanitisers in an industrial scale membrane plant
Single strain (K. B006)
3.3. The efficacy of sanitisers
Sanitiser
The efficacy of different cleaners used in the CBR 90 in reducing counts of culturable bacteria on membrane surfaces is shown in Table 4. All the cleaners were more effective on biofilms of a single strain, than those composed of the dual strains, when no sanitiser was applied, as cleaning efficiency was significantly (p = 0.038) affected by the inoculation of single or dual strains. However, the effectiveness of different cleaners in reducing culturable bacterial numbers did not differ significantly (p > 0.05). The control clean removed 70–80% of culturable cells from the membrane surfaces. The effectiveness in reducing the number of culturable cells in biofilms using mixed enzymes has been reported [28]. In our studies, QuatroZyme® , containing a mixture of enzymes, performed slightly better than the other cleaners, but still left at least 1.2 log CFU cm−2 culturable cells on the membrane surfaces. Reflux® E1000 was less effective than the control in removing the bacteria from the membrane surface.
Table 5 The efficacy of different sanitisers in reducing viable cells on cleaned membrane surfaces (means and standard deviations were calculated from triplicates).
3.2. The efficacy of cleaners
1.86 1.74 1.92 0.41
Reflux® E1000
QuatroZyme®
There is evidence elsewhere that biofilms may protect bacterial cells against CIP chemicals and that culturable bacterial cells can remain attached to dairy manufacturing surfaces following a CIP [26,27]. The cells remaining on the surface will enable rapid regeneration of biofilm once suitable conditions are restored during the processing of dairy liquids.
0.18a 0.18a 0.18a 0.27
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Alkali + sodium hypochlorite 200 ppm FAC (control)
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might result in higher residual culturable bacterial counts than those which were achieved in our trials. Ozonated water appeared to be the weakest sanitiser among those used in this study. This may be because ozonated water application must be strictly controlled, such as being used freshly made in a completely closed unit; otherwise the ozone will transform into oxygen and lose the disinfectant activity [16]. Also, the effectiveness of ozone in terms of killing microorganisms is affected by ozone concentration, strains, temperature and pH [30]. 4. Conclusions Our study demonstrated that the use of sanitisers following a CIP procedure improved the reduction of culturable bacterial cells on the membrane surfaces. The most effective sanitiser from our studies was the MIOX® EW anolyte (120 ppm FAC, pH 6.8) when compared with the control CIP clean. Sodium hypochlorite and Perform® functioned equally well when combined with Reflux E1000 for single species biofilms, and with Reflux E1000 or Quatrozyme for dual species biofilms. This study would indicate that if a dairy processor is to use a standard CIP (like the control) on membrane systems, then a further flush with MIOX® EW anolyte would reduce residual attached microbial populations further. In addition, using enzyme cleaners, followed by a sanitiser, would be even more effective though in the latter case, one would have to use the enzyme cleaners with care, as residuals may impair dairy product being produced. The active disinfectant agent in the EW is believed to be hypochlorous acid [19]. However, our results showed that the effectiveness of the MIOX® EW anolyte with 120 ppm FAC (pH 6.8) in reducing culturable bacteria was greater than sodium hypochlorite with 200 ppm FAC (pH 6.5). This indicates that there might be something else (e.g. other oxidants), not only hypochlorous acid killing the culturable cells. For the application of anolyte of EW on membranes, the presence of chlorine and the low pH are the main concerns. This aspect of membrane sensitivity requires investigation to determine the effects of using the anolyte of EW at different pH values on the life of PES membranes. Acknowledgments We would like to thank the New Zealand dairy industry for providing used membrane materials and Davey Water Products, Auckland, New Zealand, for the MIOX BPS laboratory scale equipment for this study. The Department of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand, provided the laboratory and some assistance. This study was funded by Foundation of Research, Science and Technology, New Zealand. References [1] M. Rabiller-Baudry, L. Bégoin, D. Delaunay, L. Paugam, B. Chaufer, A dual approach of membrane cleaning based on physico-chemistry and hydrodynamics: application to PES membrane of dairy industry, Chem. Eng. Process. 47 (2008) 267–275. [2] J.L. Nilsson, Fouling of an ultrafiltration membrane by a dissolved whey protein concentrate and some whey proteins, J. Membr. Sci. 36 (1988) 147–160. [3] A.D. Marshall, P.A. Munro, G. Trägårdh, The effect of protein fouling in microfiltration and ultrafiltration on permeate flux, protein retention and selectivity: a literature review, Desalination 91 (1993) 65–108.
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