Journal of Membrane Science 360 (2010) 165173

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Journal of Membrane Science

journal homepage: www.elsevier.com/locate/memsci

Biofouling formation and modeling in nanoltration membranes applied to wastewater treatment

Hanan Ivnitsky a , Dror Minz b , Larissa Kautsky b , Ami Preis a , Avi Ostfeld a , Raphael Semiat c , Carlos G. Dosoretz a,
a b c

Faculty of Civil & Environmental Engineering and Grand Water Research Institute, Technion, IIT, Haifa, Israel Institute of Soil, Water and Environmental Sciences, ARO, The Volcani Center, Bet-Dagan, Israel Faculty of Chemical Engineering and Grand Water Research Institute, Technion, IIT, Haifa, Israel

a r t i c l e

i n f o

a b s t r a c t
Biofouling development on nanoltration membranes treating tertiary efuents was studied at low (5 bar) and high (25 bar) pressures at different feedwater concentrations, temperatures and lengths of operation. The bacterial community prole composing the biofouling layer was characterized. Most of the bacterial species identied were Gram-negative, with Proteobacteria (approximately equally divided between , and subdivisions) and Bacteroidetes being the prevalent groups. At high-pressure, scaling was the primary source of fouling whereas at low-pressure, biofouling was dominant. For these conditions, an empirical approach to forecasting the contribution of biofouling resistance to total resistance was derived based on the resistance in series theory. This approach showed that biofouling becomes a dominating factor after approximately 20 L of permeate volume has been produced. A data-driven modeling algorithm for forecasting the reduction in permeate ux due to biofouling was also established. The reduction in permeate ux rates was related to the development of a fouling layer on the membrane. Pressure, total organic carbon, pH and conductivity of the feedwater were the most inuential parameters. These results are novel in the area of model tree algorithms as they apply to forecasting the development of biofouling on membranes. 2010 Elsevier B.V. All rights reserved.

Article history: Received 20 December 2009 Received in revised form 26 April 2010 Accepted 2 May 2010 Available online 11 May 2010 Keywords: Biofouling Membranes Wastewater Nanoltration Biolm Modeling

1. Introduction Although membrane biofouling is one of the most undesirable effects in pressure-driven membrane separation processes, very little is known about the fundamental nature of the process [13]. One of the biggest challenges still facing the membrane industry is the prevention of biofouling on membranes, as uncontrolled growth of a biolm can eventually lead to clogging of the membranes and impaired function of the system [4,5]. Pressure-driven crossow membrane separation systems present a differential environment for biolm development. The surface of attachment is a selective porous substratum which allows the transfer of certain molecules, whereas the concentration polarization (CP) layer generates an environment of relatively high nutrient and salt concentrations [69]. Herzberg and Elimelech [7] found a higher permeate ux decline in reverse osmosis (RO) membranes with a biolm layer than in those fouled with

Corresponding author at: Faculty of Civil & Environmental Engineering, Israel Institute of Technology, Haifa 32000, Israel. Tel.: +972 4 8294962; fax: +972 4 8228898. E-mail address: carlosd@tx.technion.ac.il (C.G. Dosoretz). 0376-7388/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2010.05.007

dead cells alone, which was mainly attributed to the increase in hydraulic resistance caused by the extra-cellular polymeric substances (EPS) surrounding the bacterial cells. Fonseca et al. [11] also noted that the observed ux decline in nanoltration (NF) membranes is associated with the excreted polymeric substances, rather than the bacterial cells themselves. Permeate ux decline has also been related to the application of high-pressure to the membranes, which generates a greater foulant-deposition rate [10]. Huang et al. [12] found that membrane ux has a great impact on the predominant populations in fouling biolms. The performance and fouling of NF and RO membranes seem to be controlled by feedwater composition, physical and chemical properties of the membrane and system operating conditions. In general, the processes and interactions causing fouling in membrane systems are likely to be complex. The composition of membrane fouling biolms has been repeatedly reported to include primarily Gram-negative bacterial species. , and Proteobacteria (including Sphingomonas and Nitrosomonas species) and Bacteroidetes (including Flavobacterium and Sphingobacterium species) have been found to be the dominant bacterial groups involved in fouling of membranes (MF, NF, RO) operated with fresh surface water and reclaimed efuents [9,1215,17]. Pang and Liu [16], studying RO membranes applied for wastewater reclamation, retrieved bacterial phylotypes related

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to the order Rhizobiales, a group of bacteria that had not been previously implicated in membrane biofouling. It appears that biolms developing on membrane separation systems follow a generic pattern despite the high diversity of bacterial species found, in which members of Proteobacteria and Bacteroidetes are the predominant species, with a minor contribution of Gram-positive and other Gram-negative bacteria [9,12]. This prole appears to be related to a broad capacity for surface colonization in these organisms, as well as adaptability to changing nutrients and ability to proliferate under oligotrophic conditions. Modeling the biofouling phenomenon, which consists of chemical, physical, and biological processes that interact on different spatial and temporal scales, is exceptionally complex. Efforts have been made to create predictive models for permeate ux and other hydraulic parameters in dense membranes based on a variety of different approaches [11,1821]. The use of data-driven techniques for modeling this multifaceted phenomenon is employed herein. Development of these methods is motivated by the rapid advances in information-processing systems along with increasing data availability, which have directed research towards the development of intelligent systems that evolve models of natural phenomena automatically [2224]. This is the discipline of datadriven modeling, which is the study of algorithms that improve automatically through experience. Applications of data-driven modeling range from data-mining schemes that discover general rules in large datasets, to information-ltering systems that automatically learn the interests of the user. The most utilized techniques of data-driven modeling are articial neural networks (ANNs), model trees, fuzzy-rule-based systems, and support vector machines. In this study, model trees are applied for forecasting the reduction in the rate of permeate ux through membranes due to biofouling formation [22,23,25]. Although researchers have used such techniques in studies of membrane fouling [24,26], they have not dealt with estimations of the biofouling problem. The overall objective of this work was to investigate biolm formation on the surface of NF membranes used for upgrading the quality of tertiary efuents under different operating conditions. The composition of the bacterial communities developed in the biofouling layer was studied by polymeric chain reactiondenaturating gradient gel electrophoresis (PCRDGGE) and sequence analysis of 16S rRNA gene fragments from DGGE bands, as a function of temperature, pressure and length of operation. An empirical series of equations, based on permeate ux in NF membranes treating tertiary wastewater, was derived to estimate the contribution to resistance of the biofouling layer developed on the membrane surface. In addition, a model algorithm for forecasting the ux reduction rate in biofouled membranes and a method for estimating the biofouling layer resistance in NF membranes treating tertiary wastewater efuents are proposed.

sponding to a linear velocity of 0.3 m/s and Re of 5300) in all cases. Before each experiment, the system was intensively cleaned with alkaline sodium hypochlorite, profusely rinsed with tap water and a new membrane assembled. Following each run, the membranes were dismounted and segmented into even slices of approximately 1 cm for the different microbiological and microscopic analyses described further on. 2.2. Feedwater Tertiary quality efuents were generated in a continuous membrane bioreactor (MBR) operating at the Technion campus: rst they were subjected to MF through at sheets with 8-m2 ltration area and 0.2-m pore size (Kubota), as previously described [9]. The MBR was fed with primary efuents from the sedimentation of raw wastewater collected from the Technion campus sewage-collection system. MBR efuents were free of suspended solids (<1 NTU) and characteristic organic matter content was (average standard deviation) 12.8 6.5 mg/L total organic carbon (TOC), ranging from 4.7 mg/L to 26.2 mg/L. Electrical conductivity (EC) and pH were in the range of 12701760 mS/cm and 6.28.2, respectively. 2.3. Chemical analysis EC and pH were measured using a Cyborscan (Eutech Instruments, Nijkerk, Netherlands). Turbidity was measured with a 2100P turbidimeter (Hach Co., Loveland, CO). TOC was measured with an N/C 2100 multi-analyzer (Analytik Jena AG, Jena, Germany). Ion content was analyzed with a Metrohm (Herisau, Switzerland) ion conductivity chromatograph model 761IC using Metrosep Cation 1-2 and Anion dual 1 columns. 2.4. Bacterial community analysis The bacterial community analyses applied in this research (DNA extraction and PCRDGGE analysis, cloning of PCR products and sequencing, phylogenetic analysis) were as described in detail in our previous work [9]. Briey, the tubular membrane slices were subsectioned into pieces of approximately 1 cm2 and subjected to DNA extraction by a modied bead-beating method; the extracted DNA was stored at 20 C until further processing. PCR amplication for DGGE was performed with the bacterial primer pair 341F, with a GC clamp at its 5 end, and 907R [27] using a T-gradient thermocycler (Biometra, Goettingen). DGGE of PCR-amplied 16S rRNA gene fragments was performed as described by Muyzer et al. [27]. Prevalent DGGE bands were carefully excised on a Dark Reader Transilluminator (Clare Chemical Research, Inc., Dolores, CO), puried, and re-amplied and veried by a second DGGE analysis. The resultant bands were excised, cloned and screened and selected plasmids were puried with the Concert Rapid Miniprep Plasmid Purication System (Gibco-Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer. Clones were sequenced using the Applied Biosystems (Foster City, CA) PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA polymerase and the T7 primer as suggested by the manufacturer. The sequencing products were analyzed with an Applied Biosystems 377 DNA sequencer. For phylogenetic analysis, 530bp nucleotide sequences were incorporated into a pre-aligned database of 16S rRNA sequences, using the aligning tool supplied by the ARB phylogenetic program package [28], and trees were generated with neighbor-joining and maximum-likelihood methods. 2.4.1. Bacterial enumeration Two procedures were applied for the enumeration of live heterotrophic bacteria accumulated on the membrane surface. The rst was based on bacterial counts determined by swabbing the

2. Materials and methods 2.1. Experimental set-up and conditions Nanoltration experiments were performed in a crossow apparatus equipped with a MIC-RO 240 module (PCI) comprising one NF-polyamide tubular membrane (AFC 30, PCI) with a ltration area of 120 cm2 (30 cm length 1.25 cm diameter) and 200 Da MWCO, as previously described [9,17]. Runs were conducted in semi-batch mode with full recirculation of the permeate and concentrate into the feed tank, the contents of which were replaced daily. Pressure was maintained at 5 bar or 25 bar and temperature was maintained at 20 C, 25 C, or 34 C, as indicated. Feedwater consisted of domestic efuents of tertiary quality, as described below. Recirculation ow rate was maintained at 132.5 L/h (corre-

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membrane surface with a sterile swab of approximately 1 cm in length at selected time points during the experiment (up to 8 days). The second consisted of elution of the accumulated bacteria at the end of each experiment from a 1-cm2 membrane coupon. In all cases, enumeration was performed by heterotrophic plate count (HPC) on R2A medium. Attached bacteria were expressed in CFU/cm2 and bacteria suspended in the feedwater in CFU/mL. Plates were incubated at 30 C for 3 days and the colonies were then counted. Swab samples were taken from a surface of approximately 1 cm2 at different places on the membrane by temporarily stopping the system and opening the module. Each sample was taken at a different place on the membrane surface. The sampling site was determined by measuring the depth of each entrance (1 cm, 2 cm, 4 cm). The samplings were performed from both sides of the membrane. No area on the membrane surface was sampled twice. Swabs were placed in phosphate-buffered saline (PBS)Tween solution (PBS + 0.05% (v/v) Tween 20), vortexed for 1 min, and the suspension was then diluted and plated. PBSTween solution contained 8.0 g NaCl, 1.0 g KH2 PO4 , 14.5 g Na2 HPO4 12H2 O and 0.5 mL Tween 20, in 1000 mL DDW; pH was adjusted to 7.2. At the end of the runs, membrane samples were subsectioned into 1-cm2 pieces and shaken (30 min, 150 rpm) with sterile glass beads (1 mm in diameter) in PBS for extraction and resuspension of the biofouling layer. The suspension was subsequently diluted and plated. Feedwater samples were diluted in sterile PBS and plated. 2.5. Model tree conguration 2.5.1. Theory of the model trees algorithm The model trees algorithm builds a rule-based predictive model using a top-down induction approach [25]. The tree is tted to a training dataset by splitting the data into homogeneous subsets based on their attributes. Thereafter, the tree is constructed with all training cases being predicted by the tree leaves (i.e., each leaf is a linear regression model which predicts continuous values for the numerical attributes). The tree is then pruned from the bottom up and transformed into a set of IfThen rules, which simplify the tree structure and thus improve its ability to classify new instances. The predictive ability of the tree is measured by the correlation coefcients of the training and cross-validation datasets. The correlation coefcient equals one in the case of a complete t between the actual values and the model predictions. 2.5.2. Model tree construction The goal is to construct a model that relates the target values of the training cases to their values described by the input attributes. The performance of the model is generally assessed by the accuracy with which it predicts the target values of unseen cases (crossvalidation dataset). Step 1. Computation of the standard deviation of the target values of cases in dataset T. Unless T contains very few cases or their values vary only slightly, it is split on the outcome of a test. Every potential test is evaluated by determining the subset of cases associated with each outcome; let Ti denote the subset of cases that have the ith outcome of the potential test. Treating the standard deviation sd(Ti ) of the target values of cases in Ti as a measure of error, the expected reduction in error (SDR) as a result of this test can be written as: SDR = sd(T ) 1 T
i

model is constructed for the cases at each node of the model tree using standard regression techniques. The tree is then transferred into a simple set of IfThen rules that simplify its structure. Step 3. The accuracy of the model is estimated on unseen cases (i.e., usually 70% of the data is used for training and for constructing the tree while 30% of the cases are put aside for cross-validation). The predictive ability of the tree is measured by r2 , dened as the proportion of the initial variance accounted for by the model (correlation coefcient of the model represented by the model tree): r2 =
2 F2 F0 2 F0

(2)

where F2 is the residual variance, i.e., the sum of squares of the 2 is the differences between computed and observed output, and F0 sum of squares of the differences of the observed output from its mean value. The correlation coefcient is computed for the training dataset and for the cross-validation dataset as well. If there is a complete match between the model values and the actual measured values, then the correlation coefcient is equal to one. Step 4. In this last step, the over-elaborated model tree structure is simplied, pruned from the bottom up, and smoothed, thereby improving its ability to classify new datasets (i.e., crossvalidation data). The model tree is simplied mainly by removing variables that contribute little to the model; in some cases, the algorithm removes all variables, leaving only a constant. The smoothing process is performed to compensate for discontinuities that will inevitably occur between adjacent linear models at the leaves of the pruned tree, particularly for some models constructed from a smaller number of training examples. In smoothing, the adjacent linear equations are updated such that the predicted outputs for the neighboring input vectors corresponding to the different equations become closer in value. 3. Results 3.1. Characteristics of the biofouling layer 3.1.1. Bacterial community The composition of the bacterial community developed on the fouling layer of NF membranes fed with tertiary efuents was studied in 25 independent experiments by PCRDGGE. A total of 26 bands were successfully excised from the DGGE gels, re-amplied, cloned and sequenced. The sequences were compared to published 16S rRNA sequences and a phylogenetic tree was constructed (Fig. 1). The microbial populations identied by sequence homology complemented our previous report comparing synthetic and real wastewater efuents [9]. Bacteria phylogenetically associated with Saprospiraceae (10245, 10632), Flavobacterium (6522, 10855, 10631), Pseudomonas (4315, 5218, 5519), Legionella (3012), Ralstonia (3713, 10246), Hydrogenophaga (3514, 9418), Bacillus (4716, 6220), Nitrospira (106291), Delftia (6421), Sphingomonas (42, 83, 10249, 111), Novosphingobium (10226), Dyella (84), Acidovorax (4917), Limnobacter (9418) and Phaeospirillum (10148) were detected (see dendogram for clone numbers and accession numbers, Fig. 1). Most species identied by sequence analysis were Gramnegative (24 of 26 sequences); the only two Gram-positive species belonged to the Firmicutes. Proteobacteria were found to be the prevalent group in all cases (18 of 26 sequences), followed by the Bacteroidetes (5 sequences). Among the Proteobacteria, species were divided more or less equally between the subdivision (7 sequences), the subdivision (6 sequences) and the subdivision (5 sequences). Three dominant Proteobacteria subgroups, related to Pseudomonas (3 sequences), Ralstonia (2 sequences) and

Ti sd(Ti )

(1)

Step 2. After examining all possible tests, the model tree chooses one that maximizes this expected error reduction (SDR). At this stage, an initial model tree has been grown and a multivariate linear

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Fig. 1. Phylogenetic tree of sequences obtained from the biofouling layer on NF membranes treating tertiary efuents under different environmental conditions, based on 530-bp 16S rRNA gene sequences. The topology of the dendogram was generated using the neighbor-joining method included in the ARB phylogenetic package. Sequences were obtained from bands excised from PCR-DGGE gels. Bar indicates an estimated 10% sequence divergence.

Sphingomonas (4 sequences), were found most often. No specic effects of the ltration conditions studied, i.e., duration, temperature and pressure, on bacterial community composition (DGGE banding patterns) could be strictly identied. The microbial populations identied in the recirculation uid were not always found in the biolm, and not all of the predominant populations on the membranes could be found in the recirculation uid at the time of sampling. The only exception was the Pseudomonas-afliated bacterial species represented by clone number 4315, which prevailed in both the recirculation uid and the membranes. 3.1.2. Enumeration of live bacteria Fig. 2 shows a typical time course of counts of live bacteria found on the membrane surface during the rst 8 days of operation at low-

pressure (5 bar), as determined by swabbing the membrane surface during a single run and culturing on R2A medium. During the rst 24 h, a two orders of magnitude increase in bacterial counts was noted (from 1.0 103 CFU/cm2 at 3 h to 1.6 105 CFU/cm2 at 24 h). Over the next 6 days, HPC increased to 1.8 106 CFU/cm2 (48 h), 1.2 107 CFU/cm2 (4 days) and 2.3 107 CFU/cm2 (7 days). As can be seen from Fig. 2, the increasing bacterial counts in the biofouling layer with time corresponded well to the decrease in permeability. Bacteria were enumerated on the membrane surface at the end of a series of multiple runs (membrane autopsy). For tertiary efuents at high-pressure (25 bar), bacterial counts were in the range of 4.1 105 CFU/cm2 to 9.9 107 CFU/cm2 , average 2.9 107 CFU/cm2 . At low-pressure (5 bar), the average was 8.0 106 CFU/cm2 , ranging from 3.0 105 CFU/cm2 to

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Fig. 2. Typical time course of live culturable bacteria found on the membrane surface at 5 bar feedwater pressure. Samples were taken by swabbing the membrane at the indicated times. Counts were performed on R2A plates. Note that the left ordinate is logarithmic. Runs were performed in semi-batch mode with full recirculation of both permeate and concentrate into the feed tank, the content of which was renewed daily (TOC was maintained at 8.6 0.6 mg/L throughout the entire period).

Fig. 4. Summary of relative permeate ux as a function of time for experiments performed at 5 bar and 25 bar.

2.4 107 CFU/cm2 . A summary of the HPC on R2A of bacteria extracted from different membranes treating tertiary efuent as a function of the corresponding TOC is presented in Fig. 3. HPC in the recirculation uid was in the range of 1.6 105 CFU/mL to 9.0 106 CFU/mL (average 1.62 106 CFU/mL) at low-pressure (5 bar), and in the range of 1.0 105 CFU/mL to 2.4 107 CFU/mL (average 5.7 106 CFU/mL) at high-pressure (25 bar). Slightly higher bacterial counts appeared in the high-pressure tests, probably due to enhanced accumulation of organic matter and drag force towards the membrane surface. The development of the biofouling layer to a convergent countable magnitude of 107 CFU/cm2 regardless of pressure, time or temperature applied, appeared to be the result of a steady nutrient supply to the biolm and the shear forces developed on the membrane surface. 3.2. Contribution of biolm development to membrane hydraulic properties A typical effect of biofouling on membrane performance can be seen in the ux proles of membranes at either 5 bar (low-pressure) or 25 bar (high-pressure) as a function of time (Fig. 4) or permeation volume (Fig. 5). As depicted in Fig. 4, a moderate ux decline was observed at 5 bar, reaching 16% after 24 h, 23% after 48 h, 32% after 72 h and 42% after 96 h. At 25 bar, the ux decline was very pronounced with an 80% decrease after 24 h in most of the experiments. Thereafter, the ux decreased at a lower rate (82% decrease

at 48 h, 85% at 72 h and 88% at 96 h). A similar ux decline prole was obtained from the plot of permeate ux vs. permeation volume (Fig. 5). Once again, a moderate ux decline was observed at low-pressure, reaching an asymptotic value of 35% ux decrease at approximately 2030 L of permeate. At high-pressure, the ux decline was very sharp, reaching a minimum value corresponding to 20% of the original ux at less than 20 L permeate volume. Similar temporal behavior as a function of operating pressure has been reported in RO membranes [10,29]. Membrane surface resistance due to fouling can be described by a series of resistance expressions [30], all of which contribute to permeate ux decline. The permeate ux can be described by: J= and Rt = RS + Rm RS = RSo + RSf + Rbf RS = RSo + RSf (4) (5) (6) P Rt (3)

where Rt is the total membrane resistance, RS is the membrane surface resistance (hydraulic resistance), RSo is CP resistance, RSf is the surface fouling resistance (clogging, adsorption, resistance due to gel formation), Rbf is the biofouling resistance, Rm is the membrane resistance, is the osmotic pressure difference, J is the permeate ux, P is the transmembrane pressure (TMP), and is the dynamic viscosity of the tertiary efuents. calculated during the experiments was of low magnitude (<6% and 4% of the applied pressure for 5 bar and 25 bar, respectively) and it can therefore be neglected. The permeate ux

Fig. 3. Summary of heterotrophic bacterial enumeration on the membrane surface as a function of TOC concentration in the feedwater. 5 bar and 25 bar denote the inlet pressure conditions. Plate counts were performed on R2A of bacteria extracted from membranes after different runs.

Fig. 5. Summary of relative permeate ux as a function of the cumulative permeation volume passed through the membrane for experiments performed at 5 bar and 25 bar.

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expression then becomes: J= P (Rm + RS ) (7)

The major contribution to the ux decline observed at highpressure within the rst 24 h can be attributed to RS , i.e., the resistance developed on the membrane surface due to the CP, gel formation and deposition (Eq. (6)). These processes are very weak at low-pressure [31] and their contribution to membrane fouling seems to be less signicant. This leads to a few assumptions: (a) RS contributes less to ux decline at low-pressure and (b) at lowpressure, the major contribution to membrane surface resistance is Rbf . Based on these ndings, Rbf can be expressed from low-pressure experiments (Fig. 5). RS = RSo + RSf is very low and can be neglected (0), RS Rbf and replacing in Eq. (7): Rbf = P Rm J (8)

Fig. 6. Estimation of biofouling layer resistance (Rbf ) on a NF membrane operating at 25 bar. The difference between the lines (Rt Rm ) and RS represents the value of Rbf . Rt and RS were calculated according to Eqs. (12) and (13), respectively. Rm was 7.69 2.7 1013 m1 .

Rbf can be found from low-pressure test experiments. Based on experimental data at 5 bar, the expression of the permeate ux decline can be described by an equation of exponential form: J = a ebVc Jo (9)

a = 0.88; b = 0.0104. Jo is the initial permeate ux with tertiary wastewater and Vc [L] is the cumulative permeate volume. The expression for Rbf then becomes: Rbf = P Jo a ebVc Rm (10)

A comparison of (Rt Rm ) and RS is presented in Fig. 6; the difference between (Rt Rm ) and RS is Rbf . Rbf begins to dominate the membrane surface resistance with time, due to biolm development, after 1520 L of permeate volume has passed through the membrane. At 25 bar, the major initial contribution to surface resistance is probably CP resistance and surface fouling resistance (clogging, adsorption, resistance due to gel formation), with biofouling contributing less. After 20 L permeation, the biolm begins its rapid development (Fig. 6) and biofouling starts to dominate the membrane surface resistance. 3.3. Implementation of the model trees technique to forecast ux reduction rate through the membranes due to biofouling The dataset used for this application utilizes physical, chemical and biological measurements obtained from experiments on biofouling development on wastewater treatment membranes. The input attributes include the following parameters: Discrete parameters: Temperature ( C): 20, 25, 34. Pressure (bar): high, 25; low, 5. Continuous parameters: TOC (mg/L). EC feed (mS/cm) (represented by Cb). Chemical parameters (mg/L): Na, K, NH4 , Ca, Mg, PO4 , SO4 , and B. The output attribute includes the following parameter: Normalized ux reduction rate through the membrane: 1 (Jt =0 Jt =24 h ) normalization factor Jt =0 24(h) (15)

The membrane resistance, Rm , was derived from tests with distilled water. The resultant experimental values of Rm were 4.08 1013 m1 to 1.15 1014 m1 (Rm = 7.69 2.7 1013 m1 ). Similar results have been found in studies with NF membranes [11]. Rm can be calculated from experiments with distilled water for each membrane and substituted into Eq. (10). Jo is measured in the rst hour (at 20 min) of the test with wastewater. The results of this study suggest that the biofouling layer develops to a relatively convergent magnitude under a broad range of conditions (1.0 107 CFU/cm2 at both low and high-pressures). These ndings lead to the assumption that biofouling formation and its development (biolm on membrane) are similar at low and high-pressures. Therefore, biofouling resistance can be calculated based on experimental data from the low-pressure test. Data at 25 bar yielded the following empirical equation for permeate ux: J = k (m enVc ) J = Jo k (m enVc ) Jo k = 0.82; m = 0.22; n = 0.175. Substituting Eq. (11) into Eq. (3), Rt becomes: Rt = P Jo k (m enVc ) (12) (11)

Substituting Eqs. (5) and (6) into Eq. (4) and then into Eqs. (10) and (12), assuming that the same resistance due to biofouling derived at low-pressure occurs at high-pressure, RS becomes: RS = Rt Rm Rbf = P Jo 1 1 k (m enVc ) a ebVc (13)

Substitution of the constants into Eq. (13) gives: RS = P Jo 1 1 0.18 + 0.82e0.175Vc 0.88e0.0104Vc (14)

where J is the ux through the membrane in L/m2 h; Jt=0 is the ux at the start of the experiments (t = 0 h)this is the highest ux value as biofouling development has not yet begun on the membrane; Jt=24 h is the ux after 24 h of the experiment (t = 24 h)this is the time point at which the value of the ux becomes relatively stable (the major ux reduction as a result of fouling has already occurred); the normalization factor is a constant used to keep the output value within the same range as the input values. This factor assists the data-driven technique in producing more robust solutions (e.g., in this application the normalization factor = 10 000). The number of cases obtained from the experiments was about 30; this low number is obviously not sufcient to implement a datadriven technique. Therefore, a synthetic set of cases was added to the real ones. Each real case was used to produce 10 synthetic cases by adding noise to the real input and output measurements. The noise was added by Monte Carlo simulations which produced syn-

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at high-pressure is one of the more inuential factors causing a high rate of ux reduction. Rule 6 suggests that at high-pressure, TOC > 10 mg/L and high levels of conductivity, there is a signicant reduction in permeate ux rate. In general, the set of accepted rules from the model tree shows that a substantial increase in ux reduction rate is caused by higher levels of TOC (over 10 mg/L), high levels of conductivity (over 1505 mg/L) and high-pressures. Similar results were observed in the evaluation of permeability reduction rates, i.e., a high rate of permeability reduction caused by higher levels of TOC (over 9.38 mg/L), high levels of conductivity (over 1564 mg/L), and high-pressures.

4. Discussion The development of biofouling on NF membranes treating tertiary wastewater was studied at low- and high-pressures, and various feedwater compositions, temperatures and lengths of operation. Proteobacteria, followed by the Bacteroidetes, were the prevalent groups found in all fouled membranes, with species belonging to Pseudomonas, Ralstonia and Sphingomonas most often found. In agreement with other studies [1316,3237], the dominant species found in our work were Gram-negative bacteria, which display wide metabolic and physiological diversity, broad surface colonization ability and adaptability to the environment, regardless of separation technology (NF or RO) or source of the applied feedwater. It should be noted that the specic role of each type of bacteria in the physiology of the biolm, as well as in membrane biofouling, remains to be elucidated. The HPCs in the foulant layer reported here are in general agreement with previous studies [9,17,38,39]. Interestingly, slightly higher average counts were detected at high compared to lowpressure. Moreover, HPC increased by approximately two orders of magnitude with the increase of initial organic carbon level in the feedwater in the range of 525 mg TOC/L. This is in agreement with the accepted notion that feedwater quality is a decisive factor in biofouling development [3,40]. Fonseca et al. [11] found that regardless of the surface-averaged polysaccharide densities observed, culturable cell densities (5 107 CFU/cm2 to 5 108 CFU/cm2 ) recovered from nal membrane autopsies were statistically similar (P < 0.05). Their reported surface cell densities, calculated from cultured samples of homogenized membrane biolms, were also within the ranges reported by us and other researchers [9,17,32,38,39]. An empirical approach to forecasting the contribution of biofouling resistance to the total membrane resistance to efuents in the entire range of studied pressures (525 bar) was derived based on the resistance in series theory. This approach showed that biofouling becomes the dominant factor after approximately 20 L of permeate volume has passed through the membrane. At high-pressure, this pattern was preceded by a drastic decrease in permeate ux (>80%) within the rst 24 h of operation, suggesting that surface fouling resistance is the dominant effect at highpressure. Experimental data at low-pressure (5 bar) in our previous publications [9,17] showed a detectable settling of microorganisms on the membrane surface after 8 h, along with the initiation of biolm layer development, and bacterial growth became signicant at 4872 h. Thus, whereas at low-pressure biofouling appears to be a major contributor to the fouling layer from the start, its contribution to the initial increase in total membrane resistance at high-pressure appears to be minor. The main aim of this empirical approach was to evaluate the direct contribution of each resistance factor to the total membrane resistance. In addition to previous empirical methods published elsewhere [8,11,29], our approach allows specic estimation of membrane biofouling resistance among all other series of resistances. However, further

Fig. 7. Set of rules produced for predicting the normalized ux reduction rate in 24 h through the membranes.

thetic cases similar to the real ones. At the end of this process, the total number of cases was approximately 350, a suitable dataset for data-driven modeling using the model trees technique. The model trees technique was implemented using 67% of the data for training and constructing the model tree and 33% of the data for cross-validation. The set of rules produced for predicting the normalized ux reduction rate in 24 h through the membranes is presented in Fig. 7: the most important input parameters with regards to ux reduction rate were pressure, TOC, pH and EC. The correlation coefcient for the prediction of the cross-validation dataset was relatively high (e.g., 93%), showing that the proposed data-driven methodology is capable of modeling the biofouling phenomenon by simulating a surrogate parameter such as ux reduction rate. Fig. 8 describes the accuracy of the predictions through a scatter plot that graphs the real target values of the cross-validation cases against the values predicted by the model. A thorough examination of the set of rules presented in Fig. 7 shows that a high concentration of inorganic ions (as EC), represented by high levels of conductivity (Cb > 1505 mS/cm, rule 9)

Fig. 8. Scatter plot of the correlation between real and modeled values in the crossvalidation.

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research with different membrane congurations, environmental conditions and efuent types is needed to improve the accuracy of this empirical forecasting approach. A data-driven technique for forecasting the reduction in permeate ux through the membranes was also developed and demonstrated. The values predicted by the model were in good agreement with experimental measurements. Pressure, TOC, pH and conductivity of the tertiary wastewater treated by the membrane were the most inuential parameters. The reduction in permeate ux rates was related to the development of a fouling layer on the membrane. These results are novel in the area of model tree algorithms and ANNs as they apply to forecasting the development of a biofouling layer on membranes. Previous work has not dealt with estimations of biofouling [22,24,26]. Nevertheless, for future applications, the number of real measurements needs to be increased and the number of synthetic ones reduced: the larger the dataset, the higher the likelihood of obtaining accurate predictions. In particular, increasing the number of training instances for the proposed model is expected to improve its prediction accuracy. Research challenges for extending this study lie in more closely incorporating the physical, chemical and biological processes involved in membrane separation systems. This can be accomplished, for example, by using a physically, chemically and biologically based model in conjunction with a data-driven modeling technique. An evolutionary algorithm could tie both together, but the computational effort needed to run such a set-up is likely to be very high. Biolm development in dense membrane systems is a critical mode of fouling that is complex to resolve and model, and may have a synergistic effect with other forms of fouling that negatively affect membrane performance. Moreover, the direct link between bacterial communities and membrane performance, as well as the implication of microbial diversity on long-term application of membranes, is still unclear and more insight into the biofouling phenomenon is needed for its effective control. Following van der Brugen et al. [5], not much work has been done on modeling fouling in nanoltration and the inuence of membrane fouling on ux is one of the main aspects that remain to be modeled. The utilization of a data-driven modeling scheme shows promising results. Enhancement of this approach can be accomplished by tailoring a physical model in the overall algorithm framework to select the most dominant input parameters and utilizing statistical or fuzzy-rule-based systems to enlarge the training dataset. Acknowledgements This work was funded by the Chief Scientist of the Ministry of Agriculture, the Grand Water Research Institute and the Fund for Promotion of Research at the Technion. References
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