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Biodegradation of phenol from saline wastewater using forward osmotic hollow fiber membrane bioreactor coupled chemostat
Accepted Manuscript Title: Biodegradation of phenol from saline wastewater using forward osmotic hollow fiber membrane bioreactor coupled chemostat Author: Prashant Praveen Duong Thi Thuy Nguyen Kai-Chee Loh PII: DOI: Reference:
S1369-703X(14)00330-1 http://dx.doi.org/doi:10.1016/j.bej.2014.11.014 BEJ 6073
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
17-7-2014 15-10-2014 18-11-2014
Please cite this article as: Prashant Praveen, Duong Thi Thuy Nguyen, Kai-Chee Loh, Biodegradation of phenol from saline wastewater using forward osmotic hollow fiber membrane bioreactor coupled chemostat, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2014.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodegradation of Phenol from Saline Wastewater using Forward Osmotic Hollow Fiber Membrane Bioreactor coupled Chemostat Prashant Praveen, Duong Thi Thuy Nguyen, Kai-Chee Loh* Department of Chemical and Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Singapore 117576 *Corresponding author: Associate Professor, Department of Chemical and Biomolecular Engineering,National University of Singapore, 4 Engineering Drive 4, Singapore 117576. Email: [email protected]; Tel.: +65 6516 2174; Fax: +65 6779 1936 Highlights •
A chemostat was coupled with forward osmotic hollow fiber membrane bioreactor
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The integrated bioreactor was used for treatment of saline phenolic wastewater
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Effects of operating parameters on bioreactor performance were examined
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Forward osmosis performance decreased sharply due to biofouling
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The severity of biofouling increased at higher permeate flux
Abstract A chemostat was coupled with a forward osmotic hollow fiber membrane bioreactor (FOHFMB) for treatment of high strength saline phenolic wastewater using Pseudomonas putida ATCC 11172. The microorganisms were protected from the inhibitory effects of phenol and sodium chloride through dilution of the feed wastewater. This resulted in high cell growth and biodegradation rates during transient operation and steady state was achieved within 20 h. Effluent from the chemostat was desalinated in the FOHFMB through forward osmosis (FO) using magnesium chloride as the draw solute (DS). Permeate flux during FO remained stable for over 70 h in the orientation with DS facing the porous side of the membranes (PRO mode of operation). Water used for dilution could be recovered using 0.8
M DS when the wastewater did not contain any sodium chloride, whereas 1.5 M DS was required to recover water from the wastewater containing 0.6 M sodium chloride. Biomass attachment on the membranes during FO operation was visualized using SEM, which showed that FO membranes was susceptible to fouling propensity and biomass deposition on the membranes was directly associated with permeate flux. Nevertheless, biofouling of membranes was reversible and membrane performance was recovered by osmotic backwashing.
Keywords: Biodegradation; Biofouling; Forward osmosis; Membrane bioreactor; Saline wastewater
Introduction Salts, especially sodium chloride, are important raw materials in chemical industries where they play an indispensable role in various manufacturing processes and other industrial operations. Some of the common industrial applications of salts are in deicing, paper manufacturing, food preservation, oil refining, tanning, water conditioning, agriculture and synthesis of useful chemicals [1, 2]. Consequently, the presence of salts in industrial wastewater is a common occurrence. In fact, it has been estimated that about 5 % of industrial effluents generated throughout the world are either saline or hyper-saline [3].
Industrial saline wastewater is also usually rich in organic matter. Biological treatment of wastewater with high salinity is difficult due to the adverse effects of salts on microbial metabolism [4]. The problem is further aggravated when the wastewater contains toxic organic compounds as the microbial growth and metabolism are inhibited by high concentrations of salts, as well as the organic pollutants [5, 6]. Although wastewater can be desalinated effectively using various techniques such as evaporation, flocculation, reverse osmosis (RO), forward osmosis (FO) or electrochemical methods [7], the salts thus recovered
will have high organic content. In particular, salts polluted with toxic aromatics may not be suitable for reuse, whereas disposal of the recovered salts may require further treatment. In order to treat saline wastewater laden with toxic organic content, the organic pollutants must be removed from the wastewater prior to desalination.
Traditional methods used to alleviate substrate inhibition, such as cell immobilization or twophase biodegradation, are not very effective in protecting microorganisms in saline environment. Therefore, the preferred strategy while biodegrading organic pollutants in saline environment is the use of halophilic microorganisms [8]. However, very few halophiles have been isolated which are capable of metabolizing toxic aromatic compounds. Moreover, these halophiles very often exhibit low tolerance to the substrate, which can result in low cell growth and biodegradation rates [4]. Another approach to treat saline wastewater is by improving salt tolerance of biodegrading microorganisms such as Pseudomonas putida through adaptation. However, salt tolerance acquired through adaptation is temporary and it is quickly lost when the salinity of the medium is decreased [3, 4]. A simple approach to mitigate inhibition arising from salts and organics is the dilution of the saline wastewater. However, dilution is not a preferred method because it requires large quantities of water, increases the process volume and increases the treatment costs [9]. The drawbacks associated with dilution can be alleviated through the use of chemostat and FO technologies. Chemostat is an established technique used for continuous cultivation of microorganisms at constant volume; FO is based on the flow of water across a selectively permeable membrane under an osmotic pressure gradient and it has recently emerged as an energy efficient desalination technique [10]. Through integrating these two techniques, the strategy is to dilute the wastewater to enable biodegrading microorganisms to metabolize the pollutant in the chemostat; the organic pollutant free effluent from the chemostat is then desalinated through FO and water used for dilution is recovered and reused. The resulting
chemostat coupled forward osmotic hollow fiber membrane bioreactor (FOHFMB) can mitigate the challenges associated with wastewater dilution, achieve high biodegradation rates and perform effective desalination [11, 12]. Moreover, FO-based desalination using low or no hydraulic pressure can have the advantages of low energy requirement, better rejection of contaminants and a lower membrane fouling tendency [13]. In this research, a chemostat-FOHFMB integrated bioreactor system was operated to demonstrate the suitability of the ‘dilute-biodegrade-desalinate’ approach in the biodegradation of high concentrations of phenol in saline wastewater. The effects of wastewater salinity, extent of dilution, FO membrane orientation, draw solute (DS) concentration and DS flow rate on the performance of the chemostat-FOHFMB system were examined, and biomass attachment on the membranes was also characterized. Phenol was chosen as the model pollutant because of its toxic and recalcitrant nature. Besides, phenol and its derivatives are found in saline wastewater emanating from petroleum, textile and leather industries [9].
Materials and Methods Microorganisms, Culture Conditions, and Chemicals P. putida ATCC 11172 was used throughout this study. Stock cultures were maintained on nutrient agar (Oxoid, Hampshire, UK) slants at 4°C. The microorganisms were grown in a chemically defined mineral medium supplemented with phenol in Erlenmeyer flasks on a shaking water bath (GFL 1092, Burgwedel, Germany) at 30 °C and 150 rpm. The composition of the mineral medium has been described elsewhere [14]. All media (except phenol), pipette tips, and Erlenmeyer flasks fitted with cotton plugs were autoclaved before use. Prior to inoculation, cells were induced in mineral medium containing 200 mg/L phenol as the sole carbon source. Activated cells in the late exponential growth phase were used as inoculum for all the experiments.
All the chemicals used in this research were of analytical grade. Phenol was dissolved in 0.02 M sodium hydroxide to prepare a stock solution of 10 g/L. Magnesium chloride was used as the DS in all the experiments, whereas sodium chloride was used to maintain wastewater salinity.
Chemostat- FOHFMB FO Membrane Contactor Cellulose acetate (CA) nanofiltration hollow fiber membranes were used for FO. The inner surface of the membranes was porous, whereas the dense outer surface of the membranes acted as the rejection layer. A detailed description of the membrane characteristics and its synthesis are given elsewhere [15]. A shell and tube membrane contactor with an inner diameter of 1.27 cm was fabricated by potting the CA hollow fiber membranes into a perfluoroalkoxy module using epoxy resins (Araldite, England). The membrane contactor contained 50 fibers with an effective length of 25 cm.
Experimental Setup Fig. 1 shows the schematic diagram of the experimental setup. The chemostat consisted of a 500 mL Erlenmeyer flask with a working volume of 250 mL. A dual channel peristaltic pump (L/S modular pump, Easy-Load II pump head, Masterflex, USA) was used to pump the feed wastewater in and out of the chemostat at a constant flow rate. Humidity saturated purified air was sparged into the chemostat at a rate of 2 gas volume per reactor volume per minute (VVM). The effluent from the chemostat was fed into one side of the FOHFMB, whereas the DS was pumped into the other side of the membrane contactor using another peristaltic pump. Note that the RO unit required for DS recycle was not operated for these experiments.
Operation The synthetic feed wastewater was prepared with 1000 mg/L phenol along with mineral salts required for cell growth. In the desalination experiments, 0.6 M sodium chloride was also
added into the wastewater to increase its salinity. The wastewater was diluted prior to its addition into the chemostat, and pumped into the chemostat at the flow rate of 60 mL/h, equivalent to a hydraulic retention time (HRT) of 4.2 h. Once the wastewater had reached the required level in the chemostat and the FOHFMB was also filled with the wastewater, the chemostat was inoculated with P. putida from the preculture to achieve an initial biomass concentration of 6-7 mg/L in the chemostat. After inoculation, the DS was pumped into the FOHFMB at a constant flow rate. Magnesium chloride concentration in the DS feed was varied between 0.4-1.5 M depending on the experimental conditions, whereas the DS flow rate was varied between 1.5-7.5 mL/min. At the end of each experimental run, the biofilms on the CA membranes in the FOHFMB were cleaned through osmotic backwashing of the membranes. In this process, 0.5 M sodium chloride as DS was pumped over the biofilms whereas pure water was pumped on the clean side of the membranes. The osmotic backwashing was carried out for 2 h, following which both the shell and the tube sides of the FOHFMB were washed with pure water for 6-8 h to remove any remaining microorganisms or salts from the membranes. The same membrane contactor was used after cleaning in all the experiments. Analytical Methods Cell density was determined by measuring the optical density (OD) of the aqueous medium at 600 nm using an ultraviolet-visible spectrophotometer (UV-1240, Shimazdu, Japan). The OD was used to compute the biomass concentration using the formula: dry cell weight (mg/L) = 385×OD600 [16]. For determining phenol concentration, 3 mL of the cell culture was filtered through 0.45 µm syringe filter (Millex, Millipore, USA) and extracted into an equal volume of dichloromethane containing 100 mg/L o-cresol as internal standard. Phenol in the extract was analyzed by gas chromatography equipped with a flame ionization detector (Clarus 600, Perkin Elmer, USA). Biofilms on the membranes were characterized using a scanning
electron microscope (SEM) (JEOL JSM-5600LV) after sputtering the membranes with platinum.
Results and Discussion Flux and Salt Rejection To investigate membrane performance during FO, water flux through the CA membranes was determined using magnesium chloride as the DS against ultrapure water. A constant DS concentration was maintained on one side of the membranes using a continuous flow of the DS without any recirculation, whereas ultrapure water on the other side of the membranes was recirculated. In the FO mode, with the DS facing the porous side of the membrane (lumen of hollow fiber membranes), water flux increased from 1.5 liters per square meter per hour (LMH) at 0.2 M DS to 5.6 LMH at 2 M DS. However, the increase in flux was nonlinear and the rate of increase in flux gradually decreased with rising magnesium chloride concentration as shown in Fig. 2. A similar trend in water flux was observed in the pressureretarded osmosis (PRO) mode when the DS solute was facing the active layer of the membranes. The flux observed in the PRO mode was 40-60% higher than those observed in the FO mode at the same DS concentrations. These results are consistent with those reported in literature and the difference in permeate flux between FO and PRO mode is attributed to internal concentration polarization (ICP) in the FO mode, resulting in lower effective osmotic pressure gradient across the active layer of the membrane [17]. The CA membranes were also tested for the rejection of magnesium chloride and phenol. While the membranes exhibited excellent performance in preventing DS solute leakage across the membrane and rejected more than 99% of the DS at the investigated experimental conditions, phenol was able to diffuse through the membranes quite easily. Fig. 3 shows phenol diffusion through the membranes at different feed phenol concentration. It can be seen that phenol rejection performance of the membranes was very poor and only 40% of phenol fed to the FO membrane module was rejected. It was also observed that phenol rejection was
almost independent of feed phenol concentration and it remained constant at 39 ± 3.6 % when phenol concentration was varied from 200 to 2000 mg/L. The poor rejection of phenol could be due to the small molecular size of phenol as compared to the membrane pore size. The average molecular mass of a phenol molecule is 94 Daltons, whereas the molecular weight cut off (MWCO) of the CA membranes was 186 Daltons [15]. Although the molecular size of anhydrous magnesium chloride is comparable to phenol, the hydrated magnesium chloride molecule is likely to be much larger resulting in better rejection by the CA membranes. The rejection of phenol by the membranes was independent of the bulk feed phenol concentration. This was expected as the water flux through the FO membranes was constant for all the experiments [18]. Effects of Salinity on Microorganisms In saline wastewater contaminated with toxic organic compounds, it is important to examine the effects of increasing concentrations of the salts and the organic pollutant to determine the extent of dilution required to alleviate the inhibitory effects of wastewater on the microorganisms. The effects of phenol on growth and metabolism of P. putida are well characterized in literature and the inhibitory limit is known to be 600 mg/L [11].
In order to understand the effects of saline environment on cell growth and to determine the salt tolerance of P. putida, batch biodegradation experiments were carried out in cell culture medium containing 200 mg/L phenol as the growth substrate, and at 0.1-0.7 M sodium chloride salinity. Fig. 4 shows that cell growth rate was not affected by the presence of sodium chloride at 0.1 M salt concentration. A specific growth rate of 0.55 h-1 was observed at 0.1 M sodium chloride, which was the same as that of the control medium prepared without any sodium chloride. However, as the salt concentration was increased further to 0.2, 0.3, 0.4 and 0.5 M, the specific growth rates gradually decreased to 0.46, 0.40, 0.33 and 0.20 h-1, respectively. It was also observed that the microorganisms exhibited a short lag phase
while growing at salt concentrations higher than 0.3 M. No cell growth was observed at 0.6 M salt whereas a quick decrease in the biomass concentration was observed immediately after inoculation at 0.7 M salt. Unlike cell growth rates, biomass yield on phenol was not affected by the changes in the salinity of the cell culture medium and it remained constant at about 0.6 g-biomass/g-phenol at 0.1-0.5 M salt. These results indicate that salt concentrations above 0.1 M affected the growth and metabolism of P. putida adversely. However, the bacteria could tolerate up to 0.5 M salinity as evident from complete biodegradation of phenol even at this salt concentration. For subsequent biodegradation experiments, the wastewater in the chemostat-FOHFMB must be diluted to bring salt concentration to below 0.5 M, whereas phenol concentration should be brought down to below 600 mg/L.
Biodegradation in Chemostat-FOHFMB Operation in PRO Mode To investigate biodegradation of phenol in the chemostat, feed wastewater containing 1000 mg/L phenol was pumped into the chemostat at a flow rate of 20 mL/h. The wastewater was diluted three times with pure water to decrease the feed phenol concentration to 350 mg/L and to reach an increased influent flow rate of 60 mL/h. The dilution rate in the chemostat was thus calculated as 0.24 h-1, corresponding to a 4.2 h HRT. Since feed phenol concentration was sub-inhibitory, P. putida in the chemostat did not experience any lag phase. The microorganisms exhibited a specific growth rate of 0.48 h-1 and the net specific growth rate was calculated to be 0.24 h-1. Due to the high cell growth rate, phenol in the chemostat was rapidly metabolized and phenol concentration dropped to zero within 20 h to reach the steady state at a biomass concentration of 170 mg/L (Fig. 5). Once steady state was reached, phenol concentration in the chemostat remained zero for the entire operating period but the biomass concentration started decreasing after 40 h. The decrease in the biomass was the result of cell aggregation as lots of aggregates were observed in the bioreactor after 40 h.
While cell growth and phenol metabolism in the chemostat was stable in the first 20 h, water flux through the CA membranes in the FOHFMB was not very consistent during this period. The initial permeate flux using 0.4 M magnesium chloride as the DS was about 35 mL/h (Fig. 6a). However, the flux decreased monotonously to about 12 mL/h within 15 h of operation. The flux remained constant for about 70 h before it decreased to even lower values. The loss of flux in the osmotic membrane bioreactor is usually attributed to two factors: concentration polarization and membrane biofouling [17, 19]. Although biomass concentration in the chemostat was low and membrane biofouling during this period was not deemed severe, P. putida exhibited a very strong tendency to attach to surfaces and to produce extracellular polymeric substances (EPS) [11]. Besides, when the microorganisms or other foulants faced the porous side of the membranes, the conditions were more favorable for fouling as these entities entered the membrane pores [20]. Further evidence of cell attachment on the membranes was obtained by comparing the biomass concentration profiles in the chemostat and in the FOHFMB. Since a significant portion of water used in the dilution of feed wastewater was being recovered through the CA membranes, it was anticipated that the effluent from the FOHFMB would have higher biomass concentration as compared to that in the chemostat. However, Fig. 6b shows that the biomass concentration profiles in the chemostat did not differ significantly from that in the FOHFMB. The expected biomass concentration in the FOHFMB, which was calculated based on the water flux at that time, was higher than the biomass concentration measured experimentally. These results indicate that the biomass present in the water filtered through the CA membranes did not remain in suspension and the biomass was retained on the membrane surfaces and pores. Similarly, it was expected that phenol concentration in the effluent from the FOHFMB would be higher than those observed in the chemostat at any time because the FO membranes could reject about 40 % phenol from permeate and the rejected
phenol was retained in the FOHFMB. On the contrary, results in Fig. 6c indicate that phenol concentration in the final effluent stream released from the FOHFMB was actually lower than those observed in the chemostat at those times. These results indicate that the FO membrane module in the integrated bioreactor setup did not just act as a filter but it also facilitated phenol biodegradation by acting as an osmotic membrane bioreactor. The FOHFMB thus improved the effluent quality by retaining high concentration of biomass for biodegradation of phenol and by improving the overall HRT in the integrated bioreactor system. It can also be inferred that biofilm development on the CA membranes was favorable for phenol biodegradation in the FOHFMB as attached cells exhibited more tolerance towards toxic substrates as compared to suspended cells because of structural and physiological heterogeneity and the protection of EPS [21]. Osmotic Backwashing At the end of every experimental run, the FO membrane module was washed to remove entrapped and lined bacteria from the membranes. During the washing process, cell culture medium was replaced by 1 M sodium chloride solution, whereas pure water was passed through the lumen side. Although this washing process was not optimized, it was observed that 3-4 h of osmotic backwashing could remove most of the biofilm. An attempt was made to estimate the concentration of microorganisms removed in the backwashing. However, the high salt concentration was detrimental for the cells (causing cell lysis) and concentrations estimated in the process were inaccurate indication of the actual biomass attached to the membranes. Although the total biomass concentration in the FOHFMB could be estimated through total protein quantification in the bioreactor, even under cell lysis conditions [22], the test was not performed as it would have required the breakage of the membrane module for the collection of biofilm samples. After osmotic backwashing, the membrane contactor was washed with pure water on both the sides for at least 12 h to negate any effects of ICP. The membrane was tested for flux against 0.5 M DS after the washing process was complete. It
was observed that the flux after washing cycle was constant at 2.9 ± 0.4 LMH. These results show the advantages of FO based systems in dislodging biofilms from membrane surfaces to clean the membrane and to recover the permeate flux. In the absence of any hydraulic pressure, the biomass on the membrane surface was not compressed to form a cake layer. Consequently, the lightly attached biomass on the membrane surfaces could easily be dislodged and washed away [23]. Operation in FO Mode Since the biofouling propensity of FO membranes is high when the system is operated in the PRO mode with the microorganisms facing the porous side of the membranes [20], flow orientation was changed so that microorganisms in the FOHFMB were facing the rejection layer of the membranes. This change in membrane orientation had no effect on the chemostat operation but it resulted in significant improvement in FOHFMB performance as the permeate flux was stable for longer time (Fig. 7a). The flux at 0.4 and 0.8 M DS concentrations remained constant at 24.4 ± 3.0 mL/h and 43.1 ± 2.7 mL/h, respectively, for over 70 h. Since the feed wastewater was pumped to the chemostat at 20 mL/h, which increased to 60 mL/h after three times dilution, the amount of water used for dilution was 40 mL/h. The water used for dilution could be completely recovered using 0.8 M DS, which indicates that this process is sustainable. The decrease in flux after about 70-80 h of operation could be attributed to concentration polarization either due to salt accumulation in the membrane pores or on the membrane surfaces. The lowering of flux could also be a result of membrane biofouling as a lot of biomass had accumulated on the membrane surfaces by the end of three days of operation. To further investigate the reason for the flux lowering in the FOHFMB, experiments were carried out at different DS flow rate to examine the effects of external concentration polarization (ECP) on FOHFMB performance. It can be seen from Fig. 7b that increasing the DS flow rate from 1.5 mL/min to 7.5 mL/min had no significant effects on the flux
performance of the FOHFMB. Therefore, it can be inferred that the dilutive ECP on the DS side was not really a major factor affecting water flux through the membranes. Similar experiments to demonstrate the effects of concentrative ECP on the wastewater side could not be conducted because the maximum influent flow rate that could be supported in the chemostat was about 2.0 mL/min. Flow rates higher than 2.0 mL/min would have resulted in cell wash out. Using 0.8 M magnesium chloride as the DS in FO mode, the flux was comparatively high and stable. Consequently, the extent of biomass accumulation on the CA membranes during FO was higher as can be seen in Fig. 8a. The biomass concentration in the effluent from the FO unit was expected to be three times higher than that observed in the chemostat at any time. However, the biomass concentrations in the chemostat and the FOHFMB were almost identical. It can thus be inferred that the biomass amounting to the difference between the expected biomass concentration in the FOHFMB and the measured biomass concentration in the FOHFMB had been deposited onto the hollow fiber membranes. Furthermore, it can also be concluded that the FO membranes did not really prevent biofilm attachment on the membranes or have low biofouling tendency. The severity of biofouling depends on the permeate flux and lower flux would usually result in low membrane biofouling and vice versa [24]. As observed earlier in Fig. 6, the presence of the FOHFMB improved the overall rate of biodegradation in the chemostat-FOHFMB bioreactor system (Fig. 8b) through the presence of a high amount of microorganisms in the shell side of the membrane module as well as on the outer surfaces of the membranes. The presence of FOHFMB also enhanced the effective HRT in the bioreactor. Consequently, the effluent from the FOHFMB had a lower phenol concentration and steady state was achieved quicker than that which would have been achieved using only the chemostat for biodegradation.
The extent of dilution was crucial in facilitating the biodegradation of phenol in the chemostat-FOHFMB system. To examine this effect, phenol biodegradation in the chemostatOHFMB systems was conducted at three times and five times dilution. At five times dilution, the effective feed phenol concentration in the chemostat was about 200 mg/L, way below the inhibitory limit. At the low phenol concentration, P. putida exhibited a specific growth rate of 0.59 h-1, much higher than the specific growth rate of 0.48 h-1 observed at three times dilution of the wastewater when effective feed phenol concentration was about 350 mg/L. The low phenol concentration and high cell growth rate at five times dilution also resulted in higher biodegradation rate and steady state was achieved within 10 h of bioreactor operation (Fig. 9). However, these changes due to the degree of dilution did not affect water flux in the FOHFMB significantly and the flux remained constant at about 40 mL/h (results not shown). These results indicate that a higher degree of dilution has the advantages of higher cell growth and biodegradation rates. Consequently, the bioreactor attains steady state faster. Furthermore, a higher specific growth rate can support a higher dilution rate in the bioreactor and the chemostat can be operated at higher influent flow rates. For example, the specific cell growth rate at 200 mg/L phenol and 0.1 M salt was 0.55 h-1 whereas it decreased to about 0.2 h-1 when salt concentration was increased to 0.5 M using 200 mg/L phenol as growth substrate. Considering a chemostat volume of 250 mL, the highest feed flow rate in the bioreactor at 0.1 and 0.5 M NaCl could be 140 mL/h and 50 mL/h, respectively. Therefore, the effects of dilution of the feed wastewater is somewhat compensated due to a higher wastewater flow rate that the bioreactor can support. However, when the extent of dilution is increased, a higher DS concentration will be required to recover the extra water used for dilution. Based on these results, it can be suggested that dilution can be considered for substrate inhibition alleviation in a chemostat-FOHFMB system, especially when the bioreactor does not need any additional water resources for operation.
Membrane Biofouling The deposition of biomass on the membrane surfaces during filtration can occur due to natural diffusion of the microorganisms from solution to the membranes or under the flow of water across the membranes. Once microorganisms have been deposited on the membranes, the severity of membrane biofouling depends on the adhesion capability of the microorganisms especially when extracellular polymeric substances (EPS) are produced by the microorganisms [25]. Microorganisms such as P. putida exhibit excellent adhesive properties and often form strong biofilms on polymeric membranes [11]. Fig. 10 shows the SEM images of P. putida attached on the outer surface of the CA membranes after 12 h of chemostat-FOHFMB operation. It can be seen that the rod shaped bacteria were not just deposited on the membranes, but these cells were wrapped in the protective layer of EPS. These biofilms grew stronger with time with the production of more EPS and the deposition of more biomass on the membranes. Therefore, it is likely that the loss in permeate flux in FOHFMB after 3-4 days of operation was caused by membrane biofouling. These results were very different from those reported in municipal wastewater treatment in submerged osmotic membrane bioreactors (OMBR), where the water flux decreased by only 20% after 14 days of operation [23]. Since the membrane fouling in the FOHFMB occurred even at a low water flux of 24 mL/h which was equivalent to 1.2 LMH, it showed that biofouling could occur at very flow flux conditions. These results also suggested that the critical flux for membrane fouling in this case was very low, or critical flux could not have been an important factor in membrane biofouling when the microorganisms had a high tendency to attach to the membrane surface as observed in case of P. putida [26]. The absence of any hydraulic pressure on the membranes during FO is believed to impart a lower fouling propensity to FO membranes as compared to traditional membrane filtration techniques such reverse osmosis and ultrafiltration [13, 27]. However, the results obtained in Figs. 6 and 8 show that the microorganisms could attach readily to the membranes even in the
absence of any hydraulic pressure and the rate at which the biomass was deposited on the membranes depended not on the pressure but on the permeate flux. This observation is consistent with those reported in literature where a gradual increase in the foulant deposition on the FO membranes and a corresponding decrease in the water flux were observed, when the DS concentration was increased [24]. While the absence of hydraulic pressure in FO operation did not prevent biomass attachment on the membranes, it did however prevent biomass compression on the membranes and the formation of a dense cake layer on the membrane surface. Consequently, the effects of membrane biofouling during FO were largely reversible and the flux could be recovered after the biofilms had been dislodged from the membranes. This indicated that the loss in permeate flux after 70 h of bioreactor operation was indeed caused by membrane biofouling. These results also suggested that FO did not prevent membrane biofouling as such, but its advantage lies in the ease of biofilm removal from the membranes and recovering membrane performance. The reversibility of the membrane biofouling in FO-based filtration has been reported earlier through osmotic backwashing or other physical cleaning techniques [23, 28]. While these techniques have been reported to have recovered up to 90% flux in case of biofouling, the flux recovered during organic/inorganic fouling could reach up to 100%. Therefore, the advantages of FO lie in the ease of biofilm removal from the membranes and in providing a sustainable separation performance. Biodegradation and Desalination in Chemostat-FOHFMB Having shown the excellent performance of the chemostat-FOHFMB system in biodegradation of phenol and recovery of excess water from the treated effluent stream, experiments were conducted to demonstrate the suitability of the bioreactor in simultaneous biodegradation of phenol and desalination of the effluent. Fig. 11a shows the cell growth and phenol removal profile in the chemostat at feed phenol concentration of 1000 mg/L and NaCl concentration of 0.6 M, diluted three times to reach an effective phenol and NaCl
concentrations of 380 mg/L and 0.2 M, respectively. The specific growth rate of P. putida in the chemostat was determined to be 0.4 h-1 during transient operation, and steady state was attained within 20 h. Since the feed wastewater contained higher salt concentration as compared to the experiments carried out earlier, the permeate flux through the CA membranes was only 34 mL/h using 0.8 M magnesium chloride as the DS. However, desalination of the treated wastewater could be achieved at a reasonable permeate flux of 49 mL/h when the DS concentration was increased to 1.5 M. Based on these results, it could be concluded that the chemostat-FOHFMB integrated bioreactor system could perform both biodegradation of organic pollutants from wastewater, as well as desalination of the wastewater. During treatment of the saline wastewater in the chemostat-FOHFMB, it was observed that the permeate flux remained stable only in the first 35 h, after which it decreased to lower values (Fig. 11b). This was different from the treatment of non-saline wastewater when flux remained stable for at least 70 h. This lowering in flux could not be attributed to membrane biofouling as the cell growth rate in the saline environment was lower. Consequently, cell attachment on the membranes would have been relatively low. Therefore, the lowering of flux could only be attributed to ICP due to the increased salinity, as well as the concentrative ECP on the shell side due to the low flow rate of the cell culture medium. Another challenge in the design of the FOHFMB is the selection and purification of the DS. In hypersaline industrial wastewater, salt concentration can be as high as 200 g/L, which is equivalent to 3 M sodium chloride [1]. Therefore, FO-based desalination of hypersaline wastewater requires DS which are capable of generating very high osmotic pressures, which can transpire to high water flux. Besides, the DS in the FOHFMB was diluted during the bioreactor operation and further treatment was required to concentrate and recycle the DS. The requirement of an additional treatment process for DS concentration, possibly using
reverse osmosis, reduces the advantages of low-cost filtration through FO membranes and the large-scale operation of the FOHFMB may not be cost-effective. However, FO-based separation has made rapid progress in past few years. There are new membranes which are able to provide higher flux at a lower osmotic pressure gradient, thereby making the osmotic filtration faster and cheaper. In addition, efforts are being made to develop novel and efficient DS which need low-cost energy recovery system [12, 29].
Conclusions A chemostat coupled with FOHFMB has been fabricated and operated for treatment of saline wastewater with sodium chloride concentrations up to 0.6 M and a phenol concentration of 1000 mg/L. The effects of dilution on cell growth and phenol metabolism in the chemostat has been examined and the effects of membrane orientation, DS concentration and DS flow rate on FOHFMB performance have also been investigated. Biomass attachment on the membranes during FO was visualized through SEM, and it was concluded that cell attachment to the membranes depended primarily on permeate flux.
Acknowledgement This research was funded by the Singapore National Research Foundation under its Competitive Research Program for the project entitled, “Advanced FO Membranes and Membrane Systems for Wastewater Treatment, Water Reuse and Seawater Desalination”(grant number: R-279-000-338-281). The authors would like to thank Prof. Neal Tai-Shung Chung for providing the FO membranes. References [1] O. Lefebvre, R. Moletta, Treatment of organic pollution in industrial saline wastewater: A literature review, Water Res., 40 (2006) 3671-3682. [2] C.R. Woolard, R.L. Irvine, Treatment of hypersaline wastewater in the sequencing batch reactor, Water Res., 29 (1995) 1159-1168.
[3] O. Lefebvre, F. Habouzit, V. Bru, J.P. Delgenes, J.J. Godon, R. Moletta, Treatment of Hypersaline Industrial Wastewater by a Microbial Consortium in a Sequencing Batch Reactor, Environ. Technol., 25 (2004) 543-553. [4] J. Liu, G.L. Wang, A novel salt-tolerant mutant YWL-01 for the treatment of saline wastewater, Water Sci. Technol., 60 (2009) 2869-2877. [5] G. Moussavi, B. Barikbin, M. Mahmoudi, The removal of high concentrations of phenol from saline wastewater using aerobic granular SBR, Chem. Eng. J., 158 (2010) 498-504. [6] P. Praveen, K.-C. Loh, Two-phase biodegradation of phenol in trioctylphosphine oxide impregnated hollow fiber membrane bioreactor, Biochem. Eng. J., 79 (2013) 274-282. [7] X.-H. Gu, J.-T. Zhou, A.-L. Zhang, G.-F. Liu, Treatment of hyper-saline wastewater loaded with phenol by the combination of adsorption and an offline bio-regeneration system, J. Chem. Technol. Biotechnol., 83 (2008) 1034-1040. [8] M. Afzal, S. Iqbal, S. Rauf, Z.M. Khalid, Characteristics of phenol biodegradation in saline solutions by monocultures of Pseudomonas aeruginosa and Pseudomonas pseudomallei, J. Hazard. Mater., 149 (2007) 60-66. [9] M. Bonfá, M. Grossman, F. Piubeli, E. Mellado, L. Durrant, Phenol degradation by halophilic bacteria isolated from hypersaline environments, Biodegradation, (2013) 1-11. [10] L.A. Hoover, W.A. Phillip, A. Tiraferri, N.Y. Yip, M. Elimelech, Forward with Osmosis: Emerging Applications for Greater Sustainability, Environ Sci. Technol., 45 (2011) 9824-9830. [11] P. Praveen, K.-C. Loh, Simultaneous extraction and biodegradation of phenol in a hollow fiber supported liquid membrane bioreactor, J. Membr. Sci., 430 (2013) 242-251. [12] K. Lutchmiah, A.R.D. Verliefde, K. Roest, L.C. Rietveld, E.R. Cornelissen, Forward osmosis for application in wastewater treatment: A review, Water Res., 58 (2014) 179-197.
[13] T.Y. Cath, A.E. Childress, M. Elimelech, Forward osmosis: Principles, applications, and recent developments, J. Membr. Sci., 281 (2006) 70-87. [14] B. Cao, A. Geng, K.C. Loh, Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes, Appl. Microbiol. Biotechnol., 81 (2008) 99-107. [15] J. Su, Q. Yang, J.F. Teo, T.-S. Chung, Cellulose acetate nanofiltration hollow fiber membranes for forward osmosis processes, J. Membr. Sci., 355 (2010) 36-44. [16] P. Praveen, K.-C. Loh, Kinetics modeling of two phase biodegradation in a hollow fiber membrane bioreactor, Sep. Purif. Technol., 122 (2014) 350-358. [17] A. Alturki, J. McDonald, S.J. Khan, F.I. Hai, W.E. Price, L.D. Nghiem, Performance of a novel osmotic membrane bioreactor (OMBR) system: Flux stability and removal of trace organics, Biores. Technol., 113 (2012) 201-206. [18] J.R. McCutcheon, R.L. McGinnis, M. Elimelech, A novel ammonia-carbon dioxide forward (direct) osmosis desalination process, Desalination, 174 (2005) 1-11. [19] W.J. Yap, J.S. Zhang, W.C.L. Lay, B. Cao, A.G. Fane, Y. Liu, State of the art of osmotic membrane bioreactors for water reclamation, Biores. Technol., 122 (2012) 217-222. [20] S. Zhao, L. Zou, D. Mulcahy, Effects of membrane orientation on process performance in forward osmosis applications, J. Membr. Sci., 382 (2011) 308-315. [21] B. Cao, P.D. Majors, B. Ahmed, R.S. Renslow, C.P. Silvia, L. Shi, S. Kjelleberg, J.K. Fredrickson, H. Beyenal, Biofilm shows spatially stratified metabolic responses to contaminant exposure, Environ. Microbiol., 14 (2012) 2901-2910. [22] Y.Z. Ding, N. Peng, Y.H. Du, L.H. Ji, B. Cao, Disruption of Putrescine Biosynthesis in Shewanella oneidensis Enhances Biofilm Cohesiveness and Performance in Cr(VI) Immobilization, Appl. Environ. Microbiol., 80 (2014) 1498-1506.
[23] A. Achilli, T.Y. Cath, E.A. Marchand, A.E. Childress, The forward osmosis membrane bioreactor: A low fouling alternative to MBR processes, Desalination, 239 (2009) 10-21. [24] Y. Wang, F. Wicaksana, C.Y. Tang, A.G. Fane, Direct Microscopic Observation of Forward Osmosis Membrane Fouling, Environ. Sci. Technol., 44 (2010) 7102-7109. [25] G. Qiu, Y.-P. Ting, Short-term fouling propensity and flux behavior in an osmotic membrane bioreactor for wastewater treatment, Desalination, 332 (2014) 91-99. [26] E.R. Cornelissen, D. Harmsen, K.F. de Korte, C.J. Ruiken, J.J. Qin, H. Oo, L.P. Wessels, Membrane fouling and process performance of forward osmosis membranes on activated sludge, J Membr. Sci., 319 (2008) 158-168. [27] B. Cao, L.A. Shi, R.N. Brown, Y.J. Xiong, J.K. Fredrickson, M.F. Romine, M.J. Marshall, M.S. Lipton, H. Beyenal, Extracellular polymeric substances from Shewanella sp HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics, Environ. Microbiol., 13 (2011) 1018-1031. [28] B.X. Mi, M. Elimelech, Organic fouling of forward osmosis membranes: Fouling reversibility and cleaning without chemical reagents, J. Membr. Sci., 348 (2010) 337-345. [29] L. Chekli, S. Phuntsho, H.K. Shon, S. Vigneswaran, J. Kandasamy, A. Chanan, A review of draw solutes in forward osmosis process and their use in modern applications, Desalin. Water Treat., 43 (2012) 167-184.
Figure and Table
F Air inlet
Effluent Air outlet
P 3F
P 3F
FOHFM B
F
2F+G
Feed wastewater
P
Chemostat 2F
DS recycle
G
DS
G
2F
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(b)
(a)
(b) 49 mL/h 34 mL/h
(a)
(b)
(c)
12 mL/h
(a)
43 mL/h
24 mL/h (b) 43 mL/h
Figure 7
(a)
(b)
Figure Captions Figure 1. Schematic diagram of the chemostat-FOHFMB Figure 2. Performance of the CA membranes in FO and PRO modes Figure 3. Phenol rejection by the CA membranes Figure 4. Effects of sodium chloride on cell growth using 200 mg/L phenol as growth substrate Figure 5. Cell growth and phenol removal profiles during biodegradation of 1000 mg/L phenol after 3 times dilution in the chemostat upon operation startup Figure 6. Chemostat-FOHFMB operation in PRO mode: (a) permeate flux using 0.4 M MgCl2 as DS;(b) Biomass concentration profiles in chemostat and FOHFMB (dashed lines show predicted values), and ; (c) phenol concentration profiles in chemostat and FOHFMB Figure 7. FOHFMB performance in FO mode: (a) effects of DS concentration on permeate flux, and;(b) effects of DS flow rate on permeate flux Figure 8. Chemostat-FOHFMB operation in FO mode: (a) biomass concentration profiles in chemostat and FOHFMB (dashed lines show predicted values), and; (b) phenol concentration profiles in chemostat and FOHFMB Figure 9. Effects of dilution on cell growth and phenol metabolism in the chemostat Figure 10. SEM images of membrane attached biofilms Figure 11. Biodegradation and desalination in chemostat-FOHFMB: (a) cell growth and phenol metabolism, and; (b) permeate flux using different DS concentration
S1369-703X(14)00330-1 http://dx.doi.org/doi:10.1016/j.bej.2014.11.014 BEJ 6073
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
17-7-2014 15-10-2014 18-11-2014
Please cite this article as: Prashant Praveen, Duong Thi Thuy Nguyen, Kai-Chee Loh, Biodegradation of phenol from saline wastewater using forward osmotic hollow fiber membrane bioreactor coupled chemostat, Biochemical Engineering Journal http://dx.doi.org/10.1016/j.bej.2014.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodegradation of Phenol from Saline Wastewater using Forward Osmotic Hollow Fiber Membrane Bioreactor coupled Chemostat Prashant Praveen, Duong Thi Thuy Nguyen, Kai-Chee Loh* Department of Chemical and Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Singapore 117576 *Corresponding author: Associate Professor, Department of Chemical and Biomolecular Engineering,National University of Singapore, 4 Engineering Drive 4, Singapore 117576. Email: [email protected]; Tel.: +65 6516 2174; Fax: +65 6779 1936 Highlights •
A chemostat was coupled with forward osmotic hollow fiber membrane bioreactor
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The integrated bioreactor was used for treatment of saline phenolic wastewater
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Effects of operating parameters on bioreactor performance were examined
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Forward osmosis performance decreased sharply due to biofouling
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The severity of biofouling increased at higher permeate flux
Abstract A chemostat was coupled with a forward osmotic hollow fiber membrane bioreactor (FOHFMB) for treatment of high strength saline phenolic wastewater using Pseudomonas putida ATCC 11172. The microorganisms were protected from the inhibitory effects of phenol and sodium chloride through dilution of the feed wastewater. This resulted in high cell growth and biodegradation rates during transient operation and steady state was achieved within 20 h. Effluent from the chemostat was desalinated in the FOHFMB through forward osmosis (FO) using magnesium chloride as the draw solute (DS). Permeate flux during FO remained stable for over 70 h in the orientation with DS facing the porous side of the membranes (PRO mode of operation). Water used for dilution could be recovered using 0.8
M DS when the wastewater did not contain any sodium chloride, whereas 1.5 M DS was required to recover water from the wastewater containing 0.6 M sodium chloride. Biomass attachment on the membranes during FO operation was visualized using SEM, which showed that FO membranes was susceptible to fouling propensity and biomass deposition on the membranes was directly associated with permeate flux. Nevertheless, biofouling of membranes was reversible and membrane performance was recovered by osmotic backwashing.
Keywords: Biodegradation; Biofouling; Forward osmosis; Membrane bioreactor; Saline wastewater
Introduction Salts, especially sodium chloride, are important raw materials in chemical industries where they play an indispensable role in various manufacturing processes and other industrial operations. Some of the common industrial applications of salts are in deicing, paper manufacturing, food preservation, oil refining, tanning, water conditioning, agriculture and synthesis of useful chemicals [1, 2]. Consequently, the presence of salts in industrial wastewater is a common occurrence. In fact, it has been estimated that about 5 % of industrial effluents generated throughout the world are either saline or hyper-saline [3].
Industrial saline wastewater is also usually rich in organic matter. Biological treatment of wastewater with high salinity is difficult due to the adverse effects of salts on microbial metabolism [4]. The problem is further aggravated when the wastewater contains toxic organic compounds as the microbial growth and metabolism are inhibited by high concentrations of salts, as well as the organic pollutants [5, 6]. Although wastewater can be desalinated effectively using various techniques such as evaporation, flocculation, reverse osmosis (RO), forward osmosis (FO) or electrochemical methods [7], the salts thus recovered
will have high organic content. In particular, salts polluted with toxic aromatics may not be suitable for reuse, whereas disposal of the recovered salts may require further treatment. In order to treat saline wastewater laden with toxic organic content, the organic pollutants must be removed from the wastewater prior to desalination.
Traditional methods used to alleviate substrate inhibition, such as cell immobilization or twophase biodegradation, are not very effective in protecting microorganisms in saline environment. Therefore, the preferred strategy while biodegrading organic pollutants in saline environment is the use of halophilic microorganisms [8]. However, very few halophiles have been isolated which are capable of metabolizing toxic aromatic compounds. Moreover, these halophiles very often exhibit low tolerance to the substrate, which can result in low cell growth and biodegradation rates [4]. Another approach to treat saline wastewater is by improving salt tolerance of biodegrading microorganisms such as Pseudomonas putida through adaptation. However, salt tolerance acquired through adaptation is temporary and it is quickly lost when the salinity of the medium is decreased [3, 4]. A simple approach to mitigate inhibition arising from salts and organics is the dilution of the saline wastewater. However, dilution is not a preferred method because it requires large quantities of water, increases the process volume and increases the treatment costs [9]. The drawbacks associated with dilution can be alleviated through the use of chemostat and FO technologies. Chemostat is an established technique used for continuous cultivation of microorganisms at constant volume; FO is based on the flow of water across a selectively permeable membrane under an osmotic pressure gradient and it has recently emerged as an energy efficient desalination technique [10]. Through integrating these two techniques, the strategy is to dilute the wastewater to enable biodegrading microorganisms to metabolize the pollutant in the chemostat; the organic pollutant free effluent from the chemostat is then desalinated through FO and water used for dilution is recovered and reused. The resulting
chemostat coupled forward osmotic hollow fiber membrane bioreactor (FOHFMB) can mitigate the challenges associated with wastewater dilution, achieve high biodegradation rates and perform effective desalination [11, 12]. Moreover, FO-based desalination using low or no hydraulic pressure can have the advantages of low energy requirement, better rejection of contaminants and a lower membrane fouling tendency [13]. In this research, a chemostat-FOHFMB integrated bioreactor system was operated to demonstrate the suitability of the ‘dilute-biodegrade-desalinate’ approach in the biodegradation of high concentrations of phenol in saline wastewater. The effects of wastewater salinity, extent of dilution, FO membrane orientation, draw solute (DS) concentration and DS flow rate on the performance of the chemostat-FOHFMB system were examined, and biomass attachment on the membranes was also characterized. Phenol was chosen as the model pollutant because of its toxic and recalcitrant nature. Besides, phenol and its derivatives are found in saline wastewater emanating from petroleum, textile and leather industries [9].
Materials and Methods Microorganisms, Culture Conditions, and Chemicals P. putida ATCC 11172 was used throughout this study. Stock cultures were maintained on nutrient agar (Oxoid, Hampshire, UK) slants at 4°C. The microorganisms were grown in a chemically defined mineral medium supplemented with phenol in Erlenmeyer flasks on a shaking water bath (GFL 1092, Burgwedel, Germany) at 30 °C and 150 rpm. The composition of the mineral medium has been described elsewhere [14]. All media (except phenol), pipette tips, and Erlenmeyer flasks fitted with cotton plugs were autoclaved before use. Prior to inoculation, cells were induced in mineral medium containing 200 mg/L phenol as the sole carbon source. Activated cells in the late exponential growth phase were used as inoculum for all the experiments.
All the chemicals used in this research were of analytical grade. Phenol was dissolved in 0.02 M sodium hydroxide to prepare a stock solution of 10 g/L. Magnesium chloride was used as the DS in all the experiments, whereas sodium chloride was used to maintain wastewater salinity.
Chemostat- FOHFMB FO Membrane Contactor Cellulose acetate (CA) nanofiltration hollow fiber membranes were used for FO. The inner surface of the membranes was porous, whereas the dense outer surface of the membranes acted as the rejection layer. A detailed description of the membrane characteristics and its synthesis are given elsewhere [15]. A shell and tube membrane contactor with an inner diameter of 1.27 cm was fabricated by potting the CA hollow fiber membranes into a perfluoroalkoxy module using epoxy resins (Araldite, England). The membrane contactor contained 50 fibers with an effective length of 25 cm.
Experimental Setup Fig. 1 shows the schematic diagram of the experimental setup. The chemostat consisted of a 500 mL Erlenmeyer flask with a working volume of 250 mL. A dual channel peristaltic pump (L/S modular pump, Easy-Load II pump head, Masterflex, USA) was used to pump the feed wastewater in and out of the chemostat at a constant flow rate. Humidity saturated purified air was sparged into the chemostat at a rate of 2 gas volume per reactor volume per minute (VVM). The effluent from the chemostat was fed into one side of the FOHFMB, whereas the DS was pumped into the other side of the membrane contactor using another peristaltic pump. Note that the RO unit required for DS recycle was not operated for these experiments.
Operation The synthetic feed wastewater was prepared with 1000 mg/L phenol along with mineral salts required for cell growth. In the desalination experiments, 0.6 M sodium chloride was also
added into the wastewater to increase its salinity. The wastewater was diluted prior to its addition into the chemostat, and pumped into the chemostat at the flow rate of 60 mL/h, equivalent to a hydraulic retention time (HRT) of 4.2 h. Once the wastewater had reached the required level in the chemostat and the FOHFMB was also filled with the wastewater, the chemostat was inoculated with P. putida from the preculture to achieve an initial biomass concentration of 6-7 mg/L in the chemostat. After inoculation, the DS was pumped into the FOHFMB at a constant flow rate. Magnesium chloride concentration in the DS feed was varied between 0.4-1.5 M depending on the experimental conditions, whereas the DS flow rate was varied between 1.5-7.5 mL/min. At the end of each experimental run, the biofilms on the CA membranes in the FOHFMB were cleaned through osmotic backwashing of the membranes. In this process, 0.5 M sodium chloride as DS was pumped over the biofilms whereas pure water was pumped on the clean side of the membranes. The osmotic backwashing was carried out for 2 h, following which both the shell and the tube sides of the FOHFMB were washed with pure water for 6-8 h to remove any remaining microorganisms or salts from the membranes. The same membrane contactor was used after cleaning in all the experiments. Analytical Methods Cell density was determined by measuring the optical density (OD) of the aqueous medium at 600 nm using an ultraviolet-visible spectrophotometer (UV-1240, Shimazdu, Japan). The OD was used to compute the biomass concentration using the formula: dry cell weight (mg/L) = 385×OD600 [16]. For determining phenol concentration, 3 mL of the cell culture was filtered through 0.45 µm syringe filter (Millex, Millipore, USA) and extracted into an equal volume of dichloromethane containing 100 mg/L o-cresol as internal standard. Phenol in the extract was analyzed by gas chromatography equipped with a flame ionization detector (Clarus 600, Perkin Elmer, USA). Biofilms on the membranes were characterized using a scanning
electron microscope (SEM) (JEOL JSM-5600LV) after sputtering the membranes with platinum.
Results and Discussion Flux and Salt Rejection To investigate membrane performance during FO, water flux through the CA membranes was determined using magnesium chloride as the DS against ultrapure water. A constant DS concentration was maintained on one side of the membranes using a continuous flow of the DS without any recirculation, whereas ultrapure water on the other side of the membranes was recirculated. In the FO mode, with the DS facing the porous side of the membrane (lumen of hollow fiber membranes), water flux increased from 1.5 liters per square meter per hour (LMH) at 0.2 M DS to 5.6 LMH at 2 M DS. However, the increase in flux was nonlinear and the rate of increase in flux gradually decreased with rising magnesium chloride concentration as shown in Fig. 2. A similar trend in water flux was observed in the pressureretarded osmosis (PRO) mode when the DS solute was facing the active layer of the membranes. The flux observed in the PRO mode was 40-60% higher than those observed in the FO mode at the same DS concentrations. These results are consistent with those reported in literature and the difference in permeate flux between FO and PRO mode is attributed to internal concentration polarization (ICP) in the FO mode, resulting in lower effective osmotic pressure gradient across the active layer of the membrane [17]. The CA membranes were also tested for the rejection of magnesium chloride and phenol. While the membranes exhibited excellent performance in preventing DS solute leakage across the membrane and rejected more than 99% of the DS at the investigated experimental conditions, phenol was able to diffuse through the membranes quite easily. Fig. 3 shows phenol diffusion through the membranes at different feed phenol concentration. It can be seen that phenol rejection performance of the membranes was very poor and only 40% of phenol fed to the FO membrane module was rejected. It was also observed that phenol rejection was
almost independent of feed phenol concentration and it remained constant at 39 ± 3.6 % when phenol concentration was varied from 200 to 2000 mg/L. The poor rejection of phenol could be due to the small molecular size of phenol as compared to the membrane pore size. The average molecular mass of a phenol molecule is 94 Daltons, whereas the molecular weight cut off (MWCO) of the CA membranes was 186 Daltons [15]. Although the molecular size of anhydrous magnesium chloride is comparable to phenol, the hydrated magnesium chloride molecule is likely to be much larger resulting in better rejection by the CA membranes. The rejection of phenol by the membranes was independent of the bulk feed phenol concentration. This was expected as the water flux through the FO membranes was constant for all the experiments [18]. Effects of Salinity on Microorganisms In saline wastewater contaminated with toxic organic compounds, it is important to examine the effects of increasing concentrations of the salts and the organic pollutant to determine the extent of dilution required to alleviate the inhibitory effects of wastewater on the microorganisms. The effects of phenol on growth and metabolism of P. putida are well characterized in literature and the inhibitory limit is known to be 600 mg/L [11].
In order to understand the effects of saline environment on cell growth and to determine the salt tolerance of P. putida, batch biodegradation experiments were carried out in cell culture medium containing 200 mg/L phenol as the growth substrate, and at 0.1-0.7 M sodium chloride salinity. Fig. 4 shows that cell growth rate was not affected by the presence of sodium chloride at 0.1 M salt concentration. A specific growth rate of 0.55 h-1 was observed at 0.1 M sodium chloride, which was the same as that of the control medium prepared without any sodium chloride. However, as the salt concentration was increased further to 0.2, 0.3, 0.4 and 0.5 M, the specific growth rates gradually decreased to 0.46, 0.40, 0.33 and 0.20 h-1, respectively. It was also observed that the microorganisms exhibited a short lag phase
while growing at salt concentrations higher than 0.3 M. No cell growth was observed at 0.6 M salt whereas a quick decrease in the biomass concentration was observed immediately after inoculation at 0.7 M salt. Unlike cell growth rates, biomass yield on phenol was not affected by the changes in the salinity of the cell culture medium and it remained constant at about 0.6 g-biomass/g-phenol at 0.1-0.5 M salt. These results indicate that salt concentrations above 0.1 M affected the growth and metabolism of P. putida adversely. However, the bacteria could tolerate up to 0.5 M salinity as evident from complete biodegradation of phenol even at this salt concentration. For subsequent biodegradation experiments, the wastewater in the chemostat-FOHFMB must be diluted to bring salt concentration to below 0.5 M, whereas phenol concentration should be brought down to below 600 mg/L.
Biodegradation in Chemostat-FOHFMB Operation in PRO Mode To investigate biodegradation of phenol in the chemostat, feed wastewater containing 1000 mg/L phenol was pumped into the chemostat at a flow rate of 20 mL/h. The wastewater was diluted three times with pure water to decrease the feed phenol concentration to 350 mg/L and to reach an increased influent flow rate of 60 mL/h. The dilution rate in the chemostat was thus calculated as 0.24 h-1, corresponding to a 4.2 h HRT. Since feed phenol concentration was sub-inhibitory, P. putida in the chemostat did not experience any lag phase. The microorganisms exhibited a specific growth rate of 0.48 h-1 and the net specific growth rate was calculated to be 0.24 h-1. Due to the high cell growth rate, phenol in the chemostat was rapidly metabolized and phenol concentration dropped to zero within 20 h to reach the steady state at a biomass concentration of 170 mg/L (Fig. 5). Once steady state was reached, phenol concentration in the chemostat remained zero for the entire operating period but the biomass concentration started decreasing after 40 h. The decrease in the biomass was the result of cell aggregation as lots of aggregates were observed in the bioreactor after 40 h.
While cell growth and phenol metabolism in the chemostat was stable in the first 20 h, water flux through the CA membranes in the FOHFMB was not very consistent during this period. The initial permeate flux using 0.4 M magnesium chloride as the DS was about 35 mL/h (Fig. 6a). However, the flux decreased monotonously to about 12 mL/h within 15 h of operation. The flux remained constant for about 70 h before it decreased to even lower values. The loss of flux in the osmotic membrane bioreactor is usually attributed to two factors: concentration polarization and membrane biofouling [17, 19]. Although biomass concentration in the chemostat was low and membrane biofouling during this period was not deemed severe, P. putida exhibited a very strong tendency to attach to surfaces and to produce extracellular polymeric substances (EPS) [11]. Besides, when the microorganisms or other foulants faced the porous side of the membranes, the conditions were more favorable for fouling as these entities entered the membrane pores [20]. Further evidence of cell attachment on the membranes was obtained by comparing the biomass concentration profiles in the chemostat and in the FOHFMB. Since a significant portion of water used in the dilution of feed wastewater was being recovered through the CA membranes, it was anticipated that the effluent from the FOHFMB would have higher biomass concentration as compared to that in the chemostat. However, Fig. 6b shows that the biomass concentration profiles in the chemostat did not differ significantly from that in the FOHFMB. The expected biomass concentration in the FOHFMB, which was calculated based on the water flux at that time, was higher than the biomass concentration measured experimentally. These results indicate that the biomass present in the water filtered through the CA membranes did not remain in suspension and the biomass was retained on the membrane surfaces and pores. Similarly, it was expected that phenol concentration in the effluent from the FOHFMB would be higher than those observed in the chemostat at any time because the FO membranes could reject about 40 % phenol from permeate and the rejected
phenol was retained in the FOHFMB. On the contrary, results in Fig. 6c indicate that phenol concentration in the final effluent stream released from the FOHFMB was actually lower than those observed in the chemostat at those times. These results indicate that the FO membrane module in the integrated bioreactor setup did not just act as a filter but it also facilitated phenol biodegradation by acting as an osmotic membrane bioreactor. The FOHFMB thus improved the effluent quality by retaining high concentration of biomass for biodegradation of phenol and by improving the overall HRT in the integrated bioreactor system. It can also be inferred that biofilm development on the CA membranes was favorable for phenol biodegradation in the FOHFMB as attached cells exhibited more tolerance towards toxic substrates as compared to suspended cells because of structural and physiological heterogeneity and the protection of EPS [21]. Osmotic Backwashing At the end of every experimental run, the FO membrane module was washed to remove entrapped and lined bacteria from the membranes. During the washing process, cell culture medium was replaced by 1 M sodium chloride solution, whereas pure water was passed through the lumen side. Although this washing process was not optimized, it was observed that 3-4 h of osmotic backwashing could remove most of the biofilm. An attempt was made to estimate the concentration of microorganisms removed in the backwashing. However, the high salt concentration was detrimental for the cells (causing cell lysis) and concentrations estimated in the process were inaccurate indication of the actual biomass attached to the membranes. Although the total biomass concentration in the FOHFMB could be estimated through total protein quantification in the bioreactor, even under cell lysis conditions [22], the test was not performed as it would have required the breakage of the membrane module for the collection of biofilm samples. After osmotic backwashing, the membrane contactor was washed with pure water on both the sides for at least 12 h to negate any effects of ICP. The membrane was tested for flux against 0.5 M DS after the washing process was complete. It
was observed that the flux after washing cycle was constant at 2.9 ± 0.4 LMH. These results show the advantages of FO based systems in dislodging biofilms from membrane surfaces to clean the membrane and to recover the permeate flux. In the absence of any hydraulic pressure, the biomass on the membrane surface was not compressed to form a cake layer. Consequently, the lightly attached biomass on the membrane surfaces could easily be dislodged and washed away [23]. Operation in FO Mode Since the biofouling propensity of FO membranes is high when the system is operated in the PRO mode with the microorganisms facing the porous side of the membranes [20], flow orientation was changed so that microorganisms in the FOHFMB were facing the rejection layer of the membranes. This change in membrane orientation had no effect on the chemostat operation but it resulted in significant improvement in FOHFMB performance as the permeate flux was stable for longer time (Fig. 7a). The flux at 0.4 and 0.8 M DS concentrations remained constant at 24.4 ± 3.0 mL/h and 43.1 ± 2.7 mL/h, respectively, for over 70 h. Since the feed wastewater was pumped to the chemostat at 20 mL/h, which increased to 60 mL/h after three times dilution, the amount of water used for dilution was 40 mL/h. The water used for dilution could be completely recovered using 0.8 M DS, which indicates that this process is sustainable. The decrease in flux after about 70-80 h of operation could be attributed to concentration polarization either due to salt accumulation in the membrane pores or on the membrane surfaces. The lowering of flux could also be a result of membrane biofouling as a lot of biomass had accumulated on the membrane surfaces by the end of three days of operation. To further investigate the reason for the flux lowering in the FOHFMB, experiments were carried out at different DS flow rate to examine the effects of external concentration polarization (ECP) on FOHFMB performance. It can be seen from Fig. 7b that increasing the DS flow rate from 1.5 mL/min to 7.5 mL/min had no significant effects on the flux
performance of the FOHFMB. Therefore, it can be inferred that the dilutive ECP on the DS side was not really a major factor affecting water flux through the membranes. Similar experiments to demonstrate the effects of concentrative ECP on the wastewater side could not be conducted because the maximum influent flow rate that could be supported in the chemostat was about 2.0 mL/min. Flow rates higher than 2.0 mL/min would have resulted in cell wash out. Using 0.8 M magnesium chloride as the DS in FO mode, the flux was comparatively high and stable. Consequently, the extent of biomass accumulation on the CA membranes during FO was higher as can be seen in Fig. 8a. The biomass concentration in the effluent from the FO unit was expected to be three times higher than that observed in the chemostat at any time. However, the biomass concentrations in the chemostat and the FOHFMB were almost identical. It can thus be inferred that the biomass amounting to the difference between the expected biomass concentration in the FOHFMB and the measured biomass concentration in the FOHFMB had been deposited onto the hollow fiber membranes. Furthermore, it can also be concluded that the FO membranes did not really prevent biofilm attachment on the membranes or have low biofouling tendency. The severity of biofouling depends on the permeate flux and lower flux would usually result in low membrane biofouling and vice versa [24]. As observed earlier in Fig. 6, the presence of the FOHFMB improved the overall rate of biodegradation in the chemostat-FOHFMB bioreactor system (Fig. 8b) through the presence of a high amount of microorganisms in the shell side of the membrane module as well as on the outer surfaces of the membranes. The presence of FOHFMB also enhanced the effective HRT in the bioreactor. Consequently, the effluent from the FOHFMB had a lower phenol concentration and steady state was achieved quicker than that which would have been achieved using only the chemostat for biodegradation.
The extent of dilution was crucial in facilitating the biodegradation of phenol in the chemostat-FOHFMB system. To examine this effect, phenol biodegradation in the chemostatOHFMB systems was conducted at three times and five times dilution. At five times dilution, the effective feed phenol concentration in the chemostat was about 200 mg/L, way below the inhibitory limit. At the low phenol concentration, P. putida exhibited a specific growth rate of 0.59 h-1, much higher than the specific growth rate of 0.48 h-1 observed at three times dilution of the wastewater when effective feed phenol concentration was about 350 mg/L. The low phenol concentration and high cell growth rate at five times dilution also resulted in higher biodegradation rate and steady state was achieved within 10 h of bioreactor operation (Fig. 9). However, these changes due to the degree of dilution did not affect water flux in the FOHFMB significantly and the flux remained constant at about 40 mL/h (results not shown). These results indicate that a higher degree of dilution has the advantages of higher cell growth and biodegradation rates. Consequently, the bioreactor attains steady state faster. Furthermore, a higher specific growth rate can support a higher dilution rate in the bioreactor and the chemostat can be operated at higher influent flow rates. For example, the specific cell growth rate at 200 mg/L phenol and 0.1 M salt was 0.55 h-1 whereas it decreased to about 0.2 h-1 when salt concentration was increased to 0.5 M using 200 mg/L phenol as growth substrate. Considering a chemostat volume of 250 mL, the highest feed flow rate in the bioreactor at 0.1 and 0.5 M NaCl could be 140 mL/h and 50 mL/h, respectively. Therefore, the effects of dilution of the feed wastewater is somewhat compensated due to a higher wastewater flow rate that the bioreactor can support. However, when the extent of dilution is increased, a higher DS concentration will be required to recover the extra water used for dilution. Based on these results, it can be suggested that dilution can be considered for substrate inhibition alleviation in a chemostat-FOHFMB system, especially when the bioreactor does not need any additional water resources for operation.
Membrane Biofouling The deposition of biomass on the membrane surfaces during filtration can occur due to natural diffusion of the microorganisms from solution to the membranes or under the flow of water across the membranes. Once microorganisms have been deposited on the membranes, the severity of membrane biofouling depends on the adhesion capability of the microorganisms especially when extracellular polymeric substances (EPS) are produced by the microorganisms [25]. Microorganisms such as P. putida exhibit excellent adhesive properties and often form strong biofilms on polymeric membranes [11]. Fig. 10 shows the SEM images of P. putida attached on the outer surface of the CA membranes after 12 h of chemostat-FOHFMB operation. It can be seen that the rod shaped bacteria were not just deposited on the membranes, but these cells were wrapped in the protective layer of EPS. These biofilms grew stronger with time with the production of more EPS and the deposition of more biomass on the membranes. Therefore, it is likely that the loss in permeate flux in FOHFMB after 3-4 days of operation was caused by membrane biofouling. These results were very different from those reported in municipal wastewater treatment in submerged osmotic membrane bioreactors (OMBR), where the water flux decreased by only 20% after 14 days of operation [23]. Since the membrane fouling in the FOHFMB occurred even at a low water flux of 24 mL/h which was equivalent to 1.2 LMH, it showed that biofouling could occur at very flow flux conditions. These results also suggested that the critical flux for membrane fouling in this case was very low, or critical flux could not have been an important factor in membrane biofouling when the microorganisms had a high tendency to attach to the membrane surface as observed in case of P. putida [26]. The absence of any hydraulic pressure on the membranes during FO is believed to impart a lower fouling propensity to FO membranes as compared to traditional membrane filtration techniques such reverse osmosis and ultrafiltration [13, 27]. However, the results obtained in Figs. 6 and 8 show that the microorganisms could attach readily to the membranes even in the
absence of any hydraulic pressure and the rate at which the biomass was deposited on the membranes depended not on the pressure but on the permeate flux. This observation is consistent with those reported in literature where a gradual increase in the foulant deposition on the FO membranes and a corresponding decrease in the water flux were observed, when the DS concentration was increased [24]. While the absence of hydraulic pressure in FO operation did not prevent biomass attachment on the membranes, it did however prevent biomass compression on the membranes and the formation of a dense cake layer on the membrane surface. Consequently, the effects of membrane biofouling during FO were largely reversible and the flux could be recovered after the biofilms had been dislodged from the membranes. This indicated that the loss in permeate flux after 70 h of bioreactor operation was indeed caused by membrane biofouling. These results also suggested that FO did not prevent membrane biofouling as such, but its advantage lies in the ease of biofilm removal from the membranes and recovering membrane performance. The reversibility of the membrane biofouling in FO-based filtration has been reported earlier through osmotic backwashing or other physical cleaning techniques [23, 28]. While these techniques have been reported to have recovered up to 90% flux in case of biofouling, the flux recovered during organic/inorganic fouling could reach up to 100%. Therefore, the advantages of FO lie in the ease of biofilm removal from the membranes and in providing a sustainable separation performance. Biodegradation and Desalination in Chemostat-FOHFMB Having shown the excellent performance of the chemostat-FOHFMB system in biodegradation of phenol and recovery of excess water from the treated effluent stream, experiments were conducted to demonstrate the suitability of the bioreactor in simultaneous biodegradation of phenol and desalination of the effluent. Fig. 11a shows the cell growth and phenol removal profile in the chemostat at feed phenol concentration of 1000 mg/L and NaCl concentration of 0.6 M, diluted three times to reach an effective phenol and NaCl
concentrations of 380 mg/L and 0.2 M, respectively. The specific growth rate of P. putida in the chemostat was determined to be 0.4 h-1 during transient operation, and steady state was attained within 20 h. Since the feed wastewater contained higher salt concentration as compared to the experiments carried out earlier, the permeate flux through the CA membranes was only 34 mL/h using 0.8 M magnesium chloride as the DS. However, desalination of the treated wastewater could be achieved at a reasonable permeate flux of 49 mL/h when the DS concentration was increased to 1.5 M. Based on these results, it could be concluded that the chemostat-FOHFMB integrated bioreactor system could perform both biodegradation of organic pollutants from wastewater, as well as desalination of the wastewater. During treatment of the saline wastewater in the chemostat-FOHFMB, it was observed that the permeate flux remained stable only in the first 35 h, after which it decreased to lower values (Fig. 11b). This was different from the treatment of non-saline wastewater when flux remained stable for at least 70 h. This lowering in flux could not be attributed to membrane biofouling as the cell growth rate in the saline environment was lower. Consequently, cell attachment on the membranes would have been relatively low. Therefore, the lowering of flux could only be attributed to ICP due to the increased salinity, as well as the concentrative ECP on the shell side due to the low flow rate of the cell culture medium. Another challenge in the design of the FOHFMB is the selection and purification of the DS. In hypersaline industrial wastewater, salt concentration can be as high as 200 g/L, which is equivalent to 3 M sodium chloride [1]. Therefore, FO-based desalination of hypersaline wastewater requires DS which are capable of generating very high osmotic pressures, which can transpire to high water flux. Besides, the DS in the FOHFMB was diluted during the bioreactor operation and further treatment was required to concentrate and recycle the DS. The requirement of an additional treatment process for DS concentration, possibly using
reverse osmosis, reduces the advantages of low-cost filtration through FO membranes and the large-scale operation of the FOHFMB may not be cost-effective. However, FO-based separation has made rapid progress in past few years. There are new membranes which are able to provide higher flux at a lower osmotic pressure gradient, thereby making the osmotic filtration faster and cheaper. In addition, efforts are being made to develop novel and efficient DS which need low-cost energy recovery system [12, 29].
Conclusions A chemostat coupled with FOHFMB has been fabricated and operated for treatment of saline wastewater with sodium chloride concentrations up to 0.6 M and a phenol concentration of 1000 mg/L. The effects of dilution on cell growth and phenol metabolism in the chemostat has been examined and the effects of membrane orientation, DS concentration and DS flow rate on FOHFMB performance have also been investigated. Biomass attachment on the membranes during FO was visualized through SEM, and it was concluded that cell attachment to the membranes depended primarily on permeate flux.
Acknowledgement This research was funded by the Singapore National Research Foundation under its Competitive Research Program for the project entitled, “Advanced FO Membranes and Membrane Systems for Wastewater Treatment, Water Reuse and Seawater Desalination”(grant number: R-279-000-338-281). The authors would like to thank Prof. Neal Tai-Shung Chung for providing the FO membranes. References [1] O. Lefebvre, R. Moletta, Treatment of organic pollution in industrial saline wastewater: A literature review, Water Res., 40 (2006) 3671-3682. [2] C.R. Woolard, R.L. Irvine, Treatment of hypersaline wastewater in the sequencing batch reactor, Water Res., 29 (1995) 1159-1168.
[3] O. Lefebvre, F. Habouzit, V. Bru, J.P. Delgenes, J.J. Godon, R. Moletta, Treatment of Hypersaline Industrial Wastewater by a Microbial Consortium in a Sequencing Batch Reactor, Environ. Technol., 25 (2004) 543-553. [4] J. Liu, G.L. Wang, A novel salt-tolerant mutant YWL-01 for the treatment of saline wastewater, Water Sci. Technol., 60 (2009) 2869-2877. [5] G. Moussavi, B. Barikbin, M. Mahmoudi, The removal of high concentrations of phenol from saline wastewater using aerobic granular SBR, Chem. Eng. J., 158 (2010) 498-504. [6] P. Praveen, K.-C. Loh, Two-phase biodegradation of phenol in trioctylphosphine oxide impregnated hollow fiber membrane bioreactor, Biochem. Eng. J., 79 (2013) 274-282. [7] X.-H. Gu, J.-T. Zhou, A.-L. Zhang, G.-F. Liu, Treatment of hyper-saline wastewater loaded with phenol by the combination of adsorption and an offline bio-regeneration system, J. Chem. Technol. Biotechnol., 83 (2008) 1034-1040. [8] M. Afzal, S. Iqbal, S. Rauf, Z.M. Khalid, Characteristics of phenol biodegradation in saline solutions by monocultures of Pseudomonas aeruginosa and Pseudomonas pseudomallei, J. Hazard. Mater., 149 (2007) 60-66. [9] M. Bonfá, M. Grossman, F. Piubeli, E. Mellado, L. Durrant, Phenol degradation by halophilic bacteria isolated from hypersaline environments, Biodegradation, (2013) 1-11. [10] L.A. Hoover, W.A. Phillip, A. Tiraferri, N.Y. Yip, M. Elimelech, Forward with Osmosis: Emerging Applications for Greater Sustainability, Environ Sci. Technol., 45 (2011) 9824-9830. [11] P. Praveen, K.-C. Loh, Simultaneous extraction and biodegradation of phenol in a hollow fiber supported liquid membrane bioreactor, J. Membr. Sci., 430 (2013) 242-251. [12] K. Lutchmiah, A.R.D. Verliefde, K. Roest, L.C. Rietveld, E.R. Cornelissen, Forward osmosis for application in wastewater treatment: A review, Water Res., 58 (2014) 179-197.
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Figure and Table
F Air inlet
Effluent Air outlet
P 3F
P 3F
FOHFM B
F
2F+G
Feed wastewater
P
Chemostat 2F
DS recycle
G
DS
G
2F
(a)
(b)
(a)
(b) 49 mL/h 34 mL/h
(a)
(b)
(c)
12 mL/h
(a)
43 mL/h
24 mL/h (b) 43 mL/h
Figure 7
(a)
(b)
Figure Captions Figure 1. Schematic diagram of the chemostat-FOHFMB Figure 2. Performance of the CA membranes in FO and PRO modes Figure 3. Phenol rejection by the CA membranes Figure 4. Effects of sodium chloride on cell growth using 200 mg/L phenol as growth substrate Figure 5. Cell growth and phenol removal profiles during biodegradation of 1000 mg/L phenol after 3 times dilution in the chemostat upon operation startup Figure 6. Chemostat-FOHFMB operation in PRO mode: (a) permeate flux using 0.4 M MgCl2 as DS;(b) Biomass concentration profiles in chemostat and FOHFMB (dashed lines show predicted values), and ; (c) phenol concentration profiles in chemostat and FOHFMB Figure 7. FOHFMB performance in FO mode: (a) effects of DS concentration on permeate flux, and;(b) effects of DS flow rate on permeate flux Figure 8. Chemostat-FOHFMB operation in FO mode: (a) biomass concentration profiles in chemostat and FOHFMB (dashed lines show predicted values), and; (b) phenol concentration profiles in chemostat and FOHFMB Figure 9. Effects of dilution on cell growth and phenol metabolism in the chemostat Figure 10. SEM images of membrane attached biofilms Figure 11. Biodegradation and desalination in chemostat-FOHFMB: (a) cell growth and phenol metabolism, and; (b) permeate flux using different DS concentration