In recent years wastewater treatments have been performed as an activated sludge process. These systems required large tanks and produced a lot of sludge . Nowadays, once of the most important processes used in municipal and industrial wastewater treatment is membrane bioreactor (MBR) technology [2-4]. First time, MBR technology was recommended in 1960 . These system combines activated sludge and membrane filtration process, and do not require to sedimentation and disinfection process in usual activated sludge methods . Some recent pollutants such as stable organic matter, heavy metals, and environmental nanoparticle pollutants can be removed by MBR . MBR performance including; higher organic loading rate, high output fluid quality, less sludge production [8-11]. However, Membrane fouling is the main defect of the MBR system, that caused a high operation cost, increase the energy demand, and increase in hydraulic resistance, reduce system efficiency and permeate flux or trans-membrane increase, and frequency of cleaning [12-14]. Fouling occurred due to activated sludge components interaction with membrane structure and happened for the reason that blockage of membrane pores by sieving and adsorption of components on the membrane surface or in membrane pores [13, 15]. The activated sludge liquor include; substrate components, cell, soluble microbial products (SMP), extracellular polymeric substances (EPS), bacteria, protein, carbohydrate, humic and fulvic acids, and there are the main membrane foulants in MBR [1, 16-19]. For reduce and control the membrane fouling phenomenon, there are three methods: membrane structure modification, variation the operation condition, and improved biomass properties [5, 10, 20]. Many researchers have been studied the adsorbent and nanoparticles effect on improving the sludge properties and membrane fouling; using the non-reactive chemical addition such as: activated carbon, zeolite, biocarriers, metal salts, poly aluminium chloride, poly ferric chloride and etc can be improved sludge characteristics (such as: size of floc, SMP, EPS, and viscosity) [6, 14, 21] ; also the other matter such as ZnO, Fe2O3, Fe3O4 with pretreatment and also non-adsorbent materials such as ozone. Crystalline zeolite nano-adsorbents with large pores and functional group such as OH can reduce 66% TMP, and adsorbed organic compounds. They reported these nano-adsorbent decreased the cake formation rate and irreversible fouling . Magdalene Tan and et al. (2015) showed both of EPS and SMP are the main causes of fouling, and an increasing amount of ZnO can reduce the EPS and SMP production rate, but did not have a significant effect on membrane fouling . However, Fe3O4 nanoparticles were not used directly in the mixed liquor suspended solids (MLSS) for a comprehensive investigation of polyvinylidene fluoride (PVDF) membrane fouling. In the previous study, Fe3O4 nanoparticles were added as a pretreatment before the MBR system, for example, Yu et al.  studied the contribution of Fe3 O4 nanoparticles to the fouling of ultrafiltration with coagulation pre-treatment. Using the obtained analysis, they concluded that increased levels of protein and polysaccharide increased the amount of TMP. The TMP system had a slowly increasing trend sever increase in TMP due to fouling of the membrane (22 kPa after 25 day). Also in another study, Fe2O3 nanoparticles were added on the membrane not mixed liquor in the MBR system. They concluded that at high mixing rate, the deposition of nanoparticles on the membrane would increase the fouling, and the presence of this nanoparticle reduces contamination and increases the membrane performance at moderate mixing rates .
Therefore, in this study, we used this nanoparticle directly in the mixed liquor suspended solid and investigated their properties on the membrane fouling and adsorption organic and inorganic components. These particles are inert and biocompatible, and also adsorb compounds with high efficiency and low concentration. Furthermore, we used these nanoparticles in the presence of PVDF membrane and were discussed its effects with different analysis such as SMP, EPS, Fourier-transform infrared spectroscopy (FTIR) analysis for cake layer, particle size distribution (PSD) of mixed liquor.
MATERIALS AND METHODS
The synthetic sewage is a model of petrochemical wastewater. Wastewater synthesized included: ethanol: 350; K2HPO4: 35; KH2PO4: 45; Urea: 560; MgSO4•7H2O: 13; CaCl2•2H2O: 7; FeCl3: 5; ZnSO4: 2; NaHCO3: 500; EDTA: 7.
As shown in Fig. 1 two membrane bioreactors were with dimensions of 10*10*35 cm, and an operating volume of 1.5 liters have been used. From the PVDF membrane was used with an effective area, mean pore size and porosity 39*10-2 m2, 0.9 µm and 73%, respectively. The synthetic wastewater of the petrochemical company is used as input to the system. In one of the bioreactors, at the first added the 0.9 g nanoparticles in the system and each time sampling 0.3 g Fe3O4 nanoparticles were added (R2), and the other was used as control (R1). The hydraulic retention time (HRT) and sludge retention time (SRT) for both bioreactors were 36 h and 30 days, respectively. The other operating conditions are given in Table 1. For evaluating the effect of Fe3O4 nanoparticles in MBR system, the amount of flux, COD, EPS, SMP, mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS) was measured; also, FTIR analysis and PSD of the sludge were taken.
MLSS, MLVSS and membrane fouling analysis
MLSS and MLVSS were estimated according to the standard methods . For membrane fouling, after operation of MBR, its flux after fouling was measured, J1. Then the cake layer on the membrane was washed with distilled water, membrane flux after washing was called J2. Finally, J3 which is membrane flux after chemical washing was measured. As there is a direct relationship between fouling mechanism and reduction of flux, resistance in the serial model is the simplest method which uses Darcy law . Total fouling, reversible, irreversible and recovery flux can be obtained by following equations relative to pure water flux from clean membrane module [23, 24].
For the investigation, the effect of the Fe3O4 nanoparticles on the MBR systems, COD standard method was measured . 2.5 ml of sample, 1.5 ml of potassium dichromate and 3.5 ml of sulfuric acid solution was poured into the vial, and placed in the termo-reactor for 2 h at 148 ◦C. Then, after cooling, the absorbance read at 600 nm. For calculating the COD removal, calibration curve used:
SMP and EPS Extraction
To analyze the amount of protein and carbohydrates, SMP and EPS were extracted from the mixed liquor. SMP was attained via centrifuging 50 ml of sludge at 12000 rpm for 10 min. Then the EPS is extracted by the thermal method. In this way, the 0.9% NaCl solution is added to the remaining sludge, and heated in a water bath at 60 ◦C for 30 min . Protein amount was measured with a modified Lowry method and carbohydrate analysis was performed by Anthron method .
Particle Size Distribution (PSD)
PSD was determined by the Fritsch “analysette 22” with a detection range of 0.01-1000 µm.
Fourier-Transform Infrared Spectroscopy (FTIR) Analysis
For FTIR analysis, 50 ml of the mixed liquor was centrifuged for 10 min at 9000 rpm and placed in an incubator at 55 ◦C for 48 h to dry.
RESULTS AND DISCUSSION
COD removal efficiency
Fig. 2 showed COD removal efficiency in both membrane bioreactors. The results obtained from the first COD removal measurement indicate that the removal efficiency in R1 and R2 were 86.92 and 98.17%, respectively. Fig. 2 shows that the presence of Fe3O4 nanoparticles improved the COD removal efficiencies, but over time the removal efficiency reduced due to saturation of the adsorption capacity (92.38% in day 23) and removal efficiency decreased. In R1, the number of COD removal increases due to the growth of microorganisms and an increase in MLSS; the increasing of MLSS caused more utilization from organic matter and COD removal increased. The use of additives such as SiO2, ZnO and Ag nanoparticles caused the COD removal efficiency to be no significant effect, more than 95% and around 95%, respectively [26, 27].
According to the prior studies, the amount of MLSS in aerobic MBR is usually reported to be between 3000-31000 mg/L . Of course, this range is not fixed in various studies. It seems that the membrane fouling decrease in lower MLSS concentration, but in MLSS concentration above 15000 mg/L, membrane fouling increases. Although the high MLSS concentration plays an effective role in increasing the removal percentage, it reduces the MBR performance . According to Fig. 3 shown in the MLSS concentration in both MBR, during the operation period, MLSS is less than 5000 mg/L, and there is no significant effect on membrane fouling. Also, the growth of the sludge in the system containing Fe3O4 nanoparticles is low compared to the without nanoparticles. It can be concluded that the presence of the nanoparticles reduces the growth rate. This nanoparticle is toxic and the presence of it near sludge cause to spoil the cell structure and also change the amount of nutrition.
SMP and EPS Changes
EPS and SMP are two main types of membrane foulants and include the sum of proteins and polysaccharides material [12,28,29]. Figs 4 and 5 show the variation of EPS and SMP in both membrane bioreactor system; the EPS and SMP concentration of R2 are less than R1. On average, EPS in R1 and R2 is respectively 231.38 and 199.7 mg/L, and also, the SMP amount is 110.8 and 69.96 mg/L. The difference in both cases can be attributed to adsorption by Fe3O4nanoparticles, as well as the shock to bacteria by these nanoparticles, which reduces their production and growth of the sludge and microorganisms [6, 30]. It seems the toxicity of nanoparticle causes the destruction of some bacteria. Also, due to the reduced adsorption capacity of nanoparticles, the production of EPS and SMP are slowly increasing trend gradually.
According to the data shown, it can be concluded that in both MBR, the protein content of EPS and SMP is greater than that of polysaccharide, and protein is identified as the main contaminant of the membrane as the system. Also, the EPS measured the cake confirmed the presence of protein in the cake layer, which Fig. 6 shows the level of EPSp in the cake layer. The amount of EPSc in the cake layer in the two membranes is not significantly different (40.75 and 39.70 mg/L in R1 and R2, respectively). But the EPSp level of cake layer in R1 is 151.87 and R2 is 180.625 mg/L). Increasing the activity of bacteria in the presence of Fe3O4 nanoparticles increases the amount of protein in the biological system. The presence of Fe3O4 nanoparticles resulted in accumulation of Fe(OH)3 as an electron receptor for aeration of bacteria in the cake layer, which leads to an increase in the protein . With regard to the above, it is expected that the membrane fouling in the R2 system is greater than R1, the results showed in the opposite of this (Fig. 7) and the flux rate in R2 is higher (on average 211 ml/m2h and 154.87 ml/m2 h in R2 and R1, respectively). According to a study by Yu et al. (2015), due to the high density of the Fe3O4 nanoparticles in the floc, the flocs in the bioreactor are easily removed and settled; and the presence of lower floc, decreasing the thickness of the cake layer formed in the R2 membrane relative to R1 membrane . Hence, the rate of flux increased in R2. Table 2 also shows that the reversible fouling induced the formation of the cake layer on the membrane surface is less in R2; in general, the rate of fouling in R1 is higher due to the blockage of the pores and presence of cake layer.
PSD in MBRs
The distribution of the sludge particle size of the two MBR is shown in Fig. 8. The particle size in R2 is more than R1, but this difference is negligible and the distribution of particle size in both MBR is inappropriate. The particle size in a maximum of points is 21.8 and 24.4 µm in R1 and R2, respectively. Yu et al. (2015) concluded that the presence of the Fe3O4 nanoparticles does not change the properties of the sludge significantly and they reported that the high density of this nanoparticles improved the separation and settling of flocs . In the other studies, they reported that particle sizes smaller than 50 µm increase the membrane fouling and, by placing it on the membrane surface, reduced the permeability . It can be considered that due to the small size of the particle in R1, the rate of fouling is greater than R2. Also, SEM images (Fig. 9) show that membrane fouling for R2 is lower than R1. For R1 all pores of the membrane were blocked but for R2 there are pores of the membrane.
To confirm the EPS in the mixed liquor, the sludge analyzed by FTIR. According to Fig. 10, the wavelengths of polysaccharides and polysaccharide-like substance, protein, and protein-like substance, humic acid were observed. The peak at the wavelength of 1053.69 cm-1 corresponded to the presence of C=O bonds (polysaccharides or polysaccharide-like substance), the wavelength of 1252.05 cm-1 related to COOH bonds (humic acid), and the wavelengths of 1404.28, 1542.70 and 1652.53 indicated the presence of Amide (Ι), (ІІ) and (ІІІ) (protein or protein-like substance), respectively [31, 32]. Also, the wavelengths of 2947.84 and 3428.03 cm-1 related to O-H and N-H bounds (polysaccharides or polysaccharide-like substance). The peak intensity of R2 is lower than R1, that shows all of the organic and inorganic component reduced.
According to the analysis done in this work, it can be concluded that:
- In R2, the amount of COD removal compared to R1 is higher, due to the adsorption of organic matter by the nanoparticle with biological treatment.
- The presence of Fe3O4 nanoparticles in a biological environment causes shock to bacteria and reduces the amount of sludge production, EPS and SMP.
- The positive effect of the presence of nanoparticles is to increase the amount of flux, although the amount of protein measured in R2 cake layer is greater than R1, the thickness of the formed cake layer is less.
- The main reason for the fouling of the membrane is blockage of membrane pores and is irreversible. The protein is a major source of contamination and has been confirmed by FTIR analysis.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest regarding the publication of this manuscript.