Abstract

This work is the first, to the best of our knowledge, to use polycaprolactone (PCL)-based membrane for the treatment of dairy wastewater. PCL is a biodegradable polymer with high biocompatibility and good oil resistance. The chlorine tolerance analysis of PCL-based membranes exhibited a good tolerance against chlorine. The PCL/TiO2 nanocomposite membrane with the addition of polyethylene glycol was prepared and tested for protein separation. The dependency of contact angle with time was analysed for the membrane, and the contact angle value reduced from 74.5 ± 2° to a steady value of 65 ± 2° in 120 s. The proteins were removed using a cross-flow filtration unit at an operating pressure of 0.4 MPa at room temperature with permeate flux of 10 L/m2 h and a relative permeate flux of about 0.10. The removal of proteins was measured qualitatively using native polyacrylamide gel electrophoresis (PAGE) and quantitatively using Lowry's test. A percentage rejection of 97.6 was obtained and the native PAGE showed the complete removal of all the major proteins present in the milk sample.

HIGHLIGHTS

  • Preparation of biodegradable polymer-based composite membrane using phase inversion technique.

  • Application of the prepared membrane for milk protein removal from synthetic dairy wastewater.

  • Qualitative analysis of protein rejection using native polyacrylamide gel electrophoresis (PAGE) and quantitative analysis using Lowry's test.

  • Chlorine tolerance analysis of the polycaprolactone membrane for the industrial application.

Graphical Abstract

Graphical Abstract
Graphical Abstract

INTRODUCTION

Among different industries, dairy industries produce a large quantity of effluent (0.2 to 10 L of effluent per litre of processed milk) together with extensive water consumption (Vourch et al. 2008). The dairy effluents are characteristically white in colour with an unpleasant odour and higher temperature with fluctuations in pH (6.5–8.0), biological oxygen demand (BOD), chemical oxygen demand (COD) and total suspended solids (TSS) (Slavov 2017). The wastewater generated from the dairy industries contain large quantities of milk constituents (Pandey et al. 2019) and thus contain high concentrations of proteins, fats, carbohydrates, grease and minerals. These organic materials contribute to high COD and BOD values and require proper treatment to improve the quality before discharge. The biodegradability of these components is complex, with easily degradable carbohydrates and comparatively less biodegradable protein and milk fats. One gram of milk protein and fat has BOD values of 1.03 g and 0.89 g and COD values of 1.36 × 10−3 g and 3 × 10−3 g, respectively (Birwal et al. 2017). The composition of untreated dairy wastewater sample reported by Gopinatha Kurup et al. consists of 360 mg/L of fat, 388 mg/L of protein and 121 mg/L of carbohydrates (Gopinatha Kurup et al. 2019). These contaminants in the dairy effluents can imbalance the ecosystem if discharged without treatment. Shete and Shinkar have reported the effect of untreated dairy effluent on water, land and atmosphere (Shete & Shinkar 2013). These effluents on discharging to a water body depletes the dissolved oxygen content of the receiving water body, increasing levels of flies and mosquitoes. It is also reported that these effluent contains heavy black flocculated sludge masses, and the rich fat content in these effluents can block the pipes of the wastewater treatment systems (Al-Wasify et al. 2017).

Compared to the other conventional techniques, including electrocoagulation and adsorption, the membrane separation process can remove these proteins and fats producing high-quality effluent. These organic materials can also be recovered feasibly by utilizing membrane separation, as protein concentration is also an exciting area under research. Eppler et al. (2011) reported that membrane separation is the standard method for the industrial production of highly concentrated protein solutions. Nevertheless, protein aggregation is a significant problem in the concentration of proteins, due to the shear stresses developed out of pumping or centrifugation (Eppler et al. 2011). The potential factors that are favourable for the application of membranes include the production of high-quality water, efficient separation, easy maintenance and compact construction (Zhao et al. 2018). However, the primary requirement for the membrane to use in long-run applications is antifouling property. The self-cleaning/antifouling property of membranes reduces the interaction of the particles (dissolved or suspended) on the membrane surface and thereby improves the membrane efficiency (Zhang et al. 2016). The other important properties of membranes, such as porosity and hydrophilicity, are vital for the treatment of wastewater. These properties and thereby performance of the membrane can be enhanced by the addition of fillers into the polymer matrix. Ma et al. studied the effect of polyethylene glycol (PEG) on polysulphone membranes, and the membrane hydrophilicity and water flux was found to be improved (Ma et al. 2011). The molecular weight of PEG also has a direct dependency on the size of the voids (Amirilargani & Mohammadi 2009).

This work focuses on the removal of less biodegradable proteins present in the dairy wastewater using a polycaprolactone (PCL)-based ultrafiltration membrane incorporated with TiO2 nanoparticles. PEG was added as a pore former in the polymer solution and the membranes were synthesised by phase inversion technique. The filtration was carried out using a cross-flow filtration at an operating pressure of 0.4 MPa and the proteins present in the samples were identified using native Polyacrylamide Gel Electrophoresis (PAGE). The percentage rejection was quantified using Lowry's test.

MATERIALS AND METHODS

The base polymer (PCL, Mn 80,000) and the additive (TiO2 nanoparticles <100 nm) were purchased from Sigma Aldrich, USA. The pore former (PEG) and the solvent (dimethyl formamide, DMF) were obtained from Merck, USA and the non-solvent used for the phase separation was deionised water. The dairy wastewater used for the study was synthetically prepared by centrifuging and diluting cow milk.

Membrane synthesis

The composite membrane is synthesized by incorporating TiO2 nanoparticles with the pore former PEG into the base polymer, PCL. TiO2 particles are very well known for the self-cleaning ability due to the antibacterial properties (Haider et al. 2017). With the incorporation of TiO2 nanoparticles, hydrophilicity and pore formation in a membrane can be achieved even at low concentrations (Nevstrueva et al. 2015). The membranes with TiO2 are also known to have improved anti-fouling resistance because of their enhanced hydrophilicity (Sotto et al. 2011). For the preparation of the casting solution, TiO2 nanoparticles were initially dispersed in the solvent by ultrasonication, and then the base polymer was dissolved by heating the solution at 70–80 °C for 2–3 h. After the complete melting of the polymer, PEG was added and stirred for another 1 h for homogeneity. Then the polymer solution was cast on a glass plate using a doctor's blade and immersed into a water bath for phase separation. Later, the formed membranes were stored in water that contained formaldehyde solution to prevent microbial growth.

In our previous publications (Nivedita & Joseph 2020), we demonstrated that the optimum concentration of TiO2 nanoparticles and PEG were 1 wt% and 6.24 wt%, respectively, by varying the composition of TiO2 nanoparticles from 0.5% to 4 wt% and PEG from 1% to 10 wt%. The properties of the optimised composite membrane were determined for porosity, hydrophilicity, tensile strength, membrane roughness, permeability and anti-fouling property using Bovine Serum Albumin (BSA) rejection. The surface morphology was already studied. The porosity was measured from the wet and dry membrane weights and the contact angle was measured using the sessile drop method. The mechanical strength of the membrane was determined using Universal Testing Machine analysis based on D882 standards. Atomic Force Microscopy (APER-A-100 SPM) was used to estimate the membrane roughness in a scan area of 10 × 10 μm. The cross-sectional morphology of the membrane was analysed using a scanning electron microscope (SEM) (JEOL JSM 6360) to obtain a clear idea about the internal structure of the membrane.

The ratio of membrane pure water flux to the operating pressure defines the permeability of the membrane. The pure water flux was calculated using Equation (1), where JW is the pure water flux (L/m2 h), Q is the volume of permeate collected (L), A is the membrane effective surface area (m2) and Δt is the sampling time interval (h).
formula
(1)
The average pore size of the synthesised membrane was calculated using the Guerout–Elford–Ferry equation (Zhao et al. 2014) presented in Equation (2).
formula
(2)
where ɛ is the membrane porosity, υ represents the viscosity of water (Pa.s), l is the membrane thickness (m), Q is the volume of permeated pure water per time (m3/s), A is the effective membrane area (m2) and ΔP is the operational pressure difference (MPa).

The chlorine tolerance of the PCL base polymer was investigated because chlorine is the most inexpensive and effective disinfectant used in wastewater treatment (Park et al. 2008). The procedure for the determination of chlorine tolerance was obtained from the literature (Lau et al. 2015). It involved soaking a neat PCL membrane in the aqueous solution of sodium hypochlorite (NaOCl) of concentration 500 ppm for 48 h. Then, the membrane was cleaned with pure water to confirm the removal of NaOCl residues. The pure water flux of the membrane before and after immersion was measured and the change was analysed to determine the tolerance of the membrane against chlorine. The contact angle was also measured to analyse the change in the membrane surface before and after the treatment. A Fourier-transform infrared spectroscopy (FTIR) analysis was carried out on the membrane before and after chlorine treatment to analyse the presence of any chemical interaction on the membrane surface.

Feed sample preparation and filtration

About 500 mL of cow milk was centrifuged at 9,392 g for 10 min at 4 °C to remove the fat layer (Sharma et al. 2017) and the serum was carefully extracted and diluted with water at a ratio of 1:3 to make a total volume of 2,000 mL. The prepared sample was filtered using the PCL/TiO2 membrane in a cross-flow filtration unit (Mass International, India) at a pressure of 0.4 MPa. The schematic diagram of the filtration system set-up used with cross-flow cell is shown in Figure 1. Prior to the experiments, all the valves were opened to relieve any pressure developed within the system. The experiments were also carried out in total recycle mode (with permeate recycling) to keep the feed concentration constant. The raw milk (feed), filtered sample (permeate) and the part of the feed that did not pass through the membrane (retentate) were collected and stored at −20 °C for analysis.

Figure 1

Schematic diagram of the cross-flow filtration unit.

Figure 1

Schematic diagram of the cross-flow filtration unit.

Native PAGE

For the analysis of the proteins, a native PAGE was carried out in a Mini-Protean II electrophoresis cell (Bio-Rad Laboratories, USA). The bands were analysed on a 10% acrylamide gel of composition as shown in Table 1.

Table 1

Composition for 10% acrylamide gel for native PAGE

Resolving gel (10%)Stacking gel (5%)
30% acrylamide bisacrylamide 30% acrylamide bisacrylamide 
mixture 2.64 mL mixture 0.5 mL 
1.5 M Tris (pH 8.8) 2 mL 0.5 M Tris (pH6.8) 0.76 mL 
10% APS 80 μL 10% APS 30 μL 
TEMED 3.2 μL TEMED 3.0 μL 
H2O 3.2 mL H2O 1.72 mL 
Resolving gel (10%)Stacking gel (5%)
30% acrylamide bisacrylamide 30% acrylamide bisacrylamide 
mixture 2.64 mL mixture 0.5 mL 
1.5 M Tris (pH 8.8) 2 mL 0.5 M Tris (pH6.8) 0.76 mL 
10% APS 80 μL 10% APS 30 μL 
TEMED 3.2 μL TEMED 3.0 μL 
H2O 3.2 mL H2O 1.72 mL 

The gel was cast and the samples with loading dye containing Bromophenol Blue (in a ratio of 5:2) were loaded into the well and into the electrophoretic tank filled with running buffer (Tris base, glycine and distilled water) at 120 V for approximately 3 h. After electrophoresis, the gels were stained with Coomassie Blue, destained using water/methanol/acetic acid mixture for the visualisation of the protein bands and compared with the molecular weight markers.

Lowry's test for protein quantification

The amount of proteins present in the feed and permeate was determined using Lowry protein assay based on the given protocol. The samples were pre-treated with copper ion in alkali solutions. Then, with the addition of Folin reagent, the amino acids present in the samples reduced the phosphomolybdate and phosphotungstic acid to produce a blue coloured solution. The colour change is proportional to the amount of protein present and was estimated by reading the absorbance at 750 nm using an ultraviolet visible spectrophotometer (Perkin Elmer, USA) against a standard BSA curve. The percentage rejection was calculated from the concentration of protein present in the feed (Cf) and the permeate (Cp) using Equation (3):
formula
(3)

RESULTS AND DISCUSSION

Membrane characteristics

Based on our recent research results, the physico-chemical properties of PCL/TiO2 nanocomposite membrane, fabricated using phase inversion technique, are shown in Table 2. The membrane exhibited 63% porosity, with an average pore diameter of 21 nm. The contact angle was measured shortly after the drop formation. Since the PCL chains are flexible, they exhibit a high percentage of elongation and lower Young's modulus, with a tensile strength of 2.5 N/mm2. The anti-fouling property of the membrane, induced by the incorporation of hydrophilic TiO2 nanoparticles was studied using BSA foulants. This was analysed based on total, reversible and irreversible fouling ratio and flux recovery of the membrane. The self-cleaning ability of the membrane caused by the non-polar hydrophobic polymer PCL was determined and reported (Nivedita & Joseph 2020). The membrane roughness parameter indicates the energy of attraction between the membrane surface and foulants. A lower roughness value traps fewer foulants inside the membrane (Guo & Kim 2017), which was confirmed from the obtained flux recovery value. From the results obtained, the synthesised nanocomposite membrane was found to be in the range of ultrafiltration having a pore size ranging from 10 to 100 nm with pure water flux less than 500 L/m2 h. The properties of bare PCL membrane are shown in Table 2 to compare with the characteristics of composite membrane. The properties of the membrane are significantly enhanced with the addition of TiO2 nanoparticles in the dope solution.

Table 2

The characteristics of bare PCL and PCL/TiO2 composite membrane

Membrane propertyBare PCL membranePCL/TiO2 composite membrane
Porosity 26 ± 2% 63 ± 2% 
Mean pore diameter ∼ 6 μm ∼ 21 nm 
Contact angle (<1 s) 87.4 ± 1° 74.5 ± 2° 
Tensile strength 2.2 N/mm2 2.5 N/mm2 
Roughness parameter 38.03 nm 59.41 nm 
Permeability 84 L/m2 h.MPa 268 L/m2 h.MPa 
Flux recovery after BSA rejection 55% 90% 
Membrane propertyBare PCL membranePCL/TiO2 composite membrane
Porosity 26 ± 2% 63 ± 2% 
Mean pore diameter ∼ 6 μm ∼ 21 nm 
Contact angle (<1 s) 87.4 ± 1° 74.5 ± 2° 
Tensile strength 2.2 N/mm2 2.5 N/mm2 
Roughness parameter 38.03 nm 59.41 nm 
Permeability 84 L/m2 h.MPa 268 L/m2 h.MPa 
Flux recovery after BSA rejection 55% 90% 

Hydrophilicity

The hydrophilicity of the synthesised membrane was determined using contact angle values. The contact angle of the membrane at an interval of 10 s was observed to determine its dependency with time because the contact angle is a function of wettability. The results are plotted in Figure 2. The contact angle values were found to decrease with time, showing the effect of the membrane on time taken for the complete wettability. The initial membrane contact angle was 74.5 ± 2° and, after 140 s, the contact angle reduced to 64.9 ± 2°. When a water droplet is placed on the membrane surface, it fills the pores of the membrane surface, which becomes locally saturated. Over time, this droplet moves into the pores and the volume of the droplet reduces. Thus, the contact angle reduces to a lower value. This wetting change is due to the membrane hydrophilicity and porosity (Milescu et al. 2019). The improvement in the hydrophilicity of the membrane reduces fouling (Purkait et al. 2018; Lakhotia et al. 2019).

Figure 2

Time dependency of contact angle of the prepared membrane.

Figure 2

Time dependency of contact angle of the prepared membrane.

Morphological analysis

The cross-section SEM images of the neat PCL membrane and PCL/TiO2 composite membrane is shown in Figure 3(a) and 3(b), respectively. Both images exhibited a porous superficial layer, a sublayer or an intermediate layer, and a bottom layer with a sponge-like pore structure. The skin layer or the porous superficial layer acts as the separation layer, and the spongy support layer provides the mechanical strength. The sublayer consisted of finger-like cavities and macrovoid structures (Liu et al. 2016). The thickness of the obtained neat PCL membrane was 54.2 μm with a sponge layer thickness of 10.9 μm. With the incorporation of PEG and TiO2 nanoparticles, a thin asymmetric layer of thickness 7 μm was found on the top surface, which results in an improved rejection rate (Ramos-Olmos et al. 2009). The total membrane thickness was 64.3 μm and the spongy layer thickness was about 33.8 μm. The increase in the skin layer thickness of membrane with TiO2 is due to the slower exchange rate of solvent and non-solvent. The transformation of membrane morphology to a more porous spongy structure is due to the delay of phase separation caused by the addition of hydrophilic materials into the polymer solution (Mbareck et al. 2009). The high viscosity of the solution with additives slows down the solvent–polymer demixing, and thereby solvent–non-solvent exchange rate is reduced (Bakeri et al. 2010).

Figure 3

Cross-section SEM image of (a) neat PCL membrane and (b) PCL-PEG-TiO2 membrane.

Figure 3

Cross-section SEM image of (a) neat PCL membrane and (b) PCL-PEG-TiO2 membrane.

Pure water flux

The pure water flux of the membrane was measured using the cross-flow filtration unit. First, the membrane was compacted at 0.8 MPa for 30 min and the pressure was reduced to 0.4 MPa to measure the pure water flux. Figure 4 presents the decline in the water flux with operating time. The application of high-pressure compresses the membrane structure, causing a reduction in pore size and thereby a reduction in flux (Srivastava et al. 2011). Within an hour of filtration, the flux reached steady state at 107 L/m2 h.

Figure 4

Decline of pure water flux and attainment of steady state with time.

Figure 4

Decline of pure water flux and attainment of steady state with time.

Chlorine tolerance

The membrane permeability and contact angle before and after the chlorine treatment was measured and is shown in Figure 5(a). The pure water flux of the membrane was found to be unaffected after high concentration chlorine exposure. The contact angle value remains the same, showing that the membrane surface was unaffected by the chlorine attack (Park et al. 2008). The FTIR spectra of the membrane before and after chlorine treatment, shown in Figure 5(b), exhibited similar peaks, confirming the absence of any degradation on the membrane surface. The occurrences of any degradation process could have been reflected on the spectra by the broadening of carbonyl band (1,600–1,800 cm−1) because of chain cleavage around the ester group (Sabino 2007).

Figure 5

Chlorine tolerance analysis of the PCL membrane (a) contact angle and permeability and (b) FTIR spectra of membranes before and after treatment.

Figure 5

Chlorine tolerance analysis of the PCL membrane (a) contact angle and permeability and (b) FTIR spectra of membranes before and after treatment.

Permeate flux and protein rejection studies

The permeate flux obtained at the operating conditions of 0.4 MPa and at room temperature declined and reached steady state at 10 L/m2 h. Figure 6 shows the reduction in relative permeate flux with time, where the flux rapidly declined initially, and then slowed to a relatively constant rate at 0.10 after 0.4 h. The considerable decrease in flux is following the flux behaviour for ultrafiltration operations. The reduction in the permeate flux is due to the presence of high concentration of proteins in the feed sample. The filtration process builds a concentration gradient between the membrane surface and the bulk fluid, resulting in concentration polarisation. Because of the larger molecular sizes, the diffusion of solute back to the solution from the membrane surface is relatively low, forming a gel layer on the membrane surface. This develops a hydraulic resistance against permeate flow. The obtained relative flux is compared with the reported commercial polysulfone membranes of 10 kDa and 20 kDa, and is found to be between 10 kDa and 20 kDa membranes (Ng et al. 2014).

Figure 6

Relative permeate flux decline using the synthesised membrane.

Figure 6

Relative permeate flux decline using the synthesised membrane.

The proteins present in milk can be broadly categorised into casein family of about 82% in composition and serum (whey) proteins of nearly 18% composition. The casein family present in milk is complex and is subdivided into four variations, namely αS1, αS2, β and k. The serum proteins are composed of 60% of β-lactoglobulin (β-lg), 22% of α-lactalbumin (α-la) and 5.5% BSA and many minor proteins (Cavallieri et al. 2007). The molecular weight of α-la, β-lg and BSA are 14 kDa, 18 kDa and 66 kDa, respectively, and the casein fractions fall in the range of about 22–26 kDa (Wróblewska & Kaliszewska 2012). All seven proteins were identified in the electrophoretic pattern obtained for the feed sample shown in line 1, Figure 7.

Figure 7

Electrophoretic pattern obtained by native PAGE of the samples before and after membrane filtration.

Figure 7

Electrophoretic pattern obtained by native PAGE of the samples before and after membrane filtration.

Line 2 and 3 in Figure 7 were permeate and retentate samples, respectively, and the marker line shows the molecular weight standards. No bands were present in line 2, showing the complete separation of all significant proteins from the milk. All the bands present in line 1 reappeared in line 3, showing the absolute rejection of proteins from the feed. This shows the ability of the membrane to retentate particles with a size greater than 14 kDa at 0.4 MPa operating pressure. Nagappan et al. reported that the retention characteristic of a commercial membrane is 20 kDa at an operating pressure of 6.9 MPa (Nagappan et al. 2018).

Using the Lowry protein assay, the concentration of proteins present in the feed and in the permeate was found to be 2,063 mg/L and 50 mg/L, respectively. This shows that the TiO2-incorporated PCL membrane exhibits a protein rejection of 97.6%, calculated using Equation (3). The remaining 2.4% shows the presence of a small amount of many minor proteins and enzymes belonging to the whey protein family.

CONCLUSION

A PCL/TiO2 nanocomposite membrane with an average pore size of 21 nm and contact angle of 74.5 ± 2° was used for the removal of proteins from natural cow milk using a cross-flow filtration unit at a pressure of 0.4 MPa. The feed sample was diluted in the ratio of 1:3. A rejection of about 97.6% was achieved from an initial feed concentration of 2,063 mg/L with a relative flux of 0.10. The native PAGE showed the removal of all major proteins with larger than 14 kDa in the sample. The similarity in contact angle values and permeability of the neat PCL membrane measured before and after the chlorine treatment showed its tolerance against chlorine attack. The results of this work suggest the possible use of the PCL/TiO2 nanocomposite membrane for the treatment of dairy wastewater. Further, this work presents a future scope in the analysis of the effect of concentration polarisation and the fouling characteristics of the membrane in long-time applications.

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

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