Coconut juice residues, discarded after processing coconut milk, were obtained from a community market located in Thasala, Nakhon Si Thammarat, Thailand. Analytical grade of ammonium sulfate [(NH₄)₂SO₄] with purity >99.99% was purchased from QRec (Thailand). Ethanol (99.8% purity) was supplied by Sigma Aldrich (Bangkok, Thailand). Granulated sugar was acquired from Mitr Phol Sugar Corp., Ltd. (Bangkok, Thailand). The acetic acid aqueous solution (5%v) was acquired from Nakornvitee Marketing Co., Ltd. (Wang Muang District, Thailand). The mixed gas of CO2/CH4 (40/60%v) was purchased from Linde (Thailand) Public Company Limited (Samut Prakan, Thailand).

Acetobacter xylinum strains obtained from the Thailand Institute of Scientific and Technological Research (TISTR), Bangkok, Thailand was used for the biosynthesis of BC. This strain is widely used as it has the best yield of BC (Kongruang, 2008). Bacterium culture on Agar plates was prepared by transferring liquid culture aseptically into Petri plates, containing NA Agar medium, and incubated at 30°C for 3 days. The 3-day-old bacteria cells were inoculated into 25 ml of starter medium of hydrosulphite of sodium (HS) (Pa'e et al., 2011). The number of viable cells in the inoculum was determined by the pour plate technique with HS medium (Kongruang, 2008; Pa'e et al., 2011). Colonies were counted after 5 days of incubation at 30°C. The substrate for biosynthesis of BC was conducted by filtrating 1,000 ml of CJR using a filtration glass set (Rocker Model VF7cat.167200-07, Taiwan). The filtrated CJR was sterilized in an autoclave at 121°C for 15 min, and then, it was cooled down to room temperature. The sterilized CJR was added into a beaker containing 1 g of ammonium sulfate, 28 g of nutrient agar, and 100 g of glucose. The substrate mixture was stirred with the hotplate stirrer at 30°C until homogenization was obtained. Then, 5 mL of acetic acid and 100 mL of 40% by volume ethanol were added (Khami et al., 2019).

Acetobacter xylinum with the amount of 1 × 10⁵ cfu/ml was added to 25 ml of the prepared substrate in the incubation plate with a diameter of 8 ± 0.3 cm. In this study, three dosages of A. xylinum were varied, 1 × 10⁵, 1 × 10⁶, 1 × 10⁷ cfu/ml, to examine its effect on bio-CA production. The production of BC was accomplished under static culture in an incubator (Binder model BD115, Germany) at 40°C for 7 days (Pa'e et al., 2011, Lestari et al., 2014). During the cultivation period, the gelation of BC was formed. The degree of gelation of BC increased with time as the substrate medium was consumed. Various degrees of thickness of the prepared membranes was achieved by varying the amount of the initially used substrate of 20, 30, and 40 ml. The bio-CA membrane was fabricated from the cultivated BC through semi-acetylation. The acetylation process depends on the accessibility of cellulose fibers and the susceptibility of individual cellulose crystallites (Barud et al., 2008). After 7 days of cultivation, BC pellicle on the substrate surface was collected and soaked in distilled water at ambient temperature for 3 days. Bacterial cell debris was removed by boiling BC with 40 ml of 2% NaOH for 3 h at 60°C. After that, 40 ml of 40% ethanol was added, and BC was further boiled for another 3 h at the same temperature. Then, BC was dehydrated in an oven at 60°C for 3 h before blending into a mixture of 40 ml of acetic acid, 50 ml of toluene, and 0.2 ml of 60% perchloric acid. To remove any impurities and remaining reagents, BC was soaked in 40 ml of 75% ethanol for 2 h. BC was rinsed off by distilled water several times until the stinky odor was vanished and finally dried in a hot air oven model M100-800 (Memmert GmbH + Co. KG, Bangkok, Thailand) at 60°C for 8 h (Khami et al., 2019). The thickness values were randomly measured using a micrometer from 10 points covering the surface area of the whole membrane. Then, the average thickness of the bio-CA membranes was estimated; the results were confirmed by the measurement from the cross-sectional area using scanning electron microscope (SEM) analysis.

Effecting factors of BC production include pH, carbon source, % Brix, A. xylinum dosage, and temperature (Phruksaphithak et al., 2019). As suggested by previous studies in our research unit (Khami et al., 2014, 2019), the conditions for the cultivation of BC selected in this study included the following: pH (4–5), Brix (5–15%), and A. xylinum dosage (1 × 10⁵-1 × 10⁷ cell/ml). The set of experiments was designed using Box–Behnken design (BBD) in the response surface methodology (RSM) (Tafreshi et al., 2017). Yields of BC in terms of the conversion of CJR into BC (BC to CJR solution weight ratio) were calculated from the results of those experiments. The results from BC yield were then put into a response optimizer to determine the optimum conditions for the biosynthesis of BC.

To study the gas permeation of CO₂ from the gas mixture using the synthesized CA membrane from CJR, the membrane separation unit, as displayed in Figure 1, was designed and constructed. The membrane separation unit was constructed from aluminum consisting of four layers. The plate at one end of the membrane unit was connected to the feed inlet and retentate outlet. The CA membrane was placed on a porous stainless-steel disc located between the feed and the permeate chamber. The permeate outlet was connected to the endplate of the permeate chamber side. Rubber O rings were used for gas leak prevention from each compartment.

From Figure 1, the gas mixture was fed to the top of the membrane separation unit through a regulator. The gas flow rate was measured and controlled using a rotameter. The separation of CO₂ from the gas mixture was studied using the gas mixture of 40/60% CO₂/CH₄ under the following conditions: a pressure range of 0.05–0.30 MPa, temperature of 30°C, CA membrane thickness of 0.03 ± 0.005, 0.04 ± 0.005, and 0.05 ± 0.005 mm.

Gas permeability and gas selectivity of pure gas was calculated using Equations (1) and (2) (Basu et al., 2010) while gas permeability and gas selectivity of mixed gas were calculated using Equations (3) and (4):

where P : the gas permeability coefficient of component i

Q: feed flow rate (cm³/s)

l : the selective layer thickness of the membrane

A: the area of the membrane

P: the pressure difference across the membrane

P_perm: permeate pressure

P_feed: feed pressure

The pure-gas selectivity (α_a/b):

where α_a/b: the selectivity (a/b)

Mixed-gas permeability of each component:

where P_i: the gas-mixed permeability coefficient of component i

x_{perm, i}: mole fraction of component i in the permeate stream

x_{feed, i}: mole fraction of component i in the feed stream

Mixed-gas selectivity

Eco-efficiency is an inclusive indicator for performance measurement of product, process, and service (PPS) system in terms of environmental impact and resource use and economic development (Liu et al., 2020). It is an effective tool for sustainable development that follows the ISO14045 standard (Environmental management: Eco-efficiency assessment of product systems) (Changwichan et al., 2018). In general, eco-efficiency was obtained from the ratio of product or service value to the environmental impact such as the consumption of energy, materials, and water or generated greenhouse gas emissions and carbon dioxide emissions. The concept equation of eco-efficiency can be calculated as Equation (5) (Heilala et al., 2014).

In this study, to apply the concept of sustainability and eco-efficiency indicators, the eco-efficiency Equation (6) was modified. The value of PPS was represented by CO₂ selectivity. The operating cost of the membrane unit was calculated from the cost of the gas mixture used in the performing experiment. The total operating cost of bio-CA membrane produced by A. xylinum including chemicals used and electricity was calculated using with following equation.

where E_d: CO₂ selectivity

E_c: Cost of bio-CA membrane production and operating of the membrane unit.

The optimum cultivation conditions were determined using the Surface optimizer. Figure 2 illustrates that, within the ranges of studied parameters, the optimum conditions of BC production were 14.9% Brix, 1 × 10⁷ cfu/ml of A. xylinum, and pH of 4. Thus, these conditions were controlled in the cultivation of BC from CJR. A. xylinum used CJR as the carbon source in the production of BC. The carbon source of CJR used in the study was 6,000–12,000 mg/l. The high-performance liquid chromatography (HPLC) analysis of the remaining substrate found the highest concentration of sucrose, glucose, fructose, lactic acid, and acetic acid at 4.99, 7,62, 14.04, 1.38, and 0.38 g/L, respectively. Throughout cultivation, pH gradually decreased with the production of lactic acid and acetic acid by A. xylinum. Therefore, pH that is suitable for BC cultivation should not be <4.

The bio-CA membranes, as shown in Figure 3, were fabricated through the acetylation from BC with the thickness of 0.03 ± 0.005, 0.04 ± 0.005, and 0.05 ± 0.005 mm. Dry weights of the CA-0.03, CA-0.04, and CA-0.05 membranes were ~0.11, 0.28, and 0.42 g, respectively.

The bio-CA membranes were obtained from the cultivation process, described earlier. In general, the thin solid membranes were flat sheet-like with some degree of roughness on the surface, as shown in Figure 3. Figure 4 shows the SEM results of the normal and cross-sectional surfaces of the bio-CA membranes with three different degrees of thickness (0.03, 0.04, and 0.05 mm). As shown in Figures 4A,C,E, the normal surface of the membranes generally featured a rough fibrous texture packed with dense randomly entangled cellulose fibers. Meanwhile, the cross-sectional results (Figures 4B,D,F) revealed a multi-stack-like structure, composing of multiple thin layers of the CA fibrous sheets overlayed and stacked up together throughout the thickness of the membranes. Variation in morphological features (e.g., density and void distribution) of the membrane was observed. This stemmed from the difference in undertaking nature in the formation of BC during the cultivation stage, resulting in the asymmetric morphology of the membrane.

The CA membrane was an organic hydrocarbon compound consisting CHO elements. Figure 5 illustrates the energy dispersive x-ray (EDX) results of the CA membranes. The element analysis revealed that the samples consisted mainly of carbon (C) and oxygen (O) with good dispersion of the components throughout the membranes. Table 1 summarizes the carbon (C) and oxygen (O) compositions of the prepared samples.

The chemical structure of the prepared membranes composed mainly of the CA was revealed in the FTIR results, as shown in Figure 6. The FTIR spectra of the sample showed peaks corresponding to the common functional groups similar to those of the CA. The presence of the peak associated with the C=O stretching of the hemicellulose at the wavelength of 1,543 cm⁻¹ was observed, altogether with the O–H stretching peak at 3,341 cm,-1 the C–O–C vibration at 1,160 cm⁻¹, and the C < H2 vibration at 1,427 cm⁻¹ of the cellulose structure, and the C=C stretching in the aromatic structure at 1,543 cm.-1 The FTIR result was in good agreement with the characteristic peaks observed in a study by Khami et al. (2019), in which the CA material was synthesized by acetylation of BC. The magnitude of transmittance in the related spectra could be different depending on the degree of substitution of the acetyl group on the cellulose main chains.

Figure 7 shows the TGA result of the CA membrane. The decomposition temperature of the material was found to be around 350°C. From room temperature to about 300°C, the TGA curve was slightly declined and almost in the plateau region, suggesting that the membrane was relatively thermally stable. This suggests the applicable range of the service temperature of the gas separation using the prepared membrane. The prepared membrane possessed similar thermal stability characteristics when compared with the CA membrane synthesized by the chemical route.

Figure 8 shows the tensile properties of the membrane samples. The average tensile strength of the membranes was in the range of 95–131 MPa. At room temperature, the membranes exhibited semi-ductile-like material characteristics under tensile. At a strain rate of 5 mm/min, the average elongation at break of the samples was ~15–17%. For the CA-0.03 membrane (0.03 mm thick), the sample showed the highest stress value of about 131 MPa. Under tensile force, the induced orientation of the CA fibers could undergo to some extent, which seemed to be more pronounced in the thicker samples (i.e., 0.04 and 0.05 mm). With a larger thickness, the laminated thin layers of the fibrous CA probably slipped from each other under shear stress, resulting in decreased strength of the sample. Without reinforcement, the tensile strength of the pristine flat-sheet membranes prepared by the proposed bioprocess in this study is relatively higher, when compared with the neat membranes synthesized via the solvent casting of the CA solution (Pak et al., 2016).

The CO₂ removal capacity of the CA membranes with three degrees of thickness of 0.03 mm (CA-0.03), 0.04 mm (CA-0.04), and 0.05 mm (CA-0.05) was tested in the gas separation unit, as described earlier. The binary mixed gas containing a fixed CO₂/CH₄ ratio (40%v CO₂) was fed into the membrane unit having the CA membrane as the separating barrier at a feed pressure ranging from 0.05 to 0.3 MPa. The CH₄ and CO₂ compositions of the permeate and the retentate were obtained from GC results. The transport of the gas components in terms of permeability and selectivity were calculated from the gas concentrations. The gas permeation results are summarized in Table 2.

For the binary gas mixture, both CO₂ and CH₄ competitively permeated under the pressure difference across the thickness of the asymmetric bio-CA membrane. Figure 9 shows the effect of feed pressure on CO₂ permeability of the gas permeate passing through the membranes with various thickness values (0.03, 0.04, and 0.05 mm). With the increasing pressure of the feed, the CO₂ permeability decreased until the pressure reached around 0.2 MPa at which the curve began to climb up. Figure 10 illustrates the effect of feed pressure on the CH₄ permeability of the gas permeate passing through the membranes with various thickness values (0.03, 0.04, and 0.05 mm). The CH₄ permeability tended to increase with the increasing feed pressure, particularly in a high-pressure region. The transport of both CO₂ and CH₄ through the dense solid CA membranes could be explained by the solution-diffusion mechanism, in which CO₂ has better solubility within the matrix relative compared to CH₄ (Ismail and Lorna, 2002). CO₂ has a greater affinity toward the CA matrix due to the polar–polar interaction between the CO₂ molecules and the acetyl/hydroxyl groups on the CA backbones. Whereas, CH₄ essentially exhibits no chemical association within the CA membrane due to its non-polar nature, and therefore, it can only be purely transported by the physical mechanism, which is limited by its low solubility on the highly polar sites in the membranes. With the incremental feed pressure, increasing the partial pressure of the CO₂ would result in higher CO₂ concentration within the CA matrix. As demonstrated in Figure 9, however, the CO₂ permeability decreased when the feed pressure increased from 0.05 to about 0.2 MPa. This could be explained by the membrane by the carrier saturation of the active sites within the CA membrane when all available carboxyl/hydroxyl pendant groups were associated with the chemical interactions with highly polar CO₂ molecules. (Zou and Winston Ho, 2006). Therefore, increasing the CO₂ partial pressure would not further increase the CO₂ flux since all the carriers have already reacted with CO₂ and attained the maximum capacities. As a result, the CO₂ permeability would drop. On the other hand, the transporting CH₄ did not have any chemical association with carriers. Thus, its sorption behavior could be explained by Henry's law in which the CH₄ flux linearly increased with the feed pressure, as shown in Figure 10.

The CO₂/CH₄ separation performance of the membrane was measured in terms of the CO₂ selectivity of the gas permeate. Figure 11 depicts the effect of operating feed pressure on the CO₂ selectivity (CO₂/CH₄ permeability ratio) of the CA membranes having different thicknesses of 0.03, 0.04, and 0.05 mm. The CO₂ selectivity drastically declined to approach very low values as the feed pressure increased. This phenomenon could be due to the plasticization effects induced by a high concentration of the sorbed CO₂ within the asymmetric CA membrane (Houde et al., 1996; Cerveira et al., 2018). The high solubility of CO₂ may potentially swell the CA polymer to such an extent that the intermolecular interactions were disrupted, which led to the enlargement of the free volume captivities (Puleo et al., 1989; Chang et al., 2019). Consequently, the transport rate of the penetrant CO₂ and CH₄ were enhanced. From SEM results, the membrane typically inherited a multi-layered flat-sheet structure with a little degree of presented voids within the matrix. These void characteristics could also affect the transport properties, such as the surface sorption–desorption behaviors, of the permeances transporting through the membrane. As discussed earlier, the structure of the obtained thick membranes generally featured well-packed layers with less presence of interlayer voids. In a low-pressure region where the plasticization had not yet been taken, high CO₂ selectivity was achieved when using a thick membrane (i.e., CA-0.05), which exhibited a well-packed structure with fewer interlayer voids. From the graph, the plasticization pressure of the CA membranes for the separation of the CO₂/CH₄ gas mixture was observed to occur at 0.1 MPa for CA-0.03, 0.15 MPa for CA-0.04, and 0.2 MPa for CA-0.05, respectively. A maximum CO₂/CH₄ selectivity value of 29.53 was achieved in the separation system containing the CA-0.05 membrane operating at a feed pressure of 0.10 MPa. The CO₂ selectivity of this study was in good agreement when compared with other related studies. Table 3 summarizes the CO₂/CH₄ selectivity results in comparison with related studies on various CA membranes for CO₂/CH₄ systems. The CO₂ selectivity value of the bio-CA membrane was also plotted among the CO₂/CH₄ selectivity results obtained from the related studies in comparison to the Robeson upper bound relationship (Robeson, 2008), as shown in Figure 12. From the graph, the best result of the CO₂/CH₄ selectivity value of 29.53 locates under, but relatively close to, the Robeson upper bounds compared with the reported results obtained from other studies utilizing flat CA membrane systems (see Table 3). As discussed earlier, the flat-sheet bio-CA membranes prepared from environmental-friendly procedures proposed in this study have shown promising CO₂ removal capability for biogas treatment applications. However, an improvement on the membrane properties could be further explored to enhance its separation performance in the direction of surpassing the upper bound curve toward the commercially desirable region.

The total production cost of bio-CA membrane made from CJR in this study was roughly calculated as follows: The total production cost of 40 bio-CA membranes with a total area of 8,039 cm² was 357.62 Baht. Thus, the lab-scale production cost of bio-CA membrane from CJR in this study was ~0.044 Baht/cm². However, the cost of these in-house membranes was still relatively high, compared with the commercial CA membranes which have an estimated price in the range of 0.001–0.008 Baht/cm² (Alibaba.com, 2021). The operating cost of the membrane separation testing in this study, estimated from the gas mixture used in each experiment, was 5.10 Baht/L. The eco-efficiencies of the CO₂ separation for three different degrees of thickness of bio-CA membranes, as calculated by Equation (6), are displayed in Figure 13. The results indicate that the eco-efficiency was drastically dependent on the pressure of the gas mixture fed into the separation unit. Moreover, the thickness of the CA membrane affected the eco-efficiency as well, in which the thicker the CA membrane, the higher the eco-efficiency. The cost for preparing the CA membranes with various thicknesses is not significantly different. While the thicker membranes could deliver higher CO₂ separation performance than the thinner ones, the thicker membranes are more economically attractive for potential CO₂ removal applications. it can be concluded from the results that the bio-CA-0.05 membrane was the most appropriate one to be applied in the membrane separation unit at the air inlet pressure of 0.05 MPa.