Effect of integrated microbial fuel cell in a two-stage vertical flow constructed wetland for piggery wastewater treatment and resource recovery by photobioreactor

The world's population is projected to reach 9.8 billion by 2050, which could hike the demand for livestock products worldwide. This surge in demand has led to increased pig farming, which may critically increase water pollution and greenhouse gas emissions (Chauhan et al. 2016; Ali et al. 2022). Large amounts of water are utilised to maintain pig farms, producing large quantities of wastewater (WW). Piggery WW contains high amounts of critical pollutants like organic matter, nitrate, phosphate, ammonium, and total suspended solids (TSSs) (Lee et al. 2020). Thus, more rigorous WW treatment is required, which increases technology costs. Therefore, the discharge of untreated piggery WW can cause environmental hazards like water eutrophication, soil contamination, and global warming (Zhang et al. 2017; Chen et al. 2021).

Therefore, sustainable decentralised treatment systems can be a solution for treating piggery WW and reusing it for various agricultural purposes. The constructed wetland (CW) is popularly used as a nature-based secondary WW treatment technology due to its treatment and cost efficiency (Sharma et al. 2022; Gogoi et al. 2024). CWs are artificially engineered systems that utilise plants, microbes, and filter media to remove pollutants from WW (Gogoi et al. 2022, 2024; Sharma et al. 2022). Among sub-surface flow CW types, vertical flow constructed wetlands (VFCW) are more commonly used than horizontal flow constructed wetlands due to better treatment efficiency and less space requirement (Sharma et al. 2022). VFCWs are usually filled with appropriate filter media like coarse sand or gravel to support plant growth and act as a filter, removing pollutants from WW. The plants with fibrous root systems are preferred for the CW vegetation, e.g., Canna indicana and Heliconia psittacorum (Gogoi et al. 2022; Sharma et al. 2022; Gogoi & Mutnuri 2024). The VFCW treatment relies on a combination of physical (sieve filtration), biological (phyto and microbial remediation), and chemical processes (adsorption and sedimentation) (Gogoi et al. 2022; Sharma et al. 2022; Gogoi & Mutnuri 2024). However, the CW faces the challenge of efficient removal of ⁠, total ammoniacal nitrogen (TAN), and phosphorous (P) pollution simultaneously (Gogoi et al. 2022; Nagarajan et al. 2022; Ma et al. 2024). Thus, the scope of CW design modification or incorporating other technology in CW, e.g., microbial fuel cell (MFC), can be studied to enhance the CW treatment efficiency.

The advancement of integrating MFC in CWs has shown a promising sustainable technology (Guadarrama-Perez et al. 2019; Sharma et al. 2022). First, it can reduce CW clogging and the need for extensive surface treatment. Second, it can reduce greenhouse gas (GHG) emissions (by 5.9–32.4% CO₂ equivalents) from CW by reducing 17.9–36.9% CH₄ and 7.2–38.7% N₂O emissions (Wang et al. 2019). Moreover, this makes it an effective and cost-efficient way to treat WW while generating electricity simultaneously (Oon et al. 2015; Munoz-Cupa et al. 2021; Roy et al. 2023). A redox gradient in the VFCW filter layers facilitates the integration of MFC (Kesarwani et al. 2023). In these systems, the anode is placed in the bottom anaerobic region of the VFCW. In contrast, the cathode is placed in the top aerobic region, where the oxygen is available by plant roots and diffusion from the atmosphere (either by ventilation pipe or movement of plants due to wind). The CW-MFC performance relies on the availability of organic matter (electron source), exoelectrogen microorganisms, and suitable electrodes (higher conductivity, robustness, and electrode potential). However, the CW-MFC still holds a scope for understanding and improvement for WW treatment and power generation efficiency through electrode design optimisation (in terms of zonation and electrode arrangement in CW), hydraulic retention time (HRT) effects on the CW-MFC, and the electrode cost.

The integrated microalgae cultivation for WW treatment and its biomass production for various purposes like biofuels (Ferreira et al. 2018, 2019), animal feed (Maizatul et al. 2017), and manure (Navarro-Lopez et al. 2020) are advancing. The microalgal photobioreactor (ABR) is artificially designed to cultivate and harvest microalgal biomass under optimally controlled conditions, e.g., raceway ponds, circular ponds, column reactors, tubular reactors, and flat panel reactors (Ali et al. 2022). Microalgae are rich sources of biomolecules like proteins, lipids, carbohydrates, amino acids, polysaccharides, carotenoids, and fibres (Koyande et al. 2019). Microalgae exhibit a higher growth rate than plants and accumulate high biomass (Abdur Razzak et al. 2023). This shows the scope of pollutant removal from WW treatment. The microalgae optimally grow in water enriched with phosphate, nitrate, ammonium, and carbon dioxide, along with optimum environmental factors like sunlight (2,700–10,000 LUX), temperature (20–25 °C) and dissolved oxygen (DO) (3–10 mg/L) (Ras et al. 2013; Gonzalez-Camejo et al. 2019; Kazbar et al. 2019). Moreover, microalgal cultivation can be integrated with WW treatment as part of a biological circular economy. Furthermore, the suitability of APB post-integration to CW-MFC can be studied for algal biomass production.

The present study aims to develop a cost-effective and environmentally friendly technology that can simultaneously treat piggery WW, generate bioelectricity, and produce microalgal biomass. The technology comprised a two-stage VFCW integrated with a MFC and microalgal photobioreactor (APB). The study investigated the optimum electrode zonation in VFCW for optimal open-circuit voltage (OCV) generation and its effect on VFCW WW treatment efficiency. The performance of this integrated system was optimised at different HRTs and WW feeds loading rates. The APB design, HRT, and other environmental parameters were investigated for optimal microalgal biomass production.

A set of similarly designed two-stage VFCWs was constructed to compare their treatment efficiency, and their statistically significant difference in percentage removal efficiency for different WW parameters was validated. Thus, one system was designated as a set I VFCW and the other as a set II VFCW. two-stage VFCWs were designed to be unsaturated vertical flow in the first stage (UVFCW) and saturated vertical flow in the second stage (SVFCW). The second-stage VFCW was saturated up to 90 cm. The saturation was achieved based on the drainage pipe height (Figure 1). The PVC pipe of diameter 15 cm was used to construct a two-stage VFCW. VFCWs comprise three distinct layers: a top filter layer, a middle transition layer, and a bottom drainage layer. The river-based gravels were used for the VFCW media, and their size and layering height distribution are shown in Supplementary Table S1. Passive aeration was achieved by laying 1-inch PVC pipes up to 80 cm of UVFCW and 39 cm of SVFCW. Two C. indica were planted in both set I and set II VFCWs.

The piggery WW was pumped into both the first-stage VFCW systems at a flow rate of 1 L/min for 2 min (2 L) a day using a peristaltic pump (NFP-03, Flowtech, India). The UVFCW effluent is fed to SVFCW by gravity flow. The samples collected from both stages were analysed for various WW parameters. The volume capacity of SVFCW was determined by measuring their water-holding capacity (water input volume–water output volume).

The cathodes (at 20 and 30 cm) and anodes (at 40 and 60 cm) were placed at different heights from the top of the SVFCW, as shown in Figure 3. Then, the VFCW-MFC were left to stabilise for 1 week with the WW feed of 2 L/day. Then, the electrodes were connected to an external digital multimeter (OWON B35 T) to measure OCV generated in various electrode combinations of cathode–anode: 20–40 cm, 20–60 cm, 30–40 cm, and 30–60 cm, respectively. The WW feed and operation were like the optimised two-stage VFCWs. Furthermore, the optimised electrode zonation was enhanced by an electrode design. A pair of circular anodes and cathodes (Figure 3(b)) were cut into four pieces equally and connected in series, respectively, as shown in Figure 3(c). Furthermore, the effect of WW feed rate of 2, 4, and 5 L per day, respectively (flow rate of 1 L/min) and HRT on WW treatment efficiency and bioelectricity generation was studied.

Initially, the study examined the optimal growth conditions for algae by testing different HRTs of 1–5 days, DO, temperature, and light intensity in an open environment in a 500 mL glass bottle. Furthermore, the optimised parameters were scaled to the pilot-scale bubble column photobioreactor type of 5 L active volume capacity (Figure 2). The inoculum of a mixed algae culture was obtained from the naturally grown piggery WW.

A 5 L pilot-scale photobioreactor was constructed using a transparent acrylic cylindrical column with a height of 91 cm and a diameter of 9 cm. The bottom of the reactor was equipped with an air bubble diffuser connected to an air pump (ACO-001) with the regulated airflow metre (LZQ-7, 2–20 L/min) for aeration and mixing (Figure 1). Two drain valves were installed at specific heights to facilitate culture management (Figures 1 and 2). The upper valve, positioned 75 cm from the bottom, allowed the retention of 5 L of culture. In contrast, the lower drain valve, located 15 cm from the bottom, enabled the retention of 1 L of culture (as inoculum for the subsequent batch) and the collection of 4 L algal biomass.

The microalgal photobioreactor's growth rate, wet weight, pH, DO, temperature, and light intensity were measured daily throughout the experiment.

The samples were collected twice weekly and analysed within 24 h throughout the study. The different parameters of the WW were analysed using standard methods, i.e., chemical oxygen demand (COD) – 5220 D closed reflux colourimetric method, orthophosphate (⁠⁠) – Vanad o-molybdophosphoric acid colourimetric method, TAN – Spectroquant^® Prove 100 spectrophotometer kit (1.00683.0001), nitrate (⁠⁠) – nitrate metre (LAQUAtwin-11-S040), total organic carbon (TOC) and total nitrogen (TN) – the Shimadzu TOC-TNM-L ROHS analyser, DO – dissolved oxygen meter (PDO-519), pH – Oakton pH 510 Series Meter P/N 54X002608) and temperature – thermometer (G H ZEAL LTD) (APHA/AWWA/WEF 2005).

The unpaired t-test statistically analysed the significant difference (at the p < 0.05 level) in treatment efficiency between two similar sets of two-stage VFCW in the GraphPad Prism software (version 9.5.0). The mean standard deviation of all the data analysed was done in Microsoft Excel software (version 2019).

The first-stage VFCW of both the sets showed higher COD and TAN removal, as shown in Figure 4. The prominent reason can be due to the better aerobic design achieved by its unsaturated design and laying passive aeration till the drainage layer, which facilitates better organic matter degradation and nitrification (Gogoi et al. 2024), whereas second-stage VFCW of both the sets showed higher removal of and _, as shown in Figure 4. The primary reason can be due to its saturated design (up to 90 cm) creating anaerobic conditions and facilitating longer contact times between WW and the system. Thus, it favours denitrification and enhanced biological phosphorus removal. The raw (R) piggery WW parameters analysed for COD, TAN, ⁠, _, and pH were found to be 3,621.3 ± 286.19, 61.38 ± 15.64, 116.33 ± 11.02, 76.9 ± 13.09 mg/L, and 4.49 ± 0.47, respectively.

The perennial plant type C. indicana was selected for the study due to its long fibrous root types, which enhance its absorption, aeration, and the rhizosphere zone. Moreover, they are adaptable to grow in water stress or water-logged soil and the tropical climate of Goa, the study site (India) (Gogoi & Mutnuri 2024). The optimised VFCW design was further studied to enhance treatment by integrating MFC and investigating bioelectricity potential simultaneously.

The optimum electrode zonation (anode-cathode) was 60–20 cm, with a maximum OCV of 150.71 ± 23.62 mV with a WW feed rate of 2 L/day, as shown in Table 1. The optimisation of electrode placement in SVFCW filter layers was done in reference to two crucial factors: electrode spacing and zonation. The electrode spacing contributes to internal resistance; hence, the more spacing between the cathode and anode, the higher the internal resistance will be. This can result in less electron flow in the circuit, hence less power generation (Kesarwani et al. 2023).

Moreover, electrode zonation is critical in MFC function; it requires an optimum redox gradient of anaerobic for the anode and aerobic for cathode placement (Kesarwani et al. 2023). The electrons generated in the anode chamber should flow to the cathode chamber (due to electrode potential gradient), which contains oxygen as an electron acceptor. Therefore, the anode chamber should be deprived of electron acceptors or oxygen abundance. Thus, proper electrode zonation is a more critical concern than electrode spacing for the efficiency of the MFC function, as shown in Table 1; irrespective of electrode spacing, the appropriate zonation (60–20 cm) showed higher OCV generation of 150.71 ± 23.62 mV.

Further, the optimum electrode pair zonation (60–20 cm) was enhanced with the multiple-series connected anode and cathode arrangement, respectively, as shown in Figure 3(c). The same raw piggery WW source and same electrode surface area (SA) divided into four multiple anodes and cathodes, respectively (Figure 3(c)), showed a higher OCV of 291.83 ± 13.53 mV (Table 2) than the intact circular electrode 150.71 ± 23.62 mV (Table 1 and Figure 3(b)). The primary reason can be the increase in electrode potential difference contributed by the pair of four multiple electrodes (connected in series) systems compared to the pair of single electrode systems, irrespective of their SA. Furthermore, the VFCW-MFC potential of WW treatment efficiency and bioelectricity generation were studied at various WW loading rates and HRT, as shown in Table 2. The 2 L/day WW loading rate and HRT of 76 h, 48 min showed the highest OCV of 291.83 ± 13.53 mV than the HRT of 38 h, 24 min (211.64 ± 43.69 mV) and 30 h, 43 min (139.5 ± 4.08 mV), signifying that the appropriate WW loading rate and more HRT contribute to higher power generation. The significant reasons can be the longer contact time of the exoelectrogen and the WW (organic matter as electron source), facilitating more electron generation. Furthermore, the higher WW loading rate can disturb or wash out the exoelectrogen biofilm (Kesarwani et al. 2023).

Table 2

Comparative WW parameters reduction by two-stage VFCWs with MFC and without MFC (control (C)) at various WW loading rates and HRT

Samples .	Average reduction observed .
	COD (mg/L) .	T̀AN (mg/L) .	(mg/L) .	TN (mg/L) .	(mg/L) .	TOC (mg/L) .	TSS (mg/L) .	pH .	mV .
WW feed per day = 2 L/day (HRT = 76 h, 48 min)
R	3,351.42 ± 1,362.08	59.86 ± 29.98	119 ± 68.20	93.18 ± 54.56	74.08 ± 21.25	1,480.33 ± 701.48	–	4.77 ± 0.82	–
C1	1,890 ± 696.46	47.86 ± 22.23	101.13 ± 49.23	73.26 ± 40.44	48.54 ± 14.31	999.93 ± 441.12	–	5.18 ± 0.84	–
C2	992.86 ± 261.19	64.86 ± 18.87	58.63 ± 23.09	62.37 ± 10.77	26.33 ± 8.77	395.37 ± 150.59	–	6.87 ± 0.06	–
CW1	1,874.29 ± 692.43	47.14 ± 19.19	96.5 ± 41.66	80.02 ± 45.89	54.13 ± 22.34	1,074.24 ± 508.84	–	5.09 ± 0.78	–
CW2-MFC	432.86 ± 291.68	68.29 ± 19.72	43.5 ± 12.37	58.19 ± 13.17	4.16 ± 1.67	67.96 ± 19.88	97.67 ± 3.3	7.65 ± 0.23	291.83 ± 13.53
WW feed per day = 4 L/day (HRT = 38 h, 24 min)
R	2,604 ± 1,104.64	37.67 ± 20.98	73 ± 55.06	31.99 ± 16.95	84.83 ± 53.14	1,211.4 ± 1,205.87	–	4.76 ± 0.61	–
C1	1,330 ± 745.35	17.67 ± 11.59	68 ± 29.23	35.81 ± 16.18	45.51 ± 22.15	841.18 ± 470.45	–	5.12 ± 1.01	–
C2	272 ± 178.24	44 ± 26.29	41.2 ± 5.22	25.24 ± 9.57	14.67 ± 6.23	102.32 ± 50.98	–	6.71 ± 0.13	–
CW1	1,518 ± 817.51	16.33 ± 5.13	67 ± 38.35	36.36 ± 13.84	38.29 ± 16.84	804.17 ± 409.78	–	4.85 ± 0.89	–
CW2-MFC	186 ± 129.34	39.67 ± 20.13	32.8 ± 13.5	22.96 ± 6.98	10.23 ± 3.41	61.21 ± 32.35	67.00 ± 8.49	6.74 ± 0.08	211.64 ± 43.69
WW feed per day = 5 L/day (HRT = 30 h, 43 min)
R	3,773.33 ± 2,777.22	55 ± 54.14	172.8 ± 58.47	62.85 ± 56.14	113.15 ± 73.94	590.7 ± 280.85	5,510.42 ± 474.36	5.44 ± 0.43	–
C1	1,396.67 ± 231.59	32 ± 68.26	179.6 ± 133.69	100.2 ± 103.5	52.85 ± 47.59	497.07 ± 183.33	84.95 ± 3.09	6.09 ± 0.51	–
C2	363.33 ± 37.86	69.75 ± 20.07	62.6 ± 7.44	29.35 ± 10.08	23.35 ± 5.08	98.24 ± 19.04	31.18 ± 3.69	6.86 ± 0.06	–
CW1	1,496.66 ± 357.26	25 ± 57.98	173.6 ± 154.16	117.64 ± 125.7	60.79 ± 51.7	564.73 ± 243.78	75.08 ± 19.75	5.68 ± 0.42	–
CW2-MFC	306.67 ± 110.15	59.5 ± 12.71	44.6 ± 13.79	32.83 ± 9.21	15.83 ± 3.94	84.25 ± 24.71	46.9 ± 13.15	7.01 ± 0.3	139.5 ± 4.08

Samples .	Average reduction observed .
	COD (mg/L) .	T̀AN (mg/L) .	(mg/L) .	TN (mg/L) .	(mg/L) .	TOC (mg/L) .	TSS (mg/L) .	pH .	mV .
WW feed per day = 2 L/day (HRT = 76 h, 48 min)
R	3,351.42 ± 1,362.08	59.86 ± 29.98	119 ± 68.20	93.18 ± 54.56	74.08 ± 21.25	1,480.33 ± 701.48	–	4.77 ± 0.82	–
C1	1,890 ± 696.46	47.86 ± 22.23	101.13 ± 49.23	73.26 ± 40.44	48.54 ± 14.31	999.93 ± 441.12	–	5.18 ± 0.84	–
C2	992.86 ± 261.19	64.86 ± 18.87	58.63 ± 23.09	62.37 ± 10.77	26.33 ± 8.77	395.37 ± 150.59	–	6.87 ± 0.06	–
CW1	1,874.29 ± 692.43	47.14 ± 19.19	96.5 ± 41.66	80.02 ± 45.89	54.13 ± 22.34	1,074.24 ± 508.84	–	5.09 ± 0.78	–
CW2-MFC	432.86 ± 291.68	68.29 ± 19.72	43.5 ± 12.37	58.19 ± 13.17	4.16 ± 1.67	67.96 ± 19.88	97.67 ± 3.3	7.65 ± 0.23	291.83 ± 13.53
WW feed per day = 4 L/day (HRT = 38 h, 24 min)
R	2,604 ± 1,104.64	37.67 ± 20.98	73 ± 55.06	31.99 ± 16.95	84.83 ± 53.14	1,211.4 ± 1,205.87	–	4.76 ± 0.61	–
C1	1,330 ± 745.35	17.67 ± 11.59	68 ± 29.23	35.81 ± 16.18	45.51 ± 22.15	841.18 ± 470.45	–	5.12 ± 1.01	–
C2	272 ± 178.24	44 ± 26.29	41.2 ± 5.22	25.24 ± 9.57	14.67 ± 6.23	102.32 ± 50.98	–	6.71 ± 0.13	–
CW1	1,518 ± 817.51	16.33 ± 5.13	67 ± 38.35	36.36 ± 13.84	38.29 ± 16.84	804.17 ± 409.78	–	4.85 ± 0.89	–
CW2-MFC	186 ± 129.34	39.67 ± 20.13	32.8 ± 13.5	22.96 ± 6.98	10.23 ± 3.41	61.21 ± 32.35	67.00 ± 8.49	6.74 ± 0.08	211.64 ± 43.69
WW feed per day = 5 L/day (HRT = 30 h, 43 min)
R	3,773.33 ± 2,777.22	55 ± 54.14	172.8 ± 58.47	62.85 ± 56.14	113.15 ± 73.94	590.7 ± 280.85	5,510.42 ± 474.36	5.44 ± 0.43	–
C1	1,396.67 ± 231.59	32 ± 68.26	179.6 ± 133.69	100.2 ± 103.5	52.85 ± 47.59	497.07 ± 183.33	84.95 ± 3.09	6.09 ± 0.51	–
C2	363.33 ± 37.86	69.75 ± 20.07	62.6 ± 7.44	29.35 ± 10.08	23.35 ± 5.08	98.24 ± 19.04	31.18 ± 3.69	6.86 ± 0.06	–
CW1	1,496.66 ± 357.26	25 ± 57.98	173.6 ± 154.16	117.64 ± 125.7	60.79 ± 51.7	564.73 ± 243.78	75.08 ± 19.75	5.68 ± 0.42	–
CW2-MFC	306.67 ± 110.15	59.5 ± 12.71	44.6 ± 13.79	32.83 ± 9.21	15.83 ± 3.94	84.25 ± 24.71	46.9 ± 13.15	7.01 ± 0.3	139.5 ± 4.08

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The enhanced removal of COD and TOC in the VFCW-MFC system may be due to the increased rate of organic matter removal by anaerobic oxidation facilitated by exoelectrogen in the anode electrode (Oon et al. 2015; Kesarwani et al. 2023). Thus, in the process, free electrons are generated and transported to the cathode via anode, which can be the significant reason for enhanced and reduction in the VFCW-MFC system (Kesarwani et al. 2023). The and removal can be due to direct and indirect processes; that is, the DO availability is reduced to water in the presence of electrons and proton (H⁺), thus limiting the DO for nitrification (produces ⁠) and phosphorus mineralisation (produces ⁠) process. In the direct method, the can act as an alternative electron acceptor and get converted into and, in the presence of hydrogen ions, can form ammonium (Kesarwani et al. 2023). Thus, this might be the reason for the increase of TAN in VFCW-MFC compared to the control system. Moreover, the enhanced organic matter degradation can also release the organically bound nitrogen to TAN (Gogoi & Mutnuri 2024). Overall, the 2 L/day with the HRT of 76 h, 48 min showed better WW treatment and bioelectricity generation efficiency.

The CW-MFC's optimised design positively impacted treating piggery WW and generating OCV using a cost-effective combination of stainless steel (cathode) and aluminium (anode) electrode materials. The study investigated the effects of electrode zonations, multiple electrode arrangements, and HRT on WW treatment and power generation. Additionally, the effluent from the CW-MFC was examined for its potential to grow green microalgae biomass in an algal photobioreactor. Furthermore, the microbial and metabolic diversity of the VFCW-MFC can be studied along with alternative sustainable electrode material to enhance the simultaneous WW treatment and bioelectricity generation on a large scale.

Initially, the TSSs were studied only for the second-stage VFCW-MFC effluent of three different flowrates. The 5 L/day flow rate showed less TSS (46.9 ± 13.15 mg/L) than the 2 L/day (97.67 ± 3.3 mg/L) and 4 L/day (67.00 ± 8.49 mg/L) flow rates, as shown in Table 2. The 5 L/day flow rate was more favourable for post-photobioreactor integration because the higher TSS can inhibit algal growth by preventing light penetration (Lee et al. 2020). Then, the TSS of the untreated piggery WW and the VFCW system (both control and VFCW-MFC) for a 5 L/day flow rate was also studied. The piggery WW showed a TSS of 590.7 ± 280.85 mg/L (Table 2). Moreover, the algae utilisable nitrogen (TAN; 59.5 ± 12.71 mg/L and ⁠, 44.6 ± 13.79 mg/L) and phosphate (15.83 ± 3.94 mg/L) source ratio was 6.58:1 (Table 2). The pH also meets the optimum range of 7.01 ± 0.3 to support the algal growth, as shown in Table 2 (Sakarika & Kornaros 2016).

Then, the mixed algal culture was scaled up to 5 L/day in a bubble column photobioreactor under the same environmental conditions (Figure 6(b)). Over the study period, the average temperature and light intensity recorded were 30.3 ± 0.44 to 38.17 ± 1.17 °C and 3,942.86 ± 496.18 to 6,471.43 ± 711.14 LUX of a 12:12 h natural light-dark cycle, respectively. The DO was maintained within the optimal range of 8.24 ± 0.15 mg/L by dosing air at a flow rate of 1 L/min for 12 h daily, with a 1 h interval (Kazbar et al. 2019).

The present study successfully developed an eco-friendly and cost-effective technology comprising an integrated VFCW-MFC and microalgal photobioreactor for piggery WW treatment and simultaneously recovering bioelectricity and microalgal biomass. The unpaired t-test statistically confirmed no significant difference (at the p < 0.05 level) in treatment efficiency between two similar sets of two-stage VFCW. The optimal single electrode pair zonation was 60 cm (anode) and 20 cm (cathode) in the two-stage VFCW, which showed the highest OCV generation of 150.71 ± 23.62 mV. Overall, the two-stage VFCW-MFC with multiple-series connected anode and cathode, respectively, showed the highest OCV of 291.83 ± 13.53 mV and WW treatment efficiency at a WW loading rate of 2 rather than 4 and 5 L/day. Comparatively, the VFCW-MFC system showed better WW treatment efficiency than the VFCW. This could be possibly due to the increased microbial population and activity on the electrode surface. This stimulates the anaerobic degradation of organic matter on the anode surface, generating free electrons transferred to the cathode. The electron abundance in the cathode enhances the reduction of and ⁠.

The two-stage VFCW-MFC effluent of 5 L/day feed favoured the algal biomass production due to less TSS compared to 2 and 4 L/day feed. The optimised bubble column photobioreactor design and the optimum environmental conditions favoured the growth of 10 types of mixed green microalgae culture. The highest growth (OD 5.22 ± 1.95) and biomass production (wet weight of 21,323.34 ± 8,316.26 mg/L) were observed on the fourth day of incubation. The study successfully demonstrated the scope of circular economy in WW treatment.

Furthermore, the optimised pilot-scale integrated treatment system will be scaled up for full-scale implementation at a piggery farm in Goa, India. The post-processing of cultivated microalgae biomass for potential use as manure and pig feed will be studied. Furthermore, a comparative study of the effect of close and open-circuit CW-MFC on WW treatment can be conducted. This research shows a promising integrated WW treatment technology for circular economy and climate resilience scope.

This work was funded by the Bilateral Portugal-India DRI/India/0609/2020- Project WCAlgae ⁺KIT. The BITS-Pilani KK Birla Goa Campus provided the field site for a pilot-scale study of the integrated treatment system.

J. G. completed the two-stage VFCW and MFC optimisation work. J.G., K. N., and A. N. did the microalgal photobioreactor designing, optimisation, analysis, and manuscript writing. Prof. S. M. and Prof. A. G. contributed to the design of the experiments, reviewed the manuscript, and supported us with their technical expertise throughout the study.

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

The authors declare there is no conflict.