EP4288390A1

EP4288390A1 - Earthen membrane based two chambered constructed wetland cum micriobial fuel cell for treatment and detoxification of waste water containing azo dye

Info

Publication number: EP4288390A1
Application number: EP22749387.1A
Authority: EP
Inventors: Asheesh Kumar YADAV; Yamini MITTAL; Suddhasatwa Basu; Pavan Kumar Togapur; Pratiksha Srivastava; Supriya Gupta
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 2021-02-08
Filing date: 2022-02-04
Publication date: 2023-12-13
Also published as: WO2022168121A1; AU2022217914A1; EP4288390A4

Abstract

The present invention relates to the treatment and mineralization/detoxification of azo dye and other pollutants containing wastewater with innovatively designed earthen membrane based two chambered constructed wetland cum microbial fuel cell (CW-MFC). The developed CW-MFC provides flexibility of treating wastewater in sequential anaerobic and aerobic regimes. This innovatively design of earthen membrane based two chambered earthen CW-MFC is able to resolve the issues that generally emerge in typical CW-MFC design which include: i) cathodic and anodic anolyte mixing; ii) high resistance development iii) less oxygen diffusion at cathode; iv) diffusion of oxygen from cathode to anode; (v) use of commercial costly chemical membrane; (iv) loss of synthesized products at cathode and many other described in this application.

Description

EARTHEN MEMBRANE BASED TWO CHAMBERED CONSTRUCTED WETLAND CUM MICRIOBIAL FUEL CELL FOR TREATMENT AND DETOXIFICATION OF WASTE WATER CONTAINING AZO DYE

FIELD OF THE INVENTION

The present invention relates to an earthen membrane based two chambered constructed wetland cum microbial fuel cell for treatment and detoxification of wastewater containing azo dye. In particular, the present invention relates to the treatment and mineralization/detoxification of azo dye and other pollutants containing wastewater with innovatively designed earthen membrane based two chambered constructed wetland cum microbial fuel cell (CW-MFC).

BACKGROUND OF THE INVENTION AND DESCRIPTION OF PRIOR ART

Azo dye is one of the major pollutants and many industrial processes are responsible for release of large amounts of azo dyes in their wastewaters. The azo dyes are used in several industries such as paper printing, food, cosmetics with textile dyeing as its largest consumer. It is estimated that one million tons of azo dyes are produced each year from different industries in wastewater. Approximately 20-30% of these dyes come from unstable dyes with on an average concentration of 2000 mg/1. Contamination of azo dye creates challenges for receiving water bodies and the environment. It affects the photosynthesis process of algae and other similar organisms in water bodies by preventing light penetration and subsequently depletes oxygen, which adversely affects the flora and fauna communities. Moreover, the partial breakdown/degradation or treatment of azo dyes leads to formation of mutagenic/carcinogenic intermediate compounds. To maintain a healthy ecosystem, it is urgent to address contamination of water bodies.

The azo dyes, constitutes of aromatic compound with one or more -N=N-bond (named as azo bond). This class of dye has been largely utilized for commercial applications i.e. 700, 000 ton per year. The azo bond linkage is responsible for the color in azo dyes due to its chromophore functional group. Also, azo bond linkage dyes are highly stable owing to its complex aromatic rings and covalent azo bonds. Considering the recalcitrant nature of azo dyes, complete dye degradation is always a challenge. In general, the degradation/ decolorization of azo bond occurs in two stages. Wherein, the first stage involves the reductive cleavage of azo dyes generally taking place in anaerobic environment which results in formation of colorless but potentially hazardous aromatic amines. However, at the same time, aromatic amines are resistant to further degradation in anaerobic environment. Since, the bacterial degradation of aromatic amines exclusively requires aerobic conditions. Hence, sequential anaerobic and aerobic conditions are proposed for complete and effective dye degradation.

There are numerous physicochemical methods that exist for azo dye contained wastewater treatment. Coagulation/flocculation-based treatment requires substantial amount of chemicals and produces sludge. Adsorption and membrane filtration techniques generate secondary sludge which further accompanies sludge handling and disposal issues. Advanced oxidation processes (such as UV/H2O2, Fenton, ozonation etc.) are quite effective but are energy and cost intensive. Whereas, biological processes have been proven as a cost effective and ecofriendly alternative to other treatment technologies.

Constructed wetlands (CW) is a conventional biological and passive treatment process, suitable for different types of industrial and municipal wastewater, has also been used for several dye wastewater treatment. However, several challenges limit the adoption of CW such as slow treatment process with largely anaerobic conditions dominated areas and high land area requirement. With the recent progress in biological processes several other technologies such as microbial fuel cells (MFCs) have proven to be sustainable treatment technology for azo dye contained wastewaters (CN102315469B; CN102593469B; CN102315469B; CN102593469B; CN102315471A). However, upscaling of MFC is still a big challenge. Other challenges like low oxygen reduction rate (ORR) at low cost cathode (such as carbon or graphite) leads to high over potentials and low kinetics in MFC systems (Microbial Electrochemical Technology, 2019, 1021-1036). Accordingly, CW was implemented with MFC technology to further intensify treatment of wastewater (Ecological Engineering, 2012, 47, 126-131; Constructed wetlands for industrial wastewater treatment, 2018, 493-510).

In CW, the natural redox gradient is in proportion with the environment for the integration of MFCs (Microbial Electrochemical Technology, 2019, 1021-1036; Chemosphere, 2020, 126926). Hence, the origination of innovative hybrid technology i.e. integration of microbial fuel cell in constructed wetland (CW-MFC) which was first reported in Proceedings of 12th International Conference on Wetland Systems for Water Pollution Control (TWA), 2010, 4-10. CW-MFC contains anaerobic and aerobic regions same as CW and MFC, embedded with electrodes (conductive material) as electron acceptors which assist in enhancement of the reaction processes (Proceedings of 12th International Conference on Wetland Systems for Water Pollution Control (IWA), 2010, 4-10). The first laboratory scale CW-MFC has been studied for methylene blue dye removal and results depicts >75% dye removal at even higher dye loads of 2000 mg/L (Ecological Engineering, 2012, 47, 126-131).

In the following year, 91.24 % decolorization rate was achieved for active brilliant red X-3B (ABRX3) azo dye and voltage generation of 610 mV with CW-MFC and noted increased decolorization was promoted by the electrodes. Furthermore, methyl orange degradation was studied in open and closed-circuit CW-MFC and removal of 75.59 % and 87.60 %, respectively was observed. Additionally, the energy recovery from azo dye ABRX3 containing wastewater with CW-MFC was performed. It resulted in 95.6 % dye decolorization with 0.852 W/m³ of power density at 300 mg/L of COD and 30 % of dye proportion.

Reference may be made to Bioresource Technology, 2019, 285, 121349 wherein investigated was the removal efficiency of real dye stuff wastewater with Fimbristylis dichotoma planted closed-circuit CW-MFC and 82.2 ± 1.7 % dye removal was achieved along with 70+2% COD reduction with maximum power density generation of 198.8 mW/m³.

Reference may be made to Science of The Total Environment, 2020, 720, 137370 wherein studied was the decolorization of Acid Orange 7 (AO7), Acid Red 18 (ARI 8) and Congo Red (CR) in combination of closed, open circuit and planted and non-planted CE-MFC and higher decolorization rate found out in closed circuit CW-MFC as ARI 8 (96%) > AO7 (67%) > CR (60%). Moreover, the decolourization rate was affected by number of azo bonds and functional group positions and presence of wetland plant enhanced the power density by 73%.

It may be noted that all the aforementioned studies report a typical single chambered CW- MFC which possesses the serious issue of lack of total azo dye mineralization/detoxification. Thus, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to provide a novel two chambered earthen membrane based CW-MFC which can lower the resistance value and hence enhance the electricity generation and treatment, which is an appropriate cost-effective technology with high efficiency towards total or very high dye mineralization and detoxification from wastewater.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is therefore to provide earthen membrane based two chambered constructed wetland cum microbial fuel cell for treatment and detoxification of wastewater containing azo dye.

Another objective of the present invention is to provide a device with very high to total azo dye mineralization and detoxification along with simultaneous electricity generation during wastewater treatment.

Still another objective of the present invention is to provide a novel two chambered earthen membrane-based CW-MFC designing which can lower the resistance value and hence enhance the electricity generation and treatment.

Yet another objective of the present invention is to provide a device which does not allow the mixing of catholyte with anolyte.

Still another objective of the present invention is to provide a sequential gravitation flow of wastewater for energy and cost saving.

Yet another objective of the present invention it to provide minimal air diffusion in anaerobic chamber at low-cost and feasible manner.

Still another objective of the present invention is to provide an alternative of commercial chemical-based membrane for treatment of azo dyes.

SUMMARY OF THE INVENTION

The above and other objectives of the present invention are achieved by providing the innovatively designed earthen membrane based two chambered CW-MFC (microcosm). The microcosm was developed for highly efficient treatment of azo dye or similar pollutants containing wastewater along with other benefits like electricity generation. An innovatively designed lab scale version of CW-MFC was fabricated with a plastic container as outer cathodic/aerobic chamber. In the middle of the plastic container, earthen pot for creating earthen membrane/layer, was placed which works as anodic chamber/anaerobic chamber. Both the chambers were filled with graphite granules (5-8 mm in diameter) for acting as conductive materials-based anode and cathode electrodes. The cathodic and anodic chambers were embedded with 112 mm x 13 mm (length x width) graphite rod as charge collector/exchanger/dispenser. Some part of the cathode charge dispenser was kept in contact with atmospheric oxygen and placed 1.5 cm above the water level. The anodic and cathodic chamber has void volume/working volume of 250 mL and 500 mL respectively. Anodic sample was collected from the upper portion of earthen pot for analysis. The charge collectors of anodic and cathodic compartment were connected with an insulated copper wire for charge (electron) movement. Moreover, both anodic and cathodic wire was connected with resistance of 2000 Q as a load. An air sparger was provided at the bottom of earthen pot in the cathodic chamber and connected with an aerator (Venus, AP-408A air pump, India). Furthermore, as a precautionary measure outer bottom portion of earthen pot was covered with high density polymer liner (HDPE liner) for preventing air diffusion form cathode chamber to earthen pot which was working as anaerobic chamber.

In an embodiment of the present invention, the two chambered constructed wetland cum microbial fuel cell provides 90-100% mineralization of azo dye which is confirmed by UV- Vis spectroscopy and gas chromatograph- mass spectrometry (GC-MS) analysis. UV-Vis spectra displayed azo bond degradation in the anodic region and whereas GC-MS confirms the mineralization in the cathodic effluent with elution of several mineralized polar compounds.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Schematic diagram of innovatively designed earthen membrane based two chambered CW-MFC with sequential anaerobic and aerobic regimes. Wherein, ‘a’ represents earthen pot/layer acting as proton exchange and oxygen/gas diffusion inhibiting membrane; ‘b’ is anode cover which restricts diffusion of air inside the anaerobic earthen pot; ‘c’ is high density polymer liner (HDPE) covering the bottom part of earthen pot so that aerator’s oxygen/air won’t diffuse inside the earthen pot; ‘d’ and ‘e’ are anode and cathode charge collectors/exchangers inserted in anode and cathode respectively; ‘f’ illustrates the aerator ring situated below the earthen pot; ‘g’ is the resistance/load connected between anode and cathode; ‘h’ is cathodic chamber and an outlet ‘i’ connected to an adjustable siphoned pipe ‘j’-

Figure 2: Shows cathodic and anodic a) dissolved oxygen profile b) pH profile of earthen CW-MFC.

Figure 3: Oxidation reduction profile of cathodic and anodic chambers of CW-MFC.

Figure 4: Percentage removal of chemical oxygen demand in anodic and cathodic effluent of microcosms.

Figure 5: Represents a) UV-Vis spectra of influent, anodic and cathodic effluent b) UV-Vis spectra of sulphanilic acid c) methyl orange standardization d) MO removal in the anodic and cathodic regions of the microcosm.

Figure 6: GC-MS chromatograph of a) anodic effluent b) cathodic effluent of CW-MFC microcosm.

Figure 7: Polarization curve of CW-MFC.

Figure 8: Voltage profile of the CW-MFC.

Figure 9: Shows the microbiological analysis a) based on phylum for anodic, cathodic region and inoculum, based on class level b) anodic region c) cathodic region d) Inoculum and e) phylogenetic tree showing the involvement of different species in dye removal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the construction, working and implementation of innovatively designed earthen membrane based two chambered CW-MFC for wastewater treatment containing azo dye to its complete mineralization and detoxification with following sequence of activities:

1. Earthen membrane based two chambered CW-MFC construction and working

2. Wastewater composition

3. Inoculation and experimental startup

4. Analysis, measurements and calculations

5. Phyto-toxicity assessment with different seeds 6. Total metagenomic study of bacterial biofilms at the anodic and cathodic region

7. Results and discussion

The invention has immense potential in the area of waste water treatment and related technologies. The developed device shall help meet the 6^th sustainable development goal of clean water and sanitation.

In this innovatively designed CW-MFC, the wastewater containing azo dye enters firstly in the lower anodic region and flows further passively up to the outlet, wherein the wastewater enters into the bottom of anaerobic chamber and then flows in up flow direction as it reaches the upper portion of anaerobic chamber. As the wastewater reaches the brim of earthen pot, it oozes out in the outer compartment through the walls of earthen pot. Afterwards, it comes out from effluent pipe which is acting as adjustable siphon tube permitting half or any desirable height of the cathodic compartment to be filled or empty. As a result, the empty upper cathodic region also allows more diffusion of oxygen which may further lead to enhancement in electricity generation and aerobic treatment of wastewater.

The current density of 442 mA/m³ and power density of 97.68 mW/m³ was achieved from earthen CW-MFC. The initial COD and azo dye (/'.<?. Methyl Orange taken as model azo dye pollutant but it will be applied for all other azo dye) provided in the wastewater was 550 mg/L and 50 mg/L, respectively. The results revealed 94.04 ± 2.87% COD removal and azo dye removal of 94.22 ± 1.33% in the cathodic effluent with HRT of 27 h. UV-Vis spectroscopy results demonstrated azo bond degradation region which is further confirmed by the presence of one of the methyl orange intermediates as sulphanilic acid ( = 248 nm) in the anodic effluent. Whereas, gas chromatograph- mass spectroscopy (GC-MS) analysis of anodic region verifies the other intermediate i.e. DMPD (m/z =136) and confirms the nearly complete mineralization in the cathodic effluent with elution of several mineralized polar compounds. Furthermore, cathodic region played role in complete mineralization of aromatic intermediates of methyl orange to less harmful and non- toxic products in the cathodic portion. Toxicity study with Vigna radiata, Triticum aestivum, Cicer arietinum indicated root growth rate of 30.72%, 13.53% and 11.62% in anodic effluent whereas 69.70%, 60.28% and 34.27% in cathodic samples respectively. Moreover, toxicity data corroborates with the reduction of phytotoxicity levels in the anodic and cathodic effluent by displaying root growth rate of 18.62 ± 10.51% and 54.75 ±14.98% respectively performed with 3 different seed types. The microbiological metagenomic analysis revealed the abundancy of phylum Bacteroidetes (19.09%) in the anodic region whereas cathodic community was found to be densely populated with phylum Proteobacteria (59.50%).

In an aspect, the study focuses on complete azo dye mineralization from wastewater in unique designed unplanted earthen membrane based two chambered constructed wetlands cum microbial fuel cell (CW-MFC). The developed CW-MFC provides flexibility of treating wastewater in sequential anaerobic and aerobic regimes. This innovatively designed earthen membrane based two chambered CW-MFC aims to resolve the issues that generally emerge in typical CW-MFC design, some of which are enlisted here: i) cathodic and anodic anolyte mixing; ii) high resistance development iii) less oxygen diffusion at cathode; iv) diffusion of oxygen from cathode to anode; (v) use of commercial costly chemical membrane; (iv) loss of synthesized products at cathode and many other described in this application.

EXAMPLES

The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

Example- 1: Earthen membrane based two chambered CW-MFC construction and working

An innovatively designed earthen membrane based two chambered CW-MFC was constructed with a plastic container of 16 cm diameter and 16 cm height working as outer cathodic or aerobic chamber. In the middle of the plastic container earthen pot for creating earthen membrane/layer, was placed which works as anodic chamber/anaerobic chamber. The earthen pot has upper, middle and lower diameter of 7 cm, 9 cm and 7 cm respectively and height of 12 cm. Both the chambers were filled with graphite granules (5-8 mm in diameter) for acting as conductive materials-based anode and cathode electrodes. The cathodic and anodic chambers were embedded with 112 mm x 13 mm (length x width) graphite rod as charge collector/exchanger/dispenser.

Some part of the cathode charge dispenser was kept in contact with atmospheric oxygen and placed 1.5 cm above the water level. The cathode charge dispenser was kept in contact with atmospheric oxygen and placed 1.5 cm above the water level is also recommended by the recent published study (Yang et al., 2019). The anodic and cathodic chamber has void volume/working volume of 250 mL and 500 mL respectively. Anodic sample was collected from the upper portion of earthen pot for analysis. The charge collectors of anodic and cathodic compartment were connected with an insulated copper wire of 1mm for charge (electron) movement. A air sparger was provided at the bottom of earthen pot in the cathodic chamber and connected with a aerator (Venus, AP-408A air pump, India). Furthermore, as a precautionary measure outer bottom portion of earthen pot was covered with high density polymer liner (HDPE liner) for preventing air diffusion form cathode chamber to earthen pot which was working as anaerobic chamber, as shown in Fig 1. Moreover, both anodic and cathodic wire was connected with resistance of 2000 Q as a load.

Example-2: Wastewater composition

A synthetic wastewater with glucose (550 mg/L) as an organic source has been used throughout the study. The synthetic wastewater constitute: 0.111 g/L NaHCCL, 0.0445 g/L KH2PO4, 0.0371 g/L MgCl₂-6H₂O, 0.0301 g/L CaCl₂-2H₂O, 0.1119 g/L (NH₄)₂SO₄, 0.0842 g/L (NH₄)₂Fe(SO₄)₂-6H₂O.The trace metal mix composed of 2.86 g/L H3BO3, 1.81 g/1 MnCl₂-4H₂O, 0.222 g/L ZnSO₄-7H₂O, 0.39 g/L Na₂Mo₄-2H₂O, 0.079 g/L CuSO₄-5H₂O and 0.05 g/L CoCl₂. Tap water has been used to prepare synthetic wastewater with aforementioned compounds and trace metal mix of 0.1 mL/L. The azo dye used in the present study was methyl orange (as a model azo dye), purchased from HI Media Pvt. Ltd., India. During the experiment, initial concentration of 50 mg/L methyl orange (MO) was mixed with synthetic wastewater, and the initial carbon concentration was 550 mg/L. The synthetic wastewater was thoroughly mixed through magnetic stirrer (T arsons, Model 4050, India) at 100-140 rpm to maintain homogeneity of influent. The entire experiment was carried out at room temperature of 27 ± 2°C.

Example-3: Inoculation and experimental startup

The CW-MFC was inoculated with the inoculum of previously operated CW-MFCs in our laboratory. Initially, the microcosm was acclimatized for 8 weeks with methyl orange containing wastewater in batch mode until stable voltage reading was recorded. Thereafter, homogeneous wastewater containing methyl orange dye with glucose was continuously fed into the bottom of anode through peristaltic pump (Watson Marlow 120S model, UK) at 24.66 mL/h. The aerator was fixed to supply air at 2.4 L/min of flow rate and set up by means of automatic on-off timer (HPA digital timer, Australia) with repetitive 70 minutes on, followed by 10 minutes off cycle. The CW-MFC was having HRT of 27 h wherein, the wastewater retains in anodic earthen pot for 9 h. The wastewater containing azo dye in the CW-MFC entered first in the lower anodic chamber with the help of peristaltic pump. Then, the wastewater flows in up flow direction as it reaches the upper portion of anode. Afterwards, it moves to cathode chamber and it comes out from effluent pipe which is acting as adjustable siphon tube and thus permitting half of the cathodic chamber to be filled and half empty. As a result, the half empty upper cathodic chamber also allows more diffusion of oxygen which may further lead to enhancement in electricity generation and wastewater treatment as shown in Fig 1. Unlike single chambered systems, in our innovatively fabricated system there won’t be any chances of mixing between the anodic and cathodic anolyte as it is a double chambered system which operates in conjunction with each other. Furthermore, the generated proton and electrons from substrate metabolism in the anodic chamber transferred to the cathode by porous earthen pot membrane and through the electric wire, respectively.

Example-4: Analysis, measurements and calculations

The electricity generation was recorded in terms of voltage on daily basis with digital handheld multimeter (Fluke 17B, USA). Once the CW-MFC was fully established the polarization curve was prepared by varying the external resistance from 90 MQ to IQ using resistor box (Model 1040, time electronics, UK) and voltage was recorded in every 15 min of time interval. Afterwards, to obtain polarization curve, graph has been plotted between voltage (V), power density (mW/m³) and current density (mA/m³) to further acquire ohmic, activation, and concentration losses information. The current density and power density were calculated by dividing with the anodic zone volume (m³) of the microcosm. Furthermore, all the collected samples were filtered with Whatman grade 1 filter paper, USA (pore size- 11 pm) before any analysis. MO concentrations in anodic and cathodic samples were measured by UV-Vis spectrophotometer (Agilent carry 100, USA). A standard curve of MO was plotted in 464 nm wavelength to determine MO concentration in samples. Further, the scanning of influent, anodic and cathodic samples were performed on UV-Vis spectrophotometer from wavelength range between 200-800 nm to determine the intermediates from azo dye decolorization and degradation. The decolorization percentage (DR) of MO from effluent of cathodic and anodic samples in comparison to influent was calculated from Eq. 1 : Znffeient absorbance — Effluent absorbance

% DR = — - — - - - X 100 Eq. 1

In Luent a&sorbance

The chemical oxygen demand (COD) was carried in accordance with closed reflux colorimetric method of APHA (American Public Health Association, 2005) at 610 nm. The percentage COD removal was calculated according to Eq. 2: 100 Eq. 2

The dissolved oxygen (DO) and oxidation reduction potential (ORP) of upper and lower anodic region and cathodic region were measured with DO and ORP probe attached in HACH meter (HQ-40D, USA). Additionally, pH was measured through pH meter (Eutech instruments cyberscan pH 1500, Canada).

The Gas chromatography (GC, QP2020 NX, Shimadzu, Japan) equipped with DB-5 capillary column (30m x 0.25 mm x 0.25 pm) coupled with MS (mass spectrometry) was used to validate the azo dye intermediates. Prior to GC-MS injection, 500 mL of anodic and cathodic samples were collected, and their organic phases were extracted in chloroform. The samples were dried with anhydrous Na2SO4 to remove moisture or water content and then filtered out (Whatman grade 1, pore size 11pm, USA). Thereafter, organic phases of the samples were fully concentrated in rotatory evaporator (FL300, JULABO, Germany) and collected with ethyl acetate solvent (HPLC grade). For GC-MS analysis ultra-pure helium gas was used as carrier. The GC-MS method was developed with 3 mL/min of purge flow and 250 °C of injector temperature. The GC column was programmed to operate at 40 °C initial oven temperature without hold, rise to 250 °C at a ramping rate of 10 °C/min and then 15 min of holding. The methyl orange intermediates were identified by referring the GC-MS spectra to the NIST (National institutes of standards and technology) library.

Example-5: Phyto-toxicity assessment with different seeds

Taking into account, the high range of sensitivity in plants towards toxicity, the Phyto- toxicity study was performed with the treated methyl orange containing wastewater in comparison to the without treated wastewater (influent). The goal of toxicity experiment was to identify the root inhibition in seedling of Cicer arietinum, Tritium aestivum and Vigna radiata. The seeds were grown in influent, anodic and cathodic compartment (effluent) wastewater and growth of root and shoot from the seeds were evaluated. To perform the toxicity experiments, petri-dishes were lined with 3 layers of paper cloth and seeds were sterilized before transferring to the petri dishes. Sterilization of seeds were carried out to remove dirt or any unwanted particles on the surface of seeds. The seeds were sterilized by washing in ethanol for 10s and then washing in 5% sodium hypochlorite for 40s with manual swirling followed by 6 times of distilled water wash and drying like reported by. The experiment was set up in triplicates for all the three types of seeds with 20 seeds in each dish and 5 mL of influent, anodic and cathodic sample per petri-dish. Besides, seeds were also kept in tap water as a control experiment. Furthermore, the addition of respective 5 mL sample was done each day in each petri dish. The seeds germination was observed daily and considered germinated when hypocotyl and radical appeared together. The root and shoot length measurement of germinated Cicer arietinum, Triticum aestivum and Vigna radiata was evaluated after 6 days. At the end of exposure period, the percentage growth rate (GR) and inhibition rate (IR) was calculated as per Eq. 3:

When the obtained value from the above equation was positive, IR was considered as inhibition rate (IR %) i.e. growth was less than tap water/control, whereas when the obtained value was negative it implies the growth (GR %) i.e. growth was more than tap water/control, where tap water was used as a reference.

Example-6: Total metagenomic study of bacterial biofilms at the anodic and cathodic region

The microbial phylogenetic analysis (16s rRNA pyrosequencing) of anodic and cathodic regions was performed in comparison to the inoculum. The biofilm formed at the anode and cathode electrodes surfaces were scraped with a sterile spatula and collected along with the wastewater from that particular regions in a 250 mL sterilized sample bottle. The scrapped biofilm with wastewater was suspended in lOOmM of Tris-EDTA (TE) buffer to avoid sample degradation. The metagenomic DNA was then extracted from the Qiagen DNA easy PowerSoil Kit (Qiagen, Germany) following the manufacturer protocol. The V3-V4 region of 16S rRNA was amplified using specific V3 Forward primer CCTACGGGNBGCASCAG and V4 Reverse primer GACTACNVGGGTATCTAATCC for diverse profiling. Example-7: Environmental conditions of the CW-MFC

The environmental conditions of the CW-MFC (anode chamber and cathode chamber) was analyzed based on parameters such as DO, pH and ORP, which are the major influential parameters in any microcosms/ biological treatment system. For, analyzing the favorable conditions for degradation of MO, all these parameters were studied throughout the study. With MO concentration of 50.0 mg/L, pH in the influent was in the range of 5.0 - 5.1. Whereas, in the lower region of anode pH was 5.51 ± 0.39 and at the upper region 5.94 ± 0.29. The pH at upper anodic region was always slightly higher than the lower region as shown in Fig 2a. It could be due to the prevailed anaerobic conditions in the lower anodic region which helped in the breaking down of co-substrate into fatty acids. The production of fatty acids in the absence of oxygen shifts the pH to acidic. Whereas, cathodic region pH value was recorded as 6.16 ± 0.36 and 6.15 ± 0.45 for lower and upper region respectively. The shift of cathodic pH to less acidic value can be attributable to the proton consumption in the cathodic region.

In addition, the DO profile of influent, anode upper and lower region, cathode upper and lower region is displayed in Fig 2b. Wherein, the DO in the initial wastewater was noted as 5.5 - 7 mg/L and in the anode upper and anode lower region, it was analysed as 1.71 ± 0.57 mg/L and 0.72 ± 0.27 mg/L respectively. Furthermore, since the cathodic compartment was equipped with the aerator DO conditions at the lower and upper regions were 6.26 ± 1 mg/L and 6.72 ± 0.52 mg/L respectively.

Additionally, the ORP of anodic and cathodic regions was estimated to analyse the favorable oxidative and reductive environment of the microcosms for pollutant degradation and subsequent electricity generation. The ORP of upper and lower of cathodic and anodic regions is depicted in Fig 3. The ORP values of upper and lower anodic region was obtained as -233.72 ± 26.68 mV and -229.43 ± 43.02 mV respectively. The values ensure the highly reductive environment in the anodic chamber. Besides, in the cathodic chamber average ORP values were found to be 37.28 ± 24.28 mV and 84.03 ± 58.80 mV for upper and lower region respectively. In the beginning of experiment, cathodic ORP was 6.2 mV for upper region and 33.3 mV for lower region; however, as the microcosm stabilized it became more positive. In the low DO conditions, usually ORP tends to be more negative as compared to high DO conditions. Hence, these values also corroborate with the fact that DO and ORP have a positive relationship. Moreover, studies revealed that azo dye reduction can only take place with ORP less than -50 mV.

Example-8: Chemical oxygen demand

After acclimatization period of 2 months, the sampling began. The CW-MFC was evaluated for COD removal and decolorization rate for approximately 270 days. The COD removal efficiency by anodic and cathodic compartments are shown in Fig 4. The initial COD concentration was 630 ± 29.48 mg/L, which was contribution of nutrients, co-substrate and MO present in the synthetic wastewater. The co-substrate (i.e. glucose) was the basic organic source/electron donor for electricity production and MO degradation. The COD removal rate in the anodic region up to 6 weeks was 60- 70 % which corresponds to the effluent value of 250 ± 20.6 mg/L. Nevertheless, after six weeks the COD removal increased to 85 %, the corresponding effluent concentration of 87 ± 17.24 mg/L. The gradual increase in COD removal can relates to incremental biofilm development as anaerobic microbes use carbon source as fuel for their growth and activity. It can also imply that most of the COD removal took place in anaerobic region may be due to highly reductive environment. The ORP conditions of less than -200 mV clearly assures the reductive environment in the anaerobic chamber as shown in Fig 3c. Also, continuous electron transfer from the anode to cathode would have accelerated metabolic growth of anaerobic bacteria and fast oxidation of substrate (i.e. glucose). Moreover, remaining 87 ± 17.24 mg/L was removed in the cathodic compartment, where COD decreased to 60 mg/L in early stages of operation and later to 20 mg/L.

Example-9: Azo dye decolorization and degradation

The decolourisation of MO at different regions of microcosm was determined with the help of MO standard curve as shown in Fig 5c. It was observed that 92.12 ± 2.62% decolourisation took place in the anodic region whereas remaining 3.30 ± 1.31 mg/L was carried out in the cathodic region. Moreover, the remaining MO concentration in the effluent was found to be 2.42 ± 1.33 mg/L as shown in Fig 5d. The major dye decolorization occurred in anodic region could be due to suitable environmental conditions such as availability of substrate and biomass, favorable redox potential. As the experiment was conducted for approximately 365 days, thus, adsorption cannot play significant impact in our system. Furthermore, for tracking the pathway of dye degradation, samples were collected from the upper anodic region and effluent at a retention time interval of 9 h and 27 h from the time of influent. Fig 5a depicts the UV-Vis spectra of influent, anodic and effluent sample which confirms the azo bond cleavage and further degradation of MO. For the influent wastewater, a strong absorbance band was observed at 464 nm, which is denotation of conjugated structure made by azo bond under the influence of electron donating dimethyl amino group. Additionally, another band was at 270 nm which relates to n to 7t* transition in aromatic rings. However, in the anodic sample, no bands were observed at 464 nm and 270 nm whereas, a new band appeared at 248 nm. The band appeared at 248 nm in anodic sample signifies that with the decolorization of dye, azo bond (-N=N-) cleavage occurred which lead to the formation of a sulphanilic acid, which was further confirmed with UV- Vis spectra of standard sulphanilic acid solution shown in Fig 5b. The degradation of dye observed in anodic effluent was due to the presence of electronegative sulphonyl group on the para position in MO which can extract the electrons from the -N=N- bond via resonance and convert it into electrophilic and reduced form. Hence, the mechanism behind reduction of azo bond was due to the consumption of electrons generated with the oxidation of biodegradable substrate catalysed by microbes.

As the anodic effluent reaches the aerobic region (i.e. cathode chamber) of the microcosm, the band observed from cathodic effluent at 248 nm were significantly decreased. The band observed was 10 times lower in absorbance than the anodic samples i.e. from ~ 3.35 to 0.36, Fig 5a. The significant decrease in absorbance from anodic to cathodic regions is also an indication of decrease or disappearance of aromatic rings in effluent. However, a sharp increase of sulphalinic acid in anodic region depicts accumulation of sulphanilic acid and further the decline of band in the cathodic region signifies its further degradation. The results are also in agreement with the general consensus that sulphalinic acid can only be degraded in the aerobic environment. Moreover, further confirmation of MO mineralization from anodic and cathodic samples were carried out with GC-MS.

The UV-Vis spectra confirmed the formation of sulphalinic acid from the anodic samples. However, the GC-MS analysis indicates the formation of N.N-dimethyl-p-henylenediamine (DMPD). DMPD was eluted at a retention time of approximately 17.29 min with m/z of 136, shown in Fig 6a. Since the boiling point of sulphalinic acid is more than the temperature limit of gas chromatography it was not detected in GC-MS analysis. Moreover, from MS fragmentations pattern, m/z peaks of characteristic fragment ion were observed at: 136, 108, 93, 88, 77, 60 and 18.

Since the anodic effluent flow down to the cathodic region, the total mineralisation of MO occurred in the cathodic region confirmed with GC-MS analysis, Fig 6b. Since the aeration at the cathode provided sufficient electron acceptor for degradation of azo dye, it was more efficient in the cathodic region. In the effluent samples no aromatic ring compound was detected, Fig 6b. The compounds detected in effluent samples were in the mineralised form such as, diethyl pyrocarbonate, 1 -Ethoxy-3 -methyl-2-butene, 1,2-Ethanediol, monoacetate, 2,3-Dimethylheptane, 2-Pentanone, 4-hydroxy-4-methyl, 1-Butylnonyl acetate, which was eluted at 3.060, 3.287, 3.753, 4.153, 7.613, 10.797, respectively. Mineralisation in the aerobic region can be due to the incorporation of oxygen atoms from O2 into the aromatic rings of intermediates before the fusion of ring, catalysed by oxidoreductase enzyme of both mono and di- oxygenases. Furthermore, when the anaerobic metabolites of azo dye are metabolized aerobically then the formation of less aromatic and polar compounds leads to the shift in the retention peak to the lower value. Similar results can be visualized for the present study through the GC-MS chromatograph Fig 6a and 6b.

Example-10: Electricity generation

The maximum current density and power density achieved was 442 mA/m³ and 97.68 mW/m³ respectively. The CW-MFC was characterized electrochemically by plotting a polarization curve as represented in Fig 7. The polarization study was conducted after achieving the steady state. In the initial phase of the experiment the external resistance of 2KQ was decided based on polarization graph. The polarization curve provides basic idea about the losses, which mainly affect the system performance such as less voltage output than the predicted thermodynamic ideal voltage. The observed losses were ohmic losses, activation losses, and mass transfer losses. The losses were happened during electrochemical reaction, mass transfer and charge transfer that occur in the anodic and cathodic regions of the microcosm. To reduce the cathodic ohmic losses, anode and cathode electrodes were placed closer to each other and hence the protons had less distance to travel. This is also in agreement with the microcosm designing in present study where protons had to travel just through the porous earthen membrane. Further, all the three losses viz. activation loss, ohmic loss and concentration loss can be understood with the polarization curve as shown in Fig 7. The activation losses are depicted by the sharp decrease in the voltage whereas ohmic loss is expressed by the slow declining nature of voltage (almost in linear pattern). Moreover, concentration losses are symbolized by rapid fall of voltage at high current density.

Furthermore, the voltage profile of the microcosm is shown in Fig 8. The highest voltage achieved in CW-MFC was 498 mV at 2000 Q resistance load. The higher voltage output may be due to the presence of MO which may acts as a redox mediator in bioelectrochemical systems. The presence of redox mediator would have also participated in an efficient electron transfer at anodic and further to cathodic region. The presence of dye also acts as an electron shuttle which can help electrogenic bacteria (EAB) to transfer electron to the electrode efficiently. Also, higher oxygen availability at cathode due to aeration may have increased the cell voltage. Additionally, it can be noticed from the voltage profile that after connecting resistance, there is increase in voltage and which further gradually decreases. The plausible reason can be high rate of breakdown of substrate in the initial 1-2 days which eventually slows down in later stage of microcosm working.

Example-11: Phyto-toxicity assessment

The phytotoxicity results of seeds revealed that influent (raw) wastewater inhibited the germination of Cicer arietinum, Triticum aestivum and Vigna radiata, when compared to the tap water. Wherein, root inhibition of 51.10%, 68.83%, and 15.13% were observed for C. arietinum, T. aestivum and V. radiata respectively. On contrary to that, increased seed germination (in both root and shoot length) was observed in the anodic and cathodic effluents as shown in Table 1. In comparison of anodic effluent to cathodic effluent root germination increased from 11.62% to 34.27%, 13.53% to 60.28%, and 30.72% to 69.70% for cases of C. arietinum, T. aestivum and V. radiata respectively. Additionally, it also signifies that toxicity level has decreased far more in the cathodic effluent as compared to the anodic effluent. Since the seeds germination were more in effluent samples than the anode upper samples. Similarly, increased shoot germination was also witnessed in all the grown seedlings in comparison to tap water. Moreover, the highest increased germination was noted in V. radiata may be due to its early seedling growth as compared to other seeds. The increased growth in anode upper and effluent wastewater clearly indicates the reduction of aromatic MO metabolites phyto-toxicity to the significant level.

Table 1. shows the Phyto-toxicity assessment with different seeds in the influent, anodic effluent, cathodic effluent and tap water (chosen as control).

Example-12: Microbial community analysis

The microbial community analysis of anodic and cathodic samples in comparison to the 5 inoculum were characterized by 16sr RNA sequencing. The inoculum for the microcosm was found to be more diverse with operational taxonomic units (OTUs) of 9403 in comparison to anodic and cathodic samples with OTUs of 6568 and 7304 respectively. The Shannon and Chao indices of inoculum was 5.84 and 1136 respectively. Whereas, the anodic and cathodic samples exhibited the Shannon and Chao indices of 5.71, 5.73, 683, 588 respectively. These 10 results suggest that cathodic and anodic samples exhibit low species diversity and richness as compared to the inoculum. Further the results also indicate that anodic and cathodic samples were enriched with selective microbial communitie. The most abundant microbial community in the anodic samples consisted of phylum Bacteroidetes (19.09%) followed by Proteobacteria (14.14%), Spirochaetes (11.80%), Chlorofelxi (7.72%), Firmicutes (7.43%), 15 Caldiserica (6.26%), Acidobacteria (5.39%), Euryarchaeota (5.10%), Patescibacteria (4.37%), Verrucomicrobia (4.08%), Elusimicrobia (2.18%), Actinobacteria (2.18%), Epsilonbacteraeota (1.16%) with other phylum of less than 1%. Whereas, cathodic community was found to be densely populated with phylum Proteobacteria (59.50%) succeeded by Bacteroidetes (12.23%), Acidobacteria (8.59%), Firmicutes (3.63%), Actinobacteria (2.14%), Verrucomicrobia (1.32%), Gemmatimonadetes (1.157%) and Planctomycetes (1.488%) as shown in Fig 9a. It can be observed from Fig 9a that some microbial community from inoculum disappeared in anodic and cathodic samples which indicates the microbial community were not tolerant to dye wastewater and may be influenced with the presence of conductive materials of electrodes and operation conditions of CW-MFC. sThe abundance of Proteobacteria, Bacteriodetes and Firmicutes in the anodic samples rather than cathodic samples is further confirmed by other studies on conventional anaerobic digestors and bioelectrochemical systems. Additionally, phylum Proteobacteria of the anodic samples were abundant with the class Deltaproteobacteria (8.45%), followed by Gammaproteobacteria (4.22%), and Alphaproteobacteria (1.45%) shown in Fig 9b. Wherein, Deltaproteobacteria were mostly dominant in the anodic samples of CW-MFC. In Deltaproteobacteria class presence of an exo-electrogenic species Geobacter (1.16%) was observed which are obligate anaerobic bacteria (i.e. prefers strictly anaerobic condition). These species can be responsible for extracellular electrons transfer during organic degradation and thus promotes electricity generation in microcosm. Additionally, the presence of genus Desulfovibrio (2.04%), Desulfobulbus (0.58%) from class Deltaproteobacteria also promotes electricity generation as it is capable of bidirectional extracellular electron transfer.

Contrarily, in cathodic sample, microbes from phylum Proteobacteria contain classes Gammaproteobacteria (36.03%) > Alphaproteobacteria (22.14%) > Deltaproteobacteria (1.32%) as shown in Fig 9c. The abundance of Gamma and Alpha Proteobacteria in cathodic samples as compared to anodic samples indicates tolerance of those microbes towards MO. These microbes were mainly related to aromatic degradation and azo reduction. Besides, the OUT abundance of genus Sphingomonas was 0.29% in anodic samples and 1.15% in cathodic samples which may be due to its involvement in aromatic ring degradation of MO in cathodic region. Furthermore, the presence of Comamonas species (0.33%) from phylum Proteobacteria in the cathodic samples could also be responsible for the degradation of aromatic amines.

In addition, the abundance of species Clostridium from phylum Firmicutes in the anodic sample was 7.43%, whereas in cathodic samples it was 3.63%. The microbes from phylum Firmicutes generally plays role in conversion of complex carbon to simpler molecules and further for electron transfer. Likewise, phylum Choloflexi was abundant in anodic samples is also responsible for converting carbohydrate into electricity. Besides, presence of phylum Planctomycetes in the cathodic region would have played role in oxygen reduction, similar to known concepts.

Furthermore, Inoculum consisted of 2.96% of phylum Acidobacteria but as it gets exposed to dye wastewater in the anodic chamber Acidobacteria increased to 5.39% and further in the cathodic chamber it reached 8.95% as shown in Fig 9d. The most abundant genus in cathodic region belong to Acidobacteria i.e. Solibacter (3.63%) species as well as other species of Acidithiobacillus, Thiomonas and Acidobacteria genera might be capable of degrading azo dyes. Moreover, some species of genus Xanthomonas, Acidithiobacillus and Thiomonas are capable of degrading sulphonated azo bonds and can functionalize as azo dye reductase. Additionally, some species of Desulf obulbus, Acidithiobacillus and Thiobacillus are also capable of degrading aniline via. benzoate degradation pathway.

Furthermore, for a better understanding a phylogenetic tree has been prepared for the anodic and cathodic microbial communities showing their involvement in different process (Fig 9e).

ADVANTAGES OF THE INVENTION

• The main advantage of the present invention is enhanced azo dye mineralization and detoxification in wastewater with the novel practical and scalable design of earthen membrane based two chambered constructed wetlands cum microbial fuel cell capable of simultaneous high electricity generation and highly efficient wastewater treatment.

• Use of low-cost earthen membrane in place of chemical commercial membrane in CW- MFC for treating wastewater and electricity to an enhanced level.

• The innovative designing of CW-MFC provides a low-cost, efficient and feasible arrangement of sequential anaerobic and aerobic treatment for wastewater for various types of pollutants including wastewaters containing azo dye.

• The innovative designing of CW-MFC provides low internal resistance and hence increased electricity production.

• Innovative design of earthen membrane based two chambered constructed wetlands cum microbial fuel cell resolved following challenges and equipped with following features:

Less oxygen diffusion from aerobic cathode chamber to anaerobic anode chamber;

Prevents the mixing of synthesized products (if any) at the cathode with anodic anaerobic chamber, thus minimize the losses and easier then recovery;

Prevents the mixing of anodic and cathodic solution which occurs in single chamber CW-MFC, thus enhance the electricity generation and treatment of wastewater containing different pollutants;

Resolve the oxygen/air diffusion issues in anode due to intermittent aeration at cathode, thus enhancing the electricity generation and treatment of wastewater containing different pollutants;

Mimics the conditions of chemical membrane based MFC at low-cost without using costly chemical membrane;

Provides the arrangement of wetting the outer surface of anode compartment for improving the electricity generation and wastewater treatment;

Lowers the distance between anode and cathode for enhancing electricity generation by reducing electrochemical losses and treatment of wastewater;

Passive gravitation flow of wastewater from anaerobic chamber to the aerobic chamber and up to the outlet;

Adjustable water level arrangement at aerobic cathode chamber for diffusion of more atmospheric air or any other purpose passively for enhancement of electricity generation and wastewater treatment.

Claims

We claim:

1. An earthen membrane based two chambered constructed wetland cum microbial fuel cell [CW-MFC] (Z) for treatment and detoxification of wastewater containing azo dye comprising:

(i) an anaerobic earthen pot (a) acting as anodic chamber and the earthen pot layer acting as proton exchange and oxygen/gas diffusion inhibiting membrane;

(ii) an anodic chamber cover (b) for restricting diffusion of atmospheric air inside the anaerobic earthen pot (a);

(iii) a high-density polyethylene liner (HDPE) (c) covering bottom part of the earthen pot (a);

(iv) a container (h) acting as cathodic chamber;

(v) anode and cathode charge collectors/exchangers (d, e) inserted in anodic and cathodic chambers respectively;

(vi) an aerator ring (f) situated below the earthen pot (a); and

(vii) a resistance/load (g) connected between anode and cathode;

(viii) an outlet (i) connected to an adjustable siphoned pipe (j).

2. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the container (h) is made of plastic material/any similar inert material.

3. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the container (h) and the earthen pot (b) contain graphite granules of size 5-8 mm and wherein the graphite granules act as cathode and anode respectively.

4. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the working volume of anodic chamber (a) is half the working volume of cathodic chamber (h).

5. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the charge collectors (d, e) are connected with an insulated electric wire, wherein the insulated electric wire is preferably copper/titanium wire.

22

6. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the resistance (g) is 1000 - 2000 Q.

7. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the aerator ring (f) is connected to an aerator.

8. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the HDPE liner (c) prevents the aerator’s air/oxygen to diffuse inside the earthen pot (a).

9. The constructed wetland cum microbial fuel cell (Z) as claimed in claim 1, wherein the constructed wetland cum microbial fuel cell (Z) provides nearly complete (90-100%) dye mineralization and detoxification with a removal efficiency of 94.04 ± 2.87% COD and 94.22 ± 1.33% azo dye.

10. The constructed wetland cum microbial fuel cell as claimed in claim, 1, wherein the azo dye is methyl orange.

11. The constructed wetland cum microbial fuel cell as claimed in claim 1, wherein the microbial fuel cell simultaneously generates electricity of 442 mA/m³ and 97.68 mW/m³ as current density and power density respectively.

12. The constructed wetland cum microbial fuel cell as claimed in claim 1, wherein the pipe (j) acts as adjustable siphon tube permitting the cathodic chamber (h) to be filled partly or partly empty, wherein the empty portion of the said cathodic chamber (h) allows more diffusion of oxygen leading to enhancement in electricity generation and aerobic treatment of wastewater.

13. A process for the treatment of wastewater containing azo dye using the constructed wetland cum microbial fuel cell as claimed in claim 1, comprising: a. entering the wastewater into lower part of the earthen pot (a) b. flowing wastewater passively in up flow direction as it reaches the upper portion of earthen pot (a) c. oozing the water out in the cathodic chamber (h) through walls of the earthen pot (a); d. flowing water out from the outlet (i).

The process as claimed in claim 13, wherein the water collected from the outlet (j) is nearly complete (90-100%) detoxified.

The process as claimed in claim 13, wherein the water collected from the outlet (j) shows enhanced growth rate of Vigna radiata, Triticum aestivum, Cicer arietinum seeds with 69.70 ± 0.30%, 60.28 ± 0.70 % and 34.27 ± 0.67% growth rate, respectively.