Photosynthetic Synergism for Sustained Power Production with

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Photosynthetic Synergism for Sustained Power Production with Microalgae and Photobacteria in a Biophotovoltaic Cell Rashmi Chandra, J. Shanthi Sravan, Manupati Hemalatha, Sai Kishore Butti, and S. Venkata Mohan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.7b00486 • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Photosynthetic Synergism for Sustained Power Production with Microalgae and Photobacteria in a Biophotovoltaic Cell Rashmi Chandra, J. Shanthi Sravan, Manupati Hemalatha, Sai Kishore Butti, S.Venkata Mohan* Bioengineering and Environmental Sciences Lab (BEES), EEFF, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India *E-mail: [email protected]; Tel: 0091- 040-27191765

Abstract Synergistic interactions between biocatalysts were evaluated for power production taking advantage of the in situ oxygen production by the photosynthetic machinery. This study documents the synergetic interaction between oxygenic photosynthesis (microalgae; cathode) and anoxygenic photosynthesis (photosynthetic bacteria; anode) towards bioenergy and biomass generation apart from wastewater treatment using a dual chambered biophotovoltaic cell. Microalgae as the biocathode negate the requirement of an external terminal electron acceptor as they can be efficient in situ oxygenators that facilitate the oxygen reduction reactions. Experiments were performed in a fed batch mode of operation using designed synthetic wastewater (DSW) at variable organic loading (OL) rates in the anodic chamber and domestic sewage as feed in the cathodic chamber with a retention time of 72 h. Operation at OL3 (1500 mg/L) showed maximum power output (0.7 V; 103 mW/m2). Anodic chamber showed a maximum substrate (COD) degradation efficiency of 81% while cathode showed a constant COD removal efficiency of 90%. Maximum microalgal lipid productivity of 20% was observed at OL4 and OL5. Biophotovoltaic fuel cell configuration (BFC) provides multiple benefits of harnessing bioelectricity and biofuels apart from wastewater treatment along with CO2 sequestration.

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Keywords: Microalgae; Bioelectrochemical systems (BES); Biomass; CO2 sequestration; Microbial fuel cell; Wastewater.

1. Introduction Photosynthesis has been the key mechanism for the origin and sustenance of life on the Earth. Photosynthesis (plants, algae and photosynthetic bacteria) is a congregation of multiple light induced biochemical reactions which primarily involves the reduction of carbon dioxide to drive photoproducts with simultaneous oxygen evolution.1 Similarly, during the anoxygenic photosynthesis organic/inorganic compounds act majorly as the terminal acceptors through a defined electron transport mechanism.1-4 The diverse photosynthetic activities function in the environment either individually or in synergy. Microalgae play an important role as primary producers in the global carbon and nitrogen cycle and have been the focus of research especially with respect to their exploitability for the production of renewable energy, chemicals and CO2 sequestration.3-9 They operate under oxygenic photosynthesis which encompasses two reaction centers viz., photosystem I (PSI; P700) and photosystem II (PSII; P680) working in succession. In the presence of sunlight, PSII feeds electrons to PSI through intermediate electron shuttlers. The net reaction is the transfer of electrons from a water molecule to NADP+ to produce energy rich ATP and NADPH.10 Oxygen evolution complex (OEC) in PSII catalyzes the water catalysis to evolve O2 which is homologous to plants, algae and cyanobacteria. Microalgae sequester CO2 towards biomass production.11,12 Integration of photoautotrophic function with algae in BES has multiple benefits in providing dissolved oxygen (DO), CO2 sequestration, nutrient removal, biomass production, etc.13-18

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Whereas, in photobacteria (photo-autotrophic/-heterotrophic) the photosynthetic machinery involves, the generation of electron (e-) from molecules other than H2O involving PSII (P870). The final electron acceptors are sulphates, metals, etc., other than O2 to produce energy. The eare driven in a cyclic manner in the PSII, producing a proton (H+) gradient in order to compensate for energy (for reduction of NAD+) by an external H+ or e- donor (e.g., H2S or an organic acid), acting as a final e- sink.19 With microbial fuel cells (MFC) gaining significance for their versatility and application feasibility,20 a strategy can be formulated to integrate both the photosynthetic machinery to produce multiple products viz., bioenergy, biofuels, chemicals, etc., associated with waste remediation and CO2 sequestration.21-24 MFC are otherwise called as microbial electrochemical system or bioelectrochemical system (BES) operate in different configurations and operational variations.25-29 With a strategy to mimic nature and integrate the anoxygenic and oxygenic photosynthesis has the potential to provide multiple advantages and drive towards selfsustainability.2-6, 30 This communication tries to exploit the synergy between two photosynthetic mechanisms based on microalgae and photobacterial, functions separated by a membrane in a Biophotovoltaic fuel cell (BFC) that can eventually produce spectrum of photo-bio-based products including bioenergy. BFC can be advantageously exploited as a platform technology for sustainable production of power with innovative integration of photosynthetic microalgae and photobacteria. Photobacteria as anodic biocatalyst can perform anoxygenic photosynthesis (without O2 generation) at anode.16,26 Oxygenic photosynthesis mechanism (with O2 generation) at cathode in BES can replace the energy intensive mechanical aeration process or toxic terminal electron acceptor required for biofuel cell functioning.11,14,16 The photo-diversity between photobacteria and 3


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microalgae can create a win-win situation, by synergistically influencing each other’s physiology and metabolism. These synergistic benefits are both environment and ecofriendly that enable the establishment of a synergistic self-sustained platform with economic feasibility. 2. Experimental Methodology 2.1. Biophotovoltaic configuration A dual chambered biophotovoltaic fuel cell configuration (BFC) was constructed by joining two tubular chambers (poly methyl methacrylate; PMMA) of similar dimensions (total/working volume-0.40/0.35 l) (Fig 1). Graphite sheets (5 x 5 cm; 10 mm thick) with a surface area of 52 cm2 were used as electrodes in both anode and cathode chambers. The electrodes were pretreated in 1% ammonium chloride (NH4Cl) solution by sonicating (10 min) and transferred to water bath for treatment (1 h at 700C) for enhancing their conductivity. A proton exchange membrane (PEM; Nafion117, Sigma-Aldrich; 5 x 5 cm) was fixed between the hollow tubes (5 cm diameter) connecting both the chambers with rubber gaskets to provide water seal between the chambers. PEM was sequentially treated in deionized water (DIW) followed by H2O2 (30 % V/V) solution, DIW, 0.5 M H2SO4, and then finally in DIW (1 h each step). Copper wires were sealed with an epoxy sealant was used to maintain contact with the electrodes (Fig. 1). Fig. 1 2.2. Photosynthetic Biocatalyst Photosynthetic bacteria and mixed microalgae were used as biocatalysts in anode and cathode chambers respectively. The synergetic association and influence on bio-electrogenesis and other metabolic processes were evaluated.

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2.2.1. Anodic biocatalyst Photosynthetic bacteria acquired from an operating laboratory scale photosynthetic fuel cell (PhFC) were used as parent inoculum. Inoculum was enriched [salt succinate broth; KH2PO4 0.33 g, MgSO4.7H2O - 0.33 g, NaCl - 0.33 g, NH4Cl - 0.5 g, CaCl2.2H2O- 0.05 g, sodium succinate- 1.0 g, yeast extract- 0.02 g, trace metal solution (ZnSO4.7H2O - 10 mg, MnCl2.4H2O3 mg; H3BO3 - 30 mg; CoCl2.6H2O- 20 mg; CuCl2.2H2O - 1 mg; NiCl2.6H2O - 2 mg; Na2MoO43 mg; distilled H2O- 1.0 L; pH 3-4)- 1 mL and FeSO4.7 H2O (0.02%) solution- 0.5 mL, distilled water- 1 L] under anaerobic conditions [4000 lux; 30◦C] for 7 days to reach the logarithmic growth phase.28 The resulting culture was inoculated to the anode chamber.

2.2.2. Cathodic biocatalyst Microalgal consortia were sampled from Pedda Cheruvu lake (Nacharam).31 Prior to inoculation, the culture was washed twice with tap water to remove debris. The resulted culture was inoculated in rectangular tank exposed to sunlight with domestic sewage (DS) and was used as biocatalyst in the cathodic chamber (10% of the volume).

2.3. Anolyte and catholyte composition Designed synthetic wastewater (DSW), with varying organic load (OL) (Glucose - (500, 1000, 1500, 2000 and 3000 mg/L)) was used as anolyte.28 Domestic sewage (DS; COD – 560 mg/L, VFA – 165 mg/L, BOD – 320 mg/L, TDS – 820 mg/L, suspended solids (SS) – 360 mg/L, total alkalinity – 140 mg/L, chlorides – 175 mg/L, nitrates – 115 mg/L, pH – 7.8) was used as catholyte which acts as nutrient and carbon source for mixotrophic algal growth.

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2.4. Operation details The startup of the BFC was initiated by inoculating the anodic chamber with the photosynthetic bacteria (10% of the reactor volume) enriched in DSW (270 mL) at an OL of 500 mg/L at pH 7.0. The cathode chamber was inoculated with microalgae (10% of reactor working volume) enriched in domestic sewage (270 mL) at pH 7.8 with organic load (OL) of 560 mg/L. Before start up, anode chamber was sealed and sparged with nitrogen gas (99.9%) for 5 min to maintain anoxygenic photosynthetic activity, whereas cathode chamber was exposed to ambient air to allow CO2 biosequestration. Both the anode and cathode chambers were operated with DSW under photo-mixotrophic conditions (3000±200 Lux) for 7 days of retention time. Anode chamber was sparged with N2 gas for 5 min after every feeding event to ensure anaerobic microenvironment. BFCwas operated in fed batch mode with defined time bound phases (settling-30 min, decanting-10 min, feeding-10 min). Before feeding, respective biocatalysts in both chambers were allowed to settle (30 min) and exhausted wastewater was replaced with fresh wastewater and the settled biomass was used in the next cycle operation. Mixing of anolyte and catholyte was ensured with peristaltic pump. Drop in power output was considered as an indicator for the feed replacement. The performance of the system was studied at variable loads of DSW (OL1-500; OL2- 1000; OL3-1500; OL4- 2000 and OL5-3000 mg COD/L) with 72 h of cycle (retention time) for six cycles each at ambient temperature (28±2◦C).

2.5. Process monitoring The photo-electrogenic output of BFC was monitored in terms of open circuit voltage (OCV) and current (i). Polarization plots were constructed with the function of current density (CD) against potential (V) and power density (PD) recorded at variable resistances (30 to 0.05 kΩ). Anodic

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potential (Ewe), Cathodic potential (Ece) and cell potentials were measured across variable resistances (30 to 0.05 kΩ). Potentiostat-galvanostat system (Autolab-PGSTAT12, Ecochemie) was used to record voltammograms by applying a potential ramp over the potential range (+0.5 to -0.5 V) considering anode as working electrode (WE) and cathode as counter electrode (CE) against reference electrode (RE; Ag/AgCl (3.5 M KCl). Chemical oxygen demand (COD) (closed refluxing method), volatile fatty acids (VFA), nitrates, phosphates and pH were periodically monitored using standard methods.32 Production of H2 was estimated using a gas sensor (ATMI GmBH Inc.). Dissolved oxygen (DO) in cathode chamber was measured by using DO sensor (HACH). At the end of each cycle, biomass of both the chambers were estimated in terms of dry cell weight (mg/L) and the total bacteriochlorophyll (Bchl) of the anodic biomass was estimated by colorimetric procedure at the end of each cycle.33-35 The experimental data presented and discussed was an average of three independent analysis.

3. Results and discussion 3.1. Synergy: Photosynthetic bacteria and Microalgae 3.1.1. Photo-bio-electricity BFC showed a marked variation on the bioelectrogenic performance with the synergistic function of anolyte organic load and catholyte dissolved oxygen. The photo-bioelectrogenic profiles showed an incremental trend with increase in anolyte OL, where the OCV increased from OL1 (500 mg COD/L; 210 mV/2.07 mA) to OL2 operation (1000 mg COD/L; 450 mV/3.47 mA) (Fig. 2a). Maximum OCV of 701 mV and current of 5.98 mA was observed with OL3 operation (1500 mg COD/L). At higher OL, decremental trend was noticed [OL4 (2000 mg COD/L; 556 mV/4.43 mA); OL5 operation (3000 mg COD/L; 300 mV/2.90 mA)]. The optimal

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OL enabled the progress of the bacterial metabolism which resulted in good bioelectrogenic activity at OL3, whereas, at higher OL the biomass was observed to decrease which might be due to the inhibition caused by the load-shock. Dissolved oxygen (DO) levels in the cathode chamber also influenced the bioelectricity generation, where the maximum DO (3.1±0.4 mg/L) recorded at OL3 operation correlated with the maximum power production. The power density profile also showed the similar trend with maximum PD at OL3 (61 mW/m2) as the higher DO levels enable efficient electron transfer towards the cathode, thereby increasing the power output (Fig. 2b). Fig. 2 Polarization profiles were recorded during the stable phase of BCF operation for all the experimental conditions (Fig. 2c). The power production efficiency, internal losses and cell design point (CDP) were evaluated through the polarisation profiles. The maximum power density (PD) was observed in the OL3 operation (103 mW/m2; 200 Ω). The PD increased from OL1 (58.5 mW/m2; 200 Ω) to OL2 (71.9 mW/m2; 200 Ω) with maximum PD at OL3 and later the BCF depicted a decremental trend in power density with OL4 (61 mW/m2; 200 Ω) and OL5 (17 mW/m2; 200 Ω). The PD profile showed concurrence with OCV/current and DO profiles. The CDP of 200 Ω also portrays the effective biocatalyst enrichment and electron discharge phenomena in the anode influenced by the in situ oxygen reduction at the cathode. The availability of in situ oxygen and the effective biocatalyst activity have influenced the internal losses or over potentials of the fuel cell.36 The readily available in situ oxygen has reduced the mass transfer losses and the activation losses in the OL3 operation as compared to the other operational conditions, where the anolyte substrate removal was observed to be maximum along with catholyte DO. The steady decrease in the voltages at higher external loads in OL3 signifies

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lower electrochemical losses compared to the sudden decrement in voltages as observed in the OL1, OL4 and OL5 operations. The subsequent decrement of CDP might be due to rapid discharge of electrons to the cathode. Operation with OL3 has resulted in maximum photobioelectrogenic activity due to the syntrophic interaction of protons (H+) and electrons (e-) generated due to effective anode biocatalytic activity and in situ oxygen generation due to microalgae in the cathode chamber of BFC.

3.1.2. Cathode - Dissolved Oxygen (DO) Oxygen liberated by oxygenic microalgal photosynthesis in the cathode chamber functions as a terminal electron acceptor (TEA) and influenced the photo-bioelectrogenic output in the BFC. The H+ and e- released during the substrate oxidation at the anode get reduced at cathode though oxygen reduction reactions.11,16;37-39 The catholyte DO varied between 2.5 to 3.5 mg/L during operation depending on the microalgae growth. System operation at OL1, OL2 and OL3 maintained DO levels at 3.0±0.3 mg/L. However, at OL4 and OL5 operation, the DO levels have shown a decremented value (2.5±0.4 mg/L) correlating to the low algal biomass due to the acidic system redox conditions developed and the limited oxygen reduction reactions. DO concentration observed at all OLs in cathode chamber is accredited due to the oxygenic photosynthetic activity of microalgae and also depending on the anodic substrate oxidation.

3.1.3. Cathode- Lipids Microalgal lipid production depends on light energy, pH, organic/inorganic nutrients and mode of cultivation.21 The system pH affects the growth kinetics of microalgae and lipid production.40 Total lipids varied between 15 to 20% of dry cell weight based on the organic load (OL1-15%;

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OL2-16%; OL3-18%; OL4-20%; OL5-20%) (Fig. 3a). The improved substrate oxidation at the anode resulted in the transfer of higher H+ concentration to cathode which resembled a redox stress on microalgae influencing the lipid production. The migration of protons (H+) was evident from the drop in pH from 7.8 to 7.1. The pH 7 provides an optimal microenvironment for the lipid accumulation in microalgae.9 The higher availability of e- and H+ at the cathode and the pH influence the membrane bound lipid productivity that are developed to maintain the ionic gradient and optimum membrane transport potential in a microalgal cell.

3.1.4. Anode-Biohydrogen and Carboxylates Photosynthetic bacteria housed in the anode chamber through anoxygenic photo-heterotrophic mode of nutrition utilize glucose as the carbon source and generates H+ and e-. The anode functions as an initial electron acceptor enabling the power generation. During operation under non-sterile conditions, the acidogenic bacteria are observed to manifest in syntrophy with the photobacteria. The presence of acidogenic bacteria might be source of VFA production at higher OLs (OL4 and OL5). Cumulative H2 production observed at OL4 and OL5 might be attributed to the reduction of excessive H+ and e- that are generated during the substrate oxidation. The reduction in power production during the operation of OL4 and OL5 correlates well with the H2 production values. The non-availability of redox equivalents at electrodes resulted in a potential drop associated with a further decrement in photo-bioelectrogenic activity.36,41 Due to this consumption, BFC did not showed H2 production till OL3 operation, but at higher OLs, cumulative H2 production was observed, which depicts the shift in biocatalyst activity from electrogenesis to H2 production (Fig. 3b). H2 production during fuel cell operation has an effect

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on power output and substrate degradation which involves hydrogenase enzyme activity towards H2 production and thereby, resulting in lower power production.41, 42 The functional role of photosynthetic bacteria in synergy with fermentative bacteria was observed in anode chamber resulting in glucose reduction as well as for VFA production along with H2.43 VFA is also considered as a good substrate for photosynthetic bacteria for its growth.36,44 During initial phase of cycle operation (24 to 48 h), VFA concentration was high followed by decrement at later phase (72 h) (Fig. 3c), attributed to its consumption. A gradual increment in VFA production was observed till OL3 followed by a rapid increment at higher OLs. Final VFA concentration was low at OL1 (40 mg/L) followed by an increment (OL2, 271 mg/L; OL3, 290 mg/L; OL4, 501 mg/L; OL5, 912 mg/L). Increasing trend in the final VFA concentration is associated with a decrement in pH. VFA accumulation was relatively higher with OL4 and OL5 as compared to OL1, OL2 and OL3. Fig. 3 3.2. Anoxygenic photosynthesis enabling power production Photo-heterotrophic bacteria, in the anode chamber, rely on light as the energy source and organic compounds as carbon source. It is evident from the BFC operation, that there exists a correlation between biomass growth, substrate utilisation and power output. The biomass growth and the bacteriochlorophyll increment were key sources for power generation. OL has shown a correlative trend with the power output. Biomass concentration during the start-up phase was 1.2 g/L. A successive increment in biomass growth was noticed till OL3 (OL1, 1.6 g/L; OL2, 1.8 g/L; OL3, 1.8 g/L) operation with a marginal decrement at OL4/OL5 (1.7 mg/L) (Fig. 4a). Availability of carbon source enhances the anolyte biomass growth resulting in improvement of the substrate metabolism and electrogenic activity. Decrement in the biomass concentration at

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OL4 and OL5 can be attributed to the pH drop due to excessive VFA accumulation at high loading rates. In accordance with biomass growth, the increment in bacteriochlorophyll was observed till OL3 (OL1, 0.5 µg/mg; OL2, 0.54 µg/mg; OL3, 0.68 µg/mg) followed by decrement at high OL (OL4, 0.5 µg/mg; OL5, 0.31 µg/mg) (Fig 4a). The decrement trend can be ascribed to the pH drop in the anode chamber and this acidic redox condition triggers the pheophytinization (demetalization) which hinders the bacterial photosynthetic activity.35,36 Treatment efficiency of BFC was evaluated by measuring COD concentration in anode chamber. At OL1, maximum COD removal efficiency of 81% was observed followed by OL2 (79%), OL3 (72%), OL4 (42%) and OL5 (20%) respectively (Fig. 4b). When the system was subjected to higher OL, the COD removal decreased due to the prevailing excess carbon and redox conditions (inhibition caused due to load-shock). The observed COD removal is in good agreement with the photo-bioelectrogenic activity. Initial system pH was set at 7.8 which showed a decrement in the due course of time towards the acidic range (Fig. 4c). As the OL increased (OL4 and OL5), fermentation end products were formed at a higher rate resulting in a sharp drop in pH towards acidic range. Shifts in redox microenvironments from basic to acidic with increasing OL developed unfavorable conditions for biomass growth and electrogenic activity. Drop in pH at anode affects the bacterial photosynthetic activity resulting in low power output.28 Fig. 4

3.3. Oxygenic photosynthesis assisting the cathodic reactions Microalgal growth and O2 evolution are the two factors that governs the synergistic power output in the designed fuel cell system. DO levels observed at different OLs correlated well with the biomass growth and power production. A progressive increment in biomass concentration was

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observed till OL3 operation (OL1, 1.2 g/L; OL2, 1.4 g/L; OL3, 1.6 g/L) (Fig. 5a). A decrement in biomass concentration was observed at higher OL (OL4, 1.2 g/L; OL5: 0.9 g/L) which can be attributed to the higher H+ mobility towards the cathode from the anode chamber as a result of higher substrate degradation. The initial pH of the catholyte was 7.85 which gradually reduced to 7.1 at the end of the cycle (72 h) (Fig 5b). The lowering pH influenced the marginal decrement in the biomass in the cathodic chamber. The sustainable advantage of microalgal cultivation is its mixotrophic operation enabling nutrient removal along with CO2 biosequestration (data not provided). Cathode chamber also depicted a good COD removal efficiency of 90%. Apart from organic carbon the removal of nutrients viz., phosphates and nitrates is an important aspect in wastewater treatment. Cathodic chamber also showed efficient nutrient removal within 72 h (Fig 5c,d). Phosphates and nitrates are utilized for growth by microalgae during their cell metabolic activities. Phosphorus mainly functions in production of phospholipids, ATP and nucleic acids while nitrates provide the essential nitrogen for the synthesis of proteins and nucleic acids.45,46 Fig. 5 3.4. Synergy effect on Photo-bioelectrochemical process 3.4.1. Half Cell Potential 3.4.1.1. Anode potential The potential differences developed between the anode (WE) and cathode (CE) represent total cell potential. The total cell potential of BFC depends on individual half-cell potentials of anode and cathode.The potential difference between anode and reference electrode (vs Ag/AgCl (3.5 M KCl)) is termed as anode potential with external resistance (30–0.05 kΩ) which helps in determining the biocatalyst interaction with anode. Anode potential was maximum at OL3 (-244 mV) followed by OL4 (-214 mV), OL2 (-187 mV), OL5 (-169 mV) and OL1 (-84 mV)

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operations (Fig. 6). An increase in the anode potential can be attributed to the reduced electrogenic activity due to the shock load. Lower anode potential creates more reduced microenvironment that favors higher electron transfer from the anolyte to anode aiding in improved bioelectrogenic activity.28 On the contrary, decrease in anode potential at higher OL relates to the non availability of electrons to the anode wherein, in this case the major proportion of electrons might have consumed towards the production of H2.

3.4.1.2. Cathode potential The potential difference between cathode and reference electrode (vs. Ag/AgCl (3.5M KCl)) represents cathode potential that determines the TEA (oxygen) reduction reactions.11 Cathode potential is an index of the reaction between the TEA and the redox equivalents and is usually positive due to the accumulation of H+ gradient on cathode. Cathode potential increased from OL1 to OL3 (OL1, 62 mV; OL2, 90 mV; OL3, 112 mV) operation, due to the neutralization of more TEA with DO levels (Fig. 6). Thereafter, a decrement in cathode potential was observed at OL4 (98 mV) and OL5 (69 mV) operation due to the lower biomass and DO affecting oxygen reduction reactions. Enhancing the oxygenic photosynthesis at cathode will contribute towards more TEA availability and thus can increase the bioelectrogenic activity. High external resistance limits the electron delivery to the cathode, while, at lower external resistance, the electron delivery to the cathode is limited by kinetic and/or mass transfer (or internal resistance). Fig. 6 3.4.2. Photo-Bio-electroanalysis Voltammogram profiles visualized significant variation in the redox currents (oxidation currents (OC) and reduction currents (RC)) with the function of different organic loads at a scan rate of 1

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mV/S (Fig. 7a). The RC were observed to increase till OL3 operation (OC: 0.10±0.19 mA; RC: 0.109±0.12 mA) followed by a decrement at higher OL (OC: 0.03±0.14 mA; RC: 0.09±0.08 mA). OC were observed to increase significantly till OL3 followed by slight decrement at OL4 and rapid decrement at OL5. The higher OC suggest the enhanced biomass growth and efficient substrate oxidation at (OL1 – OL 3) and the decrement observed at high OL was attributed to the low biomass concentration and substrate oxidation. On the contrary, RC showed minor variations at all the OL (except OL5) might be due to the good oxygen reduction reactions of at cathode. Higher reduction current at OL5 represents the reducing environment representing favorable condition for the H2 production.38,46 The observed higher redox currents at OL3 are in correlation with other parameters viz., substrate degradation efficiency, electrogenic activity, biomass growth and lipid productivity. Capacitance was also observed to increase from OL1 to OL3 (OL1, 0.556 F; OL2, 0.772 F; OL3, 0.974 F) followed by a decrement at OL4 (0.887 F) and OL5 (0.545 F), which enumerates the electron holding capacity in progressive increment of power density from OL1 to OL3 (OL1: 58.50 mW/m2, OL2: 71.95 mW/m2, OL3: 103 mW/m2) and decrement at OL4 (61.20m W/m2) and OL5 (17 mW/m2). Higher capacitance and energy conversion at OL1, OL2 and OL3 associates with the higher photo-bio-electrogenic activity resulted by the collective function of anodic and cathodic biocatalysts during BFC operation.

3.4.2.1. Derivative voltammetry (DCV) The first derivative of CV (DCV) elucidates the functional involvement of redox mediators and aids in detection of EET (extracellular electron transfer) that corresponds to the redox shuttlers. It also facilitates the interpretation of the rate of change in voltammetric current (i) with respect

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to the electrode potential E (di/dE). DCV analysis showed various quasi reversible peaks corresponding to the involvement of mediators in electron transfer (Fig. 7b). At OL1, two peaks at -0.111 V and 0.064 V were noticed corresponding to the involvement of Fe-S proteins and cytochrome-bC1, respectively. Fe-S proteins and Cytochrome-bC1 complex are component of electron transport chain which facilitates the transfer of e- in electron transport chain. At OL2 operation, two peaks with a peak potential of 0.159 V and 0.029 V corresponding to the involvement of cytochrome-bC1 and quinine, respectively was observed. OL3 operation also depicted two peaks at peak potential of 0.041 V and 0.206 V corresponding to the involvement of quinone and cytochrome-C1, respectively. However, operation at OL4 depicted a single peak with a potential of 0.017 V corresponding to the involvement of crotonyl-CoA as mediator during the redox reactions. Operation at OL5 depicted five peaks with a potential of -0.1 V, 0.135 V, -0.041 V, 0.135 V and -0.159 V corresponding to the involvement of Fe-S proteins, quinone, cytochrome-bC1 and Fe-S proteins, respectively. Quinones are the electron acceptors associated with photosynthetic processes viz., plastoquinone, phylloquinone, etc. The redox shuttlers detected during DCV analysis imply the efficient biocatalytic activity and redox reactions aiding in bioelectrogenic activity with the influence of the catholytic parameters.

3.4.2.2. Electrocatalytic kinetics Tafel plots provide a clear understanding of the electrochemical losses present in the system. Tafel slopes (Oxidation slope-βa, Reduction slope-βc) and polarization resistance (Rp) is inversely proportional to the electro-catalytic activity of the biocatalyst.16 A lower slope indicates higher electro-catalytic activity and electron transfer efficiency. The same can be related in case of polarization resistance (RP) as well. Reduction slopes were comparatively

16


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Energy & Fuels

lower than the oxidation slopes indicating the occurrence of higher reduction reactions in the BFC system (Table 1). Initially, oxidative slope (βa) was observed to be 1.163 V/dec at OL1 which showed a decrement till OL3 (0.743 V/dec) operation. Later, an increment was observed at high OL (OL4: 0.861 V/dec, OL5:1.389 V/dec) (Fig. 7c). The decrement in slope till OL3 indicates the requirement of low activation energy for oxidation reactions which is also in accordance with the observed higher bio-electrogenic activity as well as substrate degradation efficiency. The increment in slope at high OL is attributed to the requirement of higher activation energy to carry out the oxidation reactions, which is in correlation with the observed low bioelectrogenic activity and substrate degradation. However, reduction slopes (βc) were comparatively lower and observed to vary at all the OL studied. Initially, βc was 0.098 V/dec at OL1, followed by an increment at OL2 (0.133 V/dec). Thereafter, a decrement was observed till OL4 operation (0.101 V/dec) followed by an increment at higher OL (OL5: 0.115 V/dec) (Fig. 7d). The decrement in reduction slope till OL4 is attributed to the efficient oxygen reduction reactions occurring at the cathode. This is in agreement with the observed DO levels due to the efficient activity of the cathodic biocatalyst (microalgae) with in situ O2 generation as TEA. Lower βc than βa in BFC indicates higher reduction reactions compared to the corresponding substrate oxidation (low oxidation of substrate). Polarization resistance refers to the optimum resistance required for electron transfer from the biocatalyst to the electrode surface. RP was observed to decrease till OL3 operation (OL1: 3.154 Ω, OL2: 3.01 Ω, OL3: 2 Ω) followed by an increment at higher OL (OL4: 2.058 Ω, OL5: 3.921 Ω). This trend is in correlation to the high bioelectrogenic activity till OL3 operation due to less resistance for electron transfer towards electrode surface. However, at high OL, the generated redox equivalents were consumed in H2 production due to high substrate oxidation and charge

17


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Page 18 of 36

deposition. Photo-bioelectroanalysis depicted efficient synergistic biocatalytic activity and redox reactions between photosynthetic bacteria and microalgae, that enabled understanding the process and enhancing the power output during BFC operation. Fig. 7 4. Conclusions Hybrid biophotovoltaic fuel cell (BFC) operation showed four distinct inferences i) synergistic interaction of photosynthetic bacteria and microalgae improved the power output (ii) substitution of microalgae at cathode for sustainable oxygenic photosynthesis is a sustainable replacement strategy for energy intensive mechanical aeration (iii) multiple photo-products synthesis by microalgae and photobacteria also facilitating CO2 biosequestration (iv) waste remediation and bioconversion. Anoxygenic photosynthesis at anode provides a feasible microenvironment for oxidation of organic pollutants and oxygenic photosynthesis at cathode provides a reducing environment (due to presence of O2). This strategy offers a sustainable strategy with multiple benefits viz., electricity generation, CO2 reduction, lipid production, wastewater treatment and the insitu generation of O2 as TEA at low cost.

Acknowledgments The authors thank the Director, CSIR-IICT for the encouragement in carrying out this work. Funding

from

Department

of

Biotechnology

(DBT),

Government

of

India

(BT/HRD/NBA/34/01/2012), Department of Science and Technology (DST) (DST/IMRCO/New INDIGO/Bio-e-MAT/2014/(G/ii)) and CSIR-XII Task Force Projects [BioEn (CSC-0116); SETCA (CSC-0113] are gratefully acknowledged. SKB acknowledges University Grants Commission (UGC) for providing research fellowship.

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References

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9. Venkata Mohan, S.; Prathima Devi, M. CO2 supplementation to domestic wastewater enhances microalgae lipid accumulation under mixotrophic microenvironment: Effect of sparging period and interval. Bioresour. Technol. 2012, 112, 116-123. 10. Taiz, L.; Zeiger, E.; Plant Physiology. 2010, Sinauer Associates. 11. Venkata Mohan, S.; Srikanth, S.; Chiranjeevi, P.; Arora, S.; Chandra, R. Algal biocathode for in situ terminal electron acceptor (TEA) production: Synergetic association of bacteria–microalgae metabolism for the functioning of biofuel cell. Bioresour. Technol. 2014 (a), 166, 566–574. 12. Cheng, L.H.; Zhang, L.; Chen, H.L.; Gao, C.J. Carbon dioxide removal from air by microalgae cultured in a membrane-photobioreactor. Sep. Purif. Technol. 2006, 50, 324329. 13. Christenson, L.; Sims, R. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnol. Adv. 2011, 29, 686–702. 14. Xiao, L.; Zhen, H. Applications and perspectives of phototrophic microorganisms for electricity generation from organic compounds in microbial fuel cells. Renew. Sust. Energ. Rev. 2014, 550–559. 15. Campo, A.G.; Canizares, P.; Rodrigo, M.A.; Fernandez, F.J.; Lobato, J. Microbial fuel cell with an algae-assisted cathode: A preliminary assessment. J. Power Sources. 2013, 242, 638-645. 16. Subhash,

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25. Venkata Mohan, S.; Butti, S.K.; Amulya, K.; Dahiya, S.; Modestra, J.A. Waste Biorefinery: A New Paradigm for a Sustainable Bioelectro Economy. Trends Biotechnol. 2016 (b), 34 (11), 852-855. 26. Kelly, P.T.; He, Z. Understanding the application niche of microbial fuel cells in a cheese wastewater treatment process. Bioresour Technol. 2014, 157, 154–160. 27. Schroder, U.; Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Phys. Chem. Chem. Phys. 2007, 9, 2619–2629. 28. Chandra, R.; Subhash, G, V.; Venkata Mohan, S. Mixotrophic operation of photobioelectrocatalytic fuel cell under anoxygenic microenvironment enhances the light dependent bioelectrogenic activity. Bioresour. Technol. 2012, 109, 46–56. 29. Wang, H.; Ren, Z.J. A comprehensive review of microbial electrochemical systems as a platform technology. Biotechnol. Adv. 2013, 31, 796–1807. 30. Clarens, A.F.; Resurreccion, E.O.; White, M.A.; Colosi, L.M. Environmental life cycle comparison of algae to the bioenergy feed stocks. Environ. Sci. Technol. 2010, 44, 1813– 19. 31. Venkata Mohan, S.; Devi, M.P.; Mohanakrishna, G.; Amarnath, N.; Babu, M.L.; Sarma, P.N. Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment. Bioresour. Technol. 2011, 102, 1109–1117. 32. APHA, American Public Health Association/American water works Association/ Water environment federation. 20th edn. Washington DC. USA. 1998. 33. Blankenship, R. E. Medigan, M. T. Bauer, C. E. Anoxygenic photosynthetic bacteria. Dordresht. The Netherlands: Kluwer Academic. 1995, 1005-28.

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41. Tsygankov, A. A.; Minakov, E. A.; Zorin, N. A.; Gosteva, K. S.; Voronin, O. G.; Karyakin, A. A. Measuring the pH dependence of hydrogenase activities. Biochemistry. 2007, 72, 9, 968-973. 42. Liu, H.; Hu, H.; Chignell, J.; Fan, Y. Microbial electrolysis: novel technology for hydrogen production from biomass. Biofuels. 2010, 1, 129–142. 43. Venkata Mohan, S.; Nikhil, G,N.; Chiranjeevi, P.; Reddy, C,N.; Rohit, M,V.; Kumar, A,N.; Sarkar, O. Waste Biorefinery Models towards Sustainable circular Bioeconomy: Critical Review and Future Perspectives. Bioresour. Technol. 2016 (c), 215, 2-12. 44. Srikanth, S.; Venkata Mohan, S.; Devi, M. P.; Peri, D.; Sarma, P.N. Acetate and butyrate as substrates for hydrogen production through photo-fermentation: process optimization and combined performance evaluation. Int. J. Hydrogen Energ. 2009, 34 (17), 7513-7522. 45. Becker, E. W. Microalgae: Biotechnology and Microbiology Cambridge University Press. Cambridge. 1994. 46. He, D.; Bultel, Y.; Magnin, J.; Roux, C.; Willison, J. C. Hydrogen photosynthesis by Rhodobacter capsulatus and its coupling to a PEM fuel cell. J. Power Sources. 2005, 141, 19-23.

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Graphical Abstract

Dual Chambered Biophotovoltaic Fuel Cell CO2

Anoxygenic Photosynthesis by photosynthetic bacteria

Waste/ Wastewater

O2

e-

e-

Oxygenic Photosynthesis by Microalgae

H2O

CO2

ee-

e-

Photosynthetic Bacteria

Wastewater

H+

LHC

Anodic Reaction

H+

CO2

O2

H+

CathodicReaction

e-

e-

H+

Microalgae

ADP + Pi

ATP

Calvin Cycle

H+

RC eQA

eQB

e-

QBH2

e-

bc1 Complex

e-

PQ

hϑ

Anode 24e- + 24H++6CO2 H+

PS II

Cathode

ADP + Pi

H+

ATP

hϑ

PQ

OEC

2H2O

NADP e-

Cyt F

Anoxygenic photosynthesis Glucose

NADPH PQH2

O2 + 4H+

PS I

ATP Synthase

Q

Anaerobic environment

ATP Synthase

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Aerobic environment Oxygenic photosynthesis

Proton Exchange Membrane (PEM)

RC (Reaction center), QA (Quinone A), QB (Quinone B), Bc1 Complex (Quinone Complex), PS II (Photosystem II), Quinone (Q), Plastoquinone (PQ), Cytochrome f (cyt f) and Plastocyanin (PC), PS I (Photosystem I) and Ferredoxin (Fd)

Schematic Representation of dual chambered Biophotovoltaic fuel cell

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Page 26 of 36

Figures

a

b

Graphite Anode

Anode Chamber

Graphite Cathode

Cathode Chamber

PEM holder

Cap

Inlet Chamber separator

c

d

Spacing for wire outlet

Lower circulation pipe port Upper circulation pipe port

‘O’ ring for sealing

Fig.1: Schematic representation of Hybrid Biofuel Cell (BFC) operation (a) Overview (b) Vertical overview (c) Front view (d) Top view with electrode setups.

26


Page 27 of 36

14

OL 3

a

Current OCV

12

0.6

OL 4

Current (mA)

10

0.5

OL 2

8

0.4

6

0.3 OL 1

OL 5

4

0.2

2

0.1

0

0.0 0

432

864

1296

1728

2160

Time (h) 70

b

OL 3

60

Power density (mW/m2)

0.7

50 40

OL 4

30

OL 2

20 OL 1

10

OL 5

0 0

432

864

1296

1728

Time (h)

27


2160

OCV (V)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

Energy & Fuels

600 550

120

c

500

100

OL3

Potential (mV)

450

80

400

OL4

350

OL2

60

OL1

40

OL5

20

300 250 200 150 100

Power density (mW/m2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

0

50 0.000

0.005

0.010

0.015

0.020

0.025

0.030

Current density (mA)

Fig.2. (a) Comparative profiles of OCV and current against operation time. (b) Power density with the function of each varying organic load (OL) with six cycles. (c) Polarization curves (Series, 30–0.05KΩ) measured at various applied voltages generated during stabilized performance at different organic load (OL).

28


Page 29 of 36

a

20

Lipid (%)

15

10

5

0 OL1

OL2

OL3

OL4

OL5

Anolyte organic load

b

18

Cumulative H2 Production (mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

OL1 OL4

OL2 OL5

OL3

15 12 9 6 3 0 Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6

29


Energy & Fuels

OL 1

OL 3

OL 2

OL 4

OL 5 c

1200 1000 800

VFA (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

600 400 200 0 0

432

864

1296

1728

2160

Time (h)

Fig.3: (a) Variation in Lipid productivity with function of varying organic load (OL) (b) Cumulative Hydrogen production (CHP) and (c) VFA in different cycles with respect to time and function of varying organic load (OL).

30


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a Biomass Bacteriochlorophyll

Anodic Biomass (mg/L)

1.80

1.6 1.4

1.76

1.2 1.72 1.0 1.68

0.8

1.64

0.6 0.4

1.60

Bacteriochlorophyll (µg/mg)

0.2 OL1

OL2

OL3

OL 3

OL 2

OL 1

OL4

OL5

OL 4

OL 5

100 b 80

COD Removal (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

60

40

20

0 0

432

864

1296

Time (h)

31


1728

2160

Energy & Fuels

OL 1

OL 3

OL 2

OL 4

OL 5 c

8.0 7.5 7.0 6.5

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

6.0 5.5 5.0 4.5 4.0 0

432

864

1296

1728

2160

Time (h)

Fig.4: (a) Anodic biomass and bacteriochlorophyll (b) COD removal (%) and (c) pH variation with varying function of organic load (OL) with respect to time.

32


Page 33 of 36

7.9

a

b

7.8 7.7

1.2

7.6 7.5

pH

Cathodic biomass (g/L)

1.6

0.8

7.4 7.3 7.2

0.4

7.1 7.0

0.0 OL1

OL2

OL3

OL4

0

OL5

12

24

Anolyte organic load

36

48

60

72

Time (h)

100

c Nitrates and Phosphates (mg/L)

50

80

COD Removal (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

60

40

20

0

Phosphates Nitrates

d

40

30

20

10

0 0

12

24

36

48

60

72

Initial (0h)

Final (72h)

Time (h)

Time (h)

Fig.5: Cathodic biomass with varying function of organic load (OL) (b) pH variation (c) COD removal (%) and (d) Removal of Nitrates and Phosphates with respect to time in cathodic chamber.

33


Energy & Fuels

300 225

Anode/Cathode Potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

Cathode potential

OL1

OL 2

OL 3

OL 4

OL 5

150 75 0

Cathode potential Anode potential

-75 -150 -225 -300

Anode potential

OL 1

25000 15000

OL 2

5000

1000

OL 3

500

OL 4

300

OL 5

100

Resistance (KΩ)

Fig.6: Anode and cathode potential (Series, 30–0.05KΩ) measured at various applied voltages generated during stabilized performance at different organic load (OL).

34


Page 35 of 36

a

0.20

OL 1 OL 4

OL 2 OL 5

b

0.04 OL 1 OL 4

0.03

OL 3 Derivative (dI/dE)

Current (mV)

0.15 0.10 0.05 0.00 -0.05

OL 2 OL 5

OL 3

0.02 0.01 0.00 -0.01 -0.02

-0.10 -0.15 -0.6

-0.03

-0.4

-0.2

0.0

0.2

0.4

-0.6

0.6

-0.4

0.22

OL 1 OL 2 OL 3 OL 4 OL 5

0.16 0.14 0.12

0.30

0.10 0.08 0.06

0.4

0.6

d

0.25 0.20 0.15 0.10

0.04 0.02

0.05

0.00 -0.02 -0.6

0.2

OL 1 OL 2 OL 3 OL 4 OL 5

0.35

log (|/mA|)

0.18

0.0

0.40

c

0.20

-0.2

E(mV) vs Ag/AgCl (3.5M KCl)


log (|/mA|)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

0.00 -0.4

-0.2

0.0

0.2

0.4

0.6

-0.6


-0.4

-0.2

0.0

0.2

0.4

0.6


Fig.7: Electroanalytical data at varying organic loads (OL) showing (a) Cyclic Voltammogram (b) First order derivative (c) Oxidative Tafel slopes (d) Reductive Tafel slopes.

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Energy & Fuels

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Table1: Consolidated data pertaining to the performance of Hybrid Biofuel Cell (BFC) Bioelectrogenic activity

OL1 OL2 OL3 OL4 OL5

Cyclic voltammetry

Cathode

Anode

OCV (mV)

Current (mA)

Power (mW/m2)

OC (A)

RC (A)

βc (V/dec)

βa (V/dec)

Rp (Ω)

Q (C)

C (F)

COD (%)

pH

VFA (mg/L)

O2 (mg/ L)

Biomass (g/L)

COD (% removal)

pH

VFA (mg/ L)

H2 (mL )

Biomass (mg/L)

210 450 701 546 300

2.07 3.47 5.98 4.43 2.90

58.50 71.95 103.00 61.20 17.15

0.089 0.137 0.190 0.173 0.06

0.109 0.101 0.115 0.115 0.133

0.098 0.133 0.106 0.101 0.115

1.163 0.824 0.743 0.861 1.389

3.154 3.01 2.000 2.058 3.921

2.782 3.862 4.873 4.433 2.727

0.556 0.772 0.974 0.887 0.545

90 90 90 90 90

7.1 7.1 7.1 7.1 7.1

40-30 40-30 40-30 40-30 40-30

3.5 3.5 3.0 2.5 2.5

1.2 1.4 1.6 1.2 0.9

81 79 72 40 21

7.8 6.9 6 4.3 4.3

40 285 346 689 1124

0 0 0 7.5 18

1.6 1.8 1.8 1.7 1.7

CV (Cyclic voltammetry); OC (oxidation current); RC (Reduction current); C (Capacitance); Q (Charge); βc (Reduction slope); βa (Oxidation slope);Rp (Polarization resistance).

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