2.1 Construction of Microbial fuel cell set up
A cast acrylic sheets were used to create three sets of cells, each having a volume of about 250cm3. Cast acrylic sheets and silicon glue were dissolved to make a glue that was used to seal the edges of the acrylic sheet cells. A Nafion 212 proton exchange membrane connected the two cellular compartments. The membrane was initially treated with 3% H2O2 for 1 hour, followed by 2 hours in deionized/distilled water, and finally 1 hour in 0.5M H2SO4.
2.1.2 Preparation of electrodes
Both the cathode and the anode electrodes were constructed using 5cm×5cm Carbon Energy WT (Taiwan) material. They were connected externally using insulated copper wires. Cathode electrode was functionalized to conduct ORR by treating with KOH, as described earlier (Jayathilake et al., 2022). At completion, the electrodes were wired to a 680Ω resistor. Each resistor was connected in parallel configuration with a PicoLogTM 1012 (Cambridge, UK) programmable data logging voltmeter, which recorded average voltage readings at 30-second intervals.
2.2 Sample collection
2.2.1 Sediment sample collection
For the anode, Marine sediment samples were collected from the seashore, Uppu Aru Bridge, Trincomalee, Sri Lanka (8o 28/ 13.0// N, 81o 12/ 23.8// E). During the collection of samples, seawater-logged areas in the shores having a black color appearance (pyrite mineral deposition) and a distinct sulfide smell indicating anaerobiosis were selected. Samples were dug-up from approximately 15 cm from the top layer of the sediment. The reason for this is; that anaerobic sediments are a good source of exoelectrogens which can reduce manganese and iron oxides. Sediment samples were collected into 1L tubs and immediately sealed and kept at 4°C until they were used in the microbiology laboratory at the Faculty of Applied Sciences, Rajarata university of Sri Lanka
2.2.2 Marine cyanobacterial sample collection
Cyanobacterial samples were collected from the seashore location near Pulmude road, Trincomalee, Sri Lanka (8o 37/ 39.4// N, 81o 12/ 21.3// E) appeared to have a visible formation of thick cyanobacterial mats. They were collected and kept in seawater-filled large open jars, exposed to sunlight without closing the lids until laboratory use.
2.2.3 Sea water sample collection
Being a marine MFC necessitated the use of sea water in the creation of all media and solutions, which allowed for the preservation of the ideal ionic conditions present in salt water. Nilaveli beach in Trincomalee, Sri Lanka (8o 41/ 35.0// N, 81o 11/ 41.8// E) was sampled for sea water.
2.3 Culture of marine cyanobacteria in the laboratory
Amended BG11 medium which is a universal medium for the cultivation and maintenance of cyanobacteria was used. It was amended with sea water to maintain the required conditions for marine cyanobacteria (i.e. BG11 was formulated using seawater as the base, instead of using distilled water).
1L of sea water amended BG11 media was autoclaved at 121 , 15psi for 20minutes and then samples were inoculated into the medium. Erlenmeyer flasks with a volume of 250cm³ were used and inoculation was conducted. The flasks containing marine cyanobacteria mats were exposed to natural light for 12 hours and kept in the dark for the other 12 hours. Sub culturing was done after 1 week for the original cultures with visible growth of cyanobacterial biomass. For the sub culturing process, small pieces of marine cyanobacterial mat were inoculated in 100cm3 of freshly prepared BG11medium amended with sea water.
2.4 Microbial inoculation in MFCs
In the anode, marine sediment inoculations were made using the collected samples. The initial abiotic cathode chambers of the MFCs were supplied with a constant flow of oxygen with the use of an air pump at a rate of 250ml air/min. But after the inoculation of the marine cyanobacterial biomass, the oxygen supply was cut off.
The anolyte consisted of Glucose (2 g /L), NH4Cl (0.46 g /L), yeast extract (0.1 g /L), peptone (0.5 g /L), KH2PO4 (5.05 g /L), and K2HPO4 (2.84 g /L).
For the cathode chamber of the control MFC, BG 11 prepared using distilled water was added and for the other two MFCs, pre-cultured marine cyanobacterial mats (approximately 25g wet biomass) in 250cm3 modified BG11 medium was added.
The cathode chamber of all MFCs were supplied with a constant flow of oxygen using an air sparger. Once the marine cyanobacterial mats were successfully grown and spread, the oxygen supply was cut off. The two cells with the cyanobacterial cultures were illuminated using a LED table lamp of light intensity 0.003lux.
2.5 Laboratory Operation of microbial fuel cells
Sea sediment and the modified semi-defined minimum medium were introduced to the anode chamber, and then the chamber was shut and left undisturbed. When depleted, glucose was supplemented into the anolyte as the exogenous feeding substrate. Cathode compartments with marine cyanobacterial cultures were illuminated 24hours with a table lamp having a 0.003lux of light intensity.
2.6 Preparation of the exogenous feeding substrate
The collected excess marine cyanobacterial biomass from the MFC biocathode was placed in 50 ml Falcon tubes and centrifuged at 5000 rpm for 5 minutes. The biomass was milled into a pulp using a mortar and pestle. In order to perform acid hydrolysis, 10 ml of 0.1M HCl was added to the ground biomass before being sonicated for 10 minutes. The pH of the solution was measured after sonication, and 1M NaOH was added to neutralise it to pH-7. This served as the alternative feedstock (to replace glucose) for the exoelectrogens in the MFC anode.
2.7 Feedstock analysis
The sugar content of the feedstock was measured using Brix refractometer (Atago 2351 MASTER-53α) at room temperature (25±2 °C).
2.8 Performance characterization of Microbial fuel cells
For the performance characterization, polarization curves which is a plot of current density versus electrode potential were used. The system with stabilized voltages, few hours after feeding was considered as the best time to commence the polarization curve data collection. Resistors ranging from 22Ω to 1 MΩ were connected and the voltage across each resistance was measured using a multimeter. Open cell voltage; without the connection of a resister was also recorded.
Ohms law was used to calculate the current flowing across each resister.
I = V/R ------------- (1)
Where; I=Current flowing through the system in Amperes (A)
V=Voltage across each resistor in Volts (V)
R=Resistance connected in Ohms (Ω)
Next, power across each resister was calculated using the equation;
P=V*I ---------------- (2)
Where; P=dissipated power in Watts (W)
, V= Voltage across each resister in Volts (V)
I = current calculated using equation 1 in Amperes (A)
For the purpose of calculating the power and current densities, the power and current values obtained in equation 1 and 2 were normalized to the area of the electrode (25 cm²).
2.9 Characterization of bacterial biofilms from anode and biotic cathode
2.9.1 Isolation of anodic microorganisms
Anode bacteria were isolated and grown on a unique chromogenic media supplemented with saltwater. These ingredients make up the growth media: 2g/Glucose, 0.46g/L NH4Cl, 0.1/L yeast extract, 0.5/L peptone, 5.05/L K2HPO4, 2.84/L KH2PO4, 1.5/L MnO2, 0.025/L Cysteine, and 15g/L agar. In order to remove any lingering oxygen from the growing media, cysteine was used. This medium can be used to separate out the microorganisms that have demonstrated the ability to use MNO2 as the terminal electron acceptor. To wit: (Nazeer and Fernando, 2022). Bacteria were rapidly streaked in the media from the anode compartment to prevent air bubbles. To create an oxygen-free environment for incubating the petri dishes, we used an anaerobic jar (Oxoid, UK) and Gas-PakTM anaerobic sachets.
2.9.2 Identification of photosynthetic microorganisms in the biocathode
Piece of a cyanobacterial mat was observed under the light microscope. It was captured using the software called light view version 3.20.9.
2.9.3 Molecular microbiological characterization of the photosynthetic microorganisms in biocathode.
Several extractions were carried out with subcultures and an optimized Cetyltrimethylammonium bromide (CTAB) DNA extraction method (Morin et al., 2010) was used to extract the marine cyanobacterial DNA. Bands were verified from the agarose gel electrphoresis. Molecular identification of the cyanobacteria were conducted using the 16s rRNA marker gene. The full length of the 16s rRNA gene was amplified using the universal primer pair 27F and 1492R (5’ - AGA GTT TGA TCM TGG CTC AG - 3’) and 1492R (5’ - CGG TTA CCT TGT TAC GAC TT – 3). The PCR reactions were conducted under the following thermo-cycler conditions (Initial denaturation at 95⁰C for 4 min, fo30 cycles of 95⁰C for 0.5 min, 58⁰C for 1 min, 72⁰C for 0.5 min, and finally 72⁰C for 7 min). PCR products were quantified using nanodrop (Jenway, UK) and finally verified using Agarose gel electrophoresis. The PCR products were bi-directionally sequenced using the same 27F and 1492R primer pair using Sanger DNA sequencing at Eton Bioscience sequencing services, New Jersey, USA. Phylogenetic analysis and placement was conducted using the molecular phylogeny software, MEGA™ version 11.
2.10 Characterization of marine photosynthetic cathode biofilms using Scanning Electron microcopy (SEM)
The SEM was used to take a look at biofilm samples that had developed on carbon nano fibre woven cathodes, which are used in marine photosynthetic biocathodes. Carefully using a sharp blade, a 1 cm2*1 cm2 portion of biofilm was removed. Following that, it became dehydrated. Due to the cathode material's conductivity, gold plating was skipped. SEM images of fixed photosynthetic marine biocathode biofilm samples formed on carbon cathodes were captured using a Carl Zeiss EVO 18 (Germany) microscope with an optical magnification range of 20-135x, an electron magnification range of 5x-1,000,000x, a maximal digital zoom of 12x, an acceleration voltage of 10 kV, a backscattered electron detector (BSD), and an energy dispersive The microscope's sample holder is temperature-controlled (from 25 oC to 50 oC) (Asahi et al., 2015).
2.11 Cyclic Voltammetry (CV) of the control cathode and the biocathode
This method is used to better understand the electrochemical behavior of a system and the interactions between an electrode and a biofilm. An electrolysis cell, a potentiostat, a current-to-voltage converter, and a data-acquisition system make up a typical cyclic voltammetry experimental setup. (Voltammetry and Epc) The CHI 9201D Scanning electrochemical microscope was used for this study.
A 1cm*1cm piece of the working electrode, a biofilm-formed carbon electrode, was assembled with the counter and reference electrodes, both of which had been cleaned with deionized water prior to use. To make sure the system was ready for the CV of the working electrode, a voltammogram of pure electrolyte was first recorded. Using NaSO4 as the electrolyte, CV was done both before and after biofilm formation. For this cyclic voltammogram, platinum served as the counter electrode and a reference electrode of Ag/AgCl in 1M KCl.