This positive feature of silk film was then applied to develop biofilm on the anodic surface of PMFC where silk fibroin. Structural studies demonstrated rational interactions of the hydrophobic β-sheet of SF with the nanomaterials. The origin of the burst potential is attributed to the depolarization of the bacterial membrane caused by the interaction with the substrate.
V2 The potential amplitude from the base midpoint of the winding tip to the tip point. The inset graph is an enlarged segment of the response data at lower concentration (< 0.5%) alcohols. B) Effect of different solvents (2 mM) on p-PMFC response. In addition, most electrode materials such as graphite and conductive metals commonly used in fuel cell applications are insusceptible or do not encourage the growth of biofilm-forming bacteria.
- Photosynthesis and respiration in cyanobacteria
- Cyanobacteria biofilm
- Microbial fuel cell: A highlight on the basic principles and operation
- Photosynthetic microbial fuel cell (PMFC)
- Electrogenic property of cyanobacteria
- Electron transfer mediator (ETM)-based PMFC
- DET-based PMFC
- Performance and application potential of PMFC
- Electrode materials used in MFCs
- Applications of MFC for sensing alcohol
Considering the enormous potential of cyanobacteria as PMFC catalysts, and the gaps and challenges in developing such PMFC, as briefly discussed above, the following objectives have been set for the current research, embodied in this thesis. The entire dissertation is divided into four chapters, as described below, followed by a short section describing the general conclusion about the current work and the scope for future research.
- Silk fibroin extraction
- Microorganisms and cultivation
- Analysis of biofilm growth
- Microscopic studies on biofilm of different polymers
- Circular dichroism (CD) analyses
- Evaluation of hydrophobicity index (HI) of bacterial cells
- Calculation of interaction energy profile
- Contact angle measurements
- Calculation of adhesion rate constant and activation Gibbs energy of adhesion
- Statistical analysis and graphics program
Results and discussion
- Biofilm growth on polymer films
- Physico-chemical factors influencing the biofilm growth
- Cell density assay
- Interaction study between SF and QD
- Circular dichroism (CD) analysis
- Isothermal titration calorimetry (ITC) analysis
- Determination of FRET
- Preparation and characterization of nanocomposite films
- Construction and operation of PMFC
- Construction of PMFC
- Operation of PMFC
- Characterization of PMFC electrodes
- Electrochemical characterizations of electrodes
- Characterization of electrodes by FESEM
Results and discussion
- Development of biofilm on nanocomposite casted anode
- Performance of the nanocomposite anode in PMFC setup
- Characterization of anodes and electron transfer mechanism
This is due to several distinct advantages of biosensors such as higher selectivity, sensitivity and better scope for improving the properties of biorecognition elements over conventional sensors based on chemical and physical recognition (Su et al., 2011 ). The low selectivity and sensitivity of microbial biosensors is often mitigated by their significantly low assay cost, long lifetime, and robust nature to withstand environmental conditions (Lim et al., 2015). Microbial biosensors with different transduction principles have been explored, among which, MFC sensors have received increasing attention in recent years due to their simplicity of production, the possibility to operate in external environments independently, low cost of operation and sensitive signal transmission. mechanism (Dávila et al., 2011).
However, most of these MFC-based biosensors have a long response time, because the response obtained by the interaction of the target species with the catalytic cells in the MFC is usually determined by the metabolism and growth of the organisms on the electrode surface. The long response time and poor selectivity of the MFC-based biosensors discourage their applications in areas where rapid and specific detection of the targets is an important task. The requirement of cellular growth of the biocatalysts in the MFC-based sensors is another limitation.
There are numerous reports on the use of various pure or consortia bacteria as catalysts for MFCs (Abrevaya et al., 2015; Li et al., 2016). These studies are mostly limited to discovering basic information about the suitability of cyanobacteria as MFC catalysts for their potential use in electricity generation and wastewater treatment. Notably, efforts to miniaturize PMFCs are limited despite the fact that small-scale PMFCs improve mass transport and reduce internal resistance (Fraiwan et al., 2016; Lee and Choi, 2015).
However, such an effective portable low-cost alcohol detection system is not widely available in the global market. An alcohol sensor for the selective detection of methanol and ethanol is very important due to the fact that methanol is toxic in alcoholic beverages and a less efficient mixture than ethanol in fuel. The various alcohol sensors and biosensors reported so far have their own advantages (Thungon et al., 2017).
The paper as a sensor platform is known to offer many advantages, including low cost, portability, disposability, biocompatibility, ease of storage and fluid-wicking properties that eliminate the need for external pumps, relevant to current PMFC work (Kakoti et al., 2015; Martínez et al., 2010).
- Analysis of spectral properties of cyanobacteria
- MTT assay
- Measurement of membrane potential
- Microscopic imaging
- Fluorescence activated cell sorting (FACS)
- Preparation of working electrode
- Construction of PMFC and potential measurement
- Bacterial cell-electrode interaction analysis
- Fabrication of p-PFMC system
- Characterization of p-PFMC
Membrane integrity of cyanobacterial cells was monitored using an anionic membrane potential sensitive fluorescent probe DiBAC4 (3) (Huang et al., 2015). The cell suspension (OD was combined with filtered (through a 0.45 μm filter, MICRO-POR®, Genetix Biotech) 50 mM sodium succinate prepared in water and incubated for 15 min to provide energy for all ATP-dependent membrane channels and cellular components (Clementi et al., 2014). This enabled performing several experiments at different time points with greater simplicity and continuously monitoring the fluorescence kinetics of the entire population over long periods of time, which is difficult to achieve using flow cytometry (Clementi et al. ., 2014).
The cell suspensions taken in the 96-well plate were mixed with the membrane potential probe (finally 500 nM). The structural integrity of the cyanobacterial cells, untreated (control) and treated with 10% alcohols and dried overnight in the laminar hood, was visualized using atomic force microscope (AFM) (Innova, Bruker) and TEM, with an operating voltage of 100 °C. kV (JEOL MODEL: 2100F). Ferricyanide in the cathode exhibits a stable and fast cathodic response, allowing only the changes in the anode chamber to reflect the MFC performance (Abrevaya et al., 2015).
The CV profiles (scan rate of 10 mV s-1) were generated in the absence (control) and presence (1%) of alcohols from the anode, where the biofilm was maintained without substrate. A ferrocene value of -4.4 eV was used as a reference to calculate the energy of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) levels. The band gap energy levels (e.g.) of redox species were calculated based on the onset of oxidation (EOxon set) and reduction potential (ERedon set), as mentioned: (Leonat et al., 2013; Ye et al., 2012).
The resistivity (ρ, Ωm) was calculated from ρ = R A/l, where R is the resistance measured using the data logger, A is the area of the electrode and l is the distance between the copper clamps. The potential response of p-PFMC was monitored in the presence of varying concentrations of alcohols using a data logger. The potential response of p-PFMC against some potential interfering solvents was measured following the similar procedure.
Uniform drops of 2 μL of the solvents were dropped onto the cell surface and contact angles were measured by the sessile drop technique at RT after 60 s using a Holmarc model No.
Results and discussion
- Sensing of alcohols using PMFC potential burst signal
- Effect of alcohols on the integrity of cyanobacterial cells
- Effect of alcohols on the cellular morphology
- Effect of alcohols on the cell–membrane polarity
- Effect of alcohol on microbial respiration and photosynthetic reaction center: 107
- Development of p-PMFC for sensing alcohol
At 10% of the respective concentrations, the detected burst potential for ethanol was ~1.4 times higher than for methanol. To understand the phenomena of potential outbursts, we investigated the effect of alcohols on the morphological, electrochemical and relevant spectroscopic properties of the cells, as discussed in the next section. The raised and smooth height profile of the untreated cells (control) creates a ruffled membrane indicating an intact cell structure (Figure 4.4 B a and 4.5 a).
Conversely, the topography of the ethanol-treated cells was significantly distorted as revealed from the reduced height profile with uneven surface structures of the cells (Figures 4.4 B b and 4.5 b-c). The morphological structure and fluorescence intensity of the methanol-treated cells did not differ significantly from the control. Both alcohols triggered depolarization of the cell membrane, as evidenced by the increased fluorescence intensity from the treatment point (45 min).
The increase in background intensity after discarding the microtiter plate (after 45 min) in the control (without alcohol) developed due to the recovery of the dye from photobleaching. The increase in respiration of alcohol-treated cells was not associated with growth, as shown in Figure 4.9 B. The effect of alcohols at their moderate concentration (1%) and low scan rate (10 mV/s) on bioanodes under noncirculating conditions was examined by CV (Figure 4.11).
After the injection of alcohols, the number of redox peaks of the bioelectrode was not changed. The potential bursting of PMFC with addition of alcohols can also be explained on the basis of band gap change of the redox entities in the bioelectrode. The highly conductive nature of the bioelectrode interface was confirmed by the respective -ve values of the band gaps (Figure 4.11 and Table 4.1).
Disruption of membrane structure by ethanol induces passive diffusion of H+ (Madeira et al., 2010) and reduces the size of the pmf (Cartwright et al., 1986).
Thus, the coupling of the potential burst with the enhanced respiration and membrane depolarization and breakdown of the cyanobacterial cells could be logically drawn in the present case. The concept was initially investigated in a laboratory-scale PMFC to understand the precise performance of the sensor. Therefore, optimization study of the load of the bacterial cells on the paper surface can expand the area for screening a wide concentration range of samples.
A further objective was to investigate the applicability of the developed PMFC as a small disposable alcohol biosensor. Each of these functions of the nano-biocomposite matrix was properly evaluated to confirm their developed properties in the hybrid state. We were able to generate a new form of signal based on the potential burst after injection of target alcohols on Synechococcus sp.
A series of experiments were conducted to understand the effect of alcohol on the integrity of bacterial cells and the potential explosion observed. Degradation of the cell membrane increases the exposure of electron transfer proteins leading to an immediate increase in the bioanode that manifests itself in the form of a potential burst. It was observed that the injection of alcohol triggered the respiration of cyanobacterial cells.
The function of transferable p-MFC can be investigated using commonly available bacterial strains such as E. Evaluation of the suitability of bis-(1,3-dibutylbarbituric acid) trimethine oxonol, (diBA-C4(3) − ), for flow cytometric assessment of bacterial viability . Applicability of the MTT assay for measuring the viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria).
Purification and crystallization of photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002.