Microbial fuel cells (MFCs) have long been considered an attractive mean for converting various carbohydrate wastes directly into electricity using electrogenic bacterial cells in the anode compartment. Most MFCs have been operated using anaerobic or facultative aerobic bacteria which oxidize various substrates including glucose, sewage sludge and petroleum hydrocarbon . Power production by MFCs varies with bacterial cell species, specific substrate concentration, cathode catalysts and the MFC configuration .
Typically, MFCs which were operated with a mixture of bacterial cells produced higher specific power than MFCs operated by a monoculture in the anode compartment . Knowledge and understanding of the anode biofilm components, morphology formation steps and electron transfer mechanism may lead to better biofilm conductivity in MFC.
This chapter focused on biofilm definition and composition. Spectroscopic methods for anode biofilm study, including advancing real-time analysis. Power-producing bacterial cells, mechanisms of electron transfer from the biofilm to the anode and the effect of medium and pH conditions on power production.
2. Biofilm definition
The term biofilm has been proposed for a structured community of microorganisms that adheres irreversibly to surfaces (biotic or abiotic) and is enclosed in a self-developed polymeric matrix of a primarily polysaccharide material .
3. Biofilm composition
The biofilm matrix, which is a prerequisite for biofilm formation, consists of up to 97% water, 2-5% microbial cells, 3-6% extracellular polymeric substance (EPS) and ions [4-6]. The EPS may be hydrophilic or hydrophobic and is composed of 40-95% polysaccharides, 1-60% proteins, 1-10% nucleic acids and 1-40% lipids .
The EPS serves as a scaffold which holds the cell aggregates together . EPS is highly hydrated, since it can incorporate large amounts of water molecules by hydrogen bonding. The microbial consortia and the environmental conditions influence the composition of the EPS . The amount and thickness of the EPS increase with the biofilm’s age .
The EPS in thin biofilms is often rich in proteins, contrary to thicker biofilms . The EPS is more abundant in the interior of the biofilm, whereas cell densities are highest in the top layer .
As mentioned, the EPS is comprised of exopolysaccharides, proteins, nucleic acids and lipids. The exopolysaccharides in the EPS can be linear or branched, with a molecular weight of 500-2000 kDa. There are homo-polymers, e.g. cellulose, curdlan, dextran and sialic acid, but the majority are hetero-polymers composed of 2-4 types of mono-sugars such as alginate, emulsan, gellan and xanthan . Nucleic acids that are found in the EPS are extracellular DNA (eDNA) that exhibit some similarities to genomic DNA but also distinct differences. In
The biofilm is enriched with specific protein adhesins that mediate known molecular binding mechanisms for irreversible attachment. In addition, membrane transport proteins such as porins and extracellular enzymes are up-regulated . Biofilm formation occurs in a sequential process of: (i) transport of microbes to a surface by chemotaxis or Brownian motion; (ii) initial attachment; (iii) irreversible attachment of bacteria and formation of microcolonies; (iv) biofilm maturation; and (v) detachment .
The substratum characteristics may influence biofilm formation and morphology. Most investigators have found that microorganisms attach more rapidly to hydrophobic, nonpolar surfaces such as teflon and other plastics than to hydrophilic materials such as glass or metals . Furthermore, microbial colonization increases with the increase in surface roughness . The biofilm architecture changes constantly, due to external and internal processes . The biofilm thickness may be affected by the number and species of microorganisms. Biofilms of pure cultures of either
Cell surface-associated proteins such as pili, flagella, curli and amyloid fibers are believed to be important factors for biofilm formation . Cell-to-cell signaling (quorum sensing) has been demonstrated to play a role in biofilm formation.
In conclusion, knowledge and understanding of the biofilm components, morphology and formation steps may lead to better biofilm conductivity in microbial fuel cells (MFC).
4. Spectroscopic methods for anode biofilm study, including advancing real-time analysis
During the last decade, most efforts in microbial fuel cell (MFC) research focused on modifications of MFC design and electrode materials, with little investigation of the properties of the anode microorganisms that are essential for maximal current production. However, understanding the functions of microbial cell surfaces requires knowledge of their chemical structural, physical properties and biological processes.
Perhaps the most challenging effort to improve MFC power production lay in the fundamental understanding of the biofilm’s chemical, physical and biological characteristics. Study of the local properties of an anode biofilm is an even a greater challenge, due to the low concentration of bacterial cells. In addition to conventional methods such as scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), there exist modern methods such as Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). These latter are nondestructive methods which can probe biofilms down to single-cell surfaces with high resolution, and thereby promote better understanding of biofilm formation, functionality and activity.
In this chapter we will discuss the principle, the advantages and the disadvantages of each of the methods and the application of these techniques in MFCs.
4.1. Confocal laser scanning microscopy
Confocal laser scanning microscopy (CLSM) is often used in optical imaging of sliced micro-fluidic velocity fields by mapping the investigated focal plane. CLSM enables obtaining a series of optical sections of intact undisturbed biological samples as thin as 0.3 µm. Commonly used analyses that rely on staining techniques are applied to determine the architecture, spatial distribution and viability profile in microbial biofilms. The most popular application of CLSM is for identification of live and dead bacteria. Simultaneous measurements of anodic biofilms during the MFC’s operation may be obscured by the need to apply labeling materials. This method is therefore used more commonly as a useful
4.2. Scanning electron microscopy
Scanning electron microscopy (SEM) analysis provides an excellent magnification technique that uses a condensed electron beam to scan a sampled object and magnify specific regions of its surface area. Highly resolved images can be produced by SEM to provide morphological details of the surface, information on the three-dimensional topography as well as an elemental composition analysis map of the same unit area when coupled with an x-ray elemental detection sensor. SEM is therefore instrumental in a broad spectrum of scientific and industrial applications.
The main shortcomings of SEM are the relatively low resolution compared to transmission electron microscopy (TEM), which is usually higher than a few tens of nanometers and the need for a surface-conducting coating layer to avoid local charging and heating effects. Moreover, SEM is an ultra-low pressure technique. All samples must therefore withstand the low pressure inside the SEM vacuum chamber .
4.3. Raman spectroscopy
The Raman spectroscopy method is based on inelastic scattering of monochromatic photons, usually from a laser source. In this process, the frequency of the back-scattered photons changes their frequency due to interaction with a sample. The process begins when photons of the laser light are absorbed by the sample and are then re-emitted at a longer wavelength compared with the laser’s original monochromatic frequency. This phenomenon is called the Raman frequency shift effect. This shift provides information on vibrational, rotational and other low frequency transitions at the molecular level. Raman spectroscopy is typically applied in the study of solid, liquid and gaseous samples.
The main strength of the Raman technique is its high sensitivity, the fact that it does not require any staining and its ability to generate detailed chemical and spatial information with a resolution below the diffraction limit. A combination of Raman spectroscopy with optical microscopy provides a powerful source of information which is both detailed and sensitive and can be obtained with a spatial resolution below the diffraction limit [26, 27]. The main disadvantages of the Raman technique is the weak Raman effect, which requires sensitive and highly optimized instrumentation for detection. Furthermore, fluorescence of the sample or impurities within it can mask the Raman spectrum. Sample heating through the intense laser radiation can damage the sample and distort the Raman spectrum .
4.4. Atomic force microscopy
Atomic force microscopy (AFM) provides one of the highest topographical profile imaging of a sampled surface, sometimes even on a nanometric scale. It does so by measuring interaction or repulsion forces between a sharp nano-size probe and a surface. The nitration distances between the tip and the sample can be as small as a few tenths of a nanometer. The AFM tip can be used either in a contact mode where the tip actually touches the surface, or in a non-contact mode where van der Waals interactions produce forces across the short distance between the tip and the surface. The forces produced by each mode of operation can be recorded to give topographic, conductive, magnetic, mechanical (friction forces) or potential images of the surface.
In fact, AFM imaging does not require any surface modification that may damage or change the sample. Most importantly, AFM techniques are well suited to work in ambient air or in a liquid environment, including physiological conditions . Therefore, the study of electrodes, biological samples, biomolecules and even living organisms can be measured rather easily by AFM. Moreover, advanced surface-imaging tools offer more than just force measurements. It can be applied to probe physical properties such as molecular interactions, surface hydrophobicity, surface charges, mechanical properties and local electrochemical properties . For example, AFM enables imagining of biofilms and the EPS distribution within the biofilm .
The disadvantage of AFM is that the scanned area of the AFM image is rather small (few tens of microns), and it is very sensitive to externally generated electrical and mechanical noises .
5. Power-producing bacterial cells
Power-producing bacterial species that have exoelectrogenic activity without exogenous mediators include:
Pure cultures are suitable for basic study of MFCs, since they allow analysis of electrochemical and biochemical processes as well as high reproducibility. However, mixed cultures are more suitable for industrial applications because no sterilization is required and it is not necessary to maintain anaerobic conditions via an external N2 flow. Mixed bacteria usually produce higher power densities in MFCs than pure bacterial strains. It is important to note that the power density depends on the specific MFC configuration, electrode spacing, bacterial cell species, growth medium and the cathode catalysts [1, 2]. Thus, power densities produced in a MFC using a specific bacterium cannot be directly compared with another bacterium unless all other parameters are identical.
Exoelectrogenic bacteria transfer electrons to anodes either directly or via self-produced mediators. In the direct mechanism, electron transfer occurs via membrane-associated C-cytochrome or through conductive pili or appendages . In mediated mechanisms, electron transfer between the bacterial cell and the anode surface occurs through self-produced soluble redox compounds such as flavins or pyocyanin . Well-known exoelectrogens include
6. Mechanisms of electron transfer from the biofilm to the anode
C-type cytochromes have been considered as one of the most important electron transfer strategies in current generation by exoelectrogens. C-cytochromes are heme-containing proteins which are widespread in most bacteria.
Cytochrome genes were examined during extracellular electron transport in MEC. A total of 21 cytochrome genes were detected. Four bacterial genera contain the cytochrome genes:
Okamoto et al identified voltammetry signals of outer membrane C-cytochrome in monolayer biofilms of
Immunogold labeling of the outer-surface C-type cytochrome OmcZ showed that when
Eaktasang et al. electrochemically oxidized the surface of a graphite felt electrode with a strong acid in order to stimulate current production in the MFC. FT-IR was used to examine the chemical property changes in the graphite felt surfaces which were induced by its electrochemical oxidation using acid treatment. Current production in the MFC equipped with the surface-modified graphite anode was about 40% higher than that obtained from the MFC of a bare graphite anode. FT-IR spectra of surface-modified felt and bare graphite felt showed a notable broad band at 3264 cm-1 ascribed to stretching vibration of OH within –COOH, a broad peak at 1560cm-1 ascribed to C=O of ketone and carboxyl groups, and a peak at 1419 cm-1 ascribed to bending vibration of –OH. The relative intensities of the abovementioned peaks increased significantly after electrochemical oxidation treatment of graphite felt, indicating that alcohol and carboxyl functional groups formed on the surface of the graphite-felt hydrogen bonding with peptide bonds in the bacterial cytochrome. They also demonstrated that the carboxylic acid terminus of gold-modified electrodes can facilitate the binding with cytochrome on its surface, and as a result, current production increased due to the enhanced transfer of electrons from the interior of the cell .
Genes encoding proteins with a PilZ domain were deleted from the G. sulfurreducens genome in an attempt to study the importance of pili to biofilm conductivity. The mutant strain designated as strain CL-1 produced more pili than the wild type strain and formed 6-fold more conductive biofilms than the wild type strain. Heme-staining revealed a higher abundance of cytochrome with a molecular weight consistent with OmcS in CL-1 and Western blot analysis with OmcS-specific antiserum confirmed higher production of OmcS in CL-1. Immunogold labeling coupled with TEM demonstrated that OmcS was localized on the pili of CL-1 . Multilayer biofilms of
The AFM technique, together with electrochemical measurements by scanning tunneling microscopy (STM), allowed mapping of conducting substrates and microbial pili in MFCs studies. These two techniques enabled identification of the pili on
7. The effect of medium and pH conditions on power production
The effects of anodic pH on electricity production in two-chamber MFCs inoculated with anaerobic activated sludge were examined. The maximum power density was 1170±58 mWm2 with a current density of 0.18 mA×cm-2 at pH 9.0, which was 29% and 89% higher than in MFCs operated at pH 7.0 and 5.0, respectively. Electrochemical measurements demonstrated that the environmental pH may influence the electron transfer kinetics of the anodic biofilms. At pH 9.0, the apparent electron transfer rate constant and exchange current density were greater, whereas charge transfer resistance was smaller than under other pH conditions. SEM analysis reveled better biofilm formation at pH 9.0 compared to pH 7.0 and 5.0. The biofilm at pH 9.0 showed increased electron transfer efficiency with respect to the electrocatalytic current, electron transfer rate, exchange current density, and charge transfer resistances, compared with biofilms at pH 7.0 and 5.0. These results demonstrate that electrochemical interactions between bacteria and electrodes in MFCs are greatly enhanced under alkaline conditions, which can an important reason for the improved current production in MFCs . It should be noted that the cathodic potentials were almost identical under all pH conditions, whereas the anodic potentials varied. The anode that operated at pH 9.0 had the most negative individual potentials in the entire current range, whereas the anode at pH 5.0 had the most positive individual potentials. The variations in the power outputs thus resulted from the anodes under different pH conditions .
The performance of two dual-chambered mediators-less MFCs was evaluated at different sludge loading rates and pH environments. A maximum volumetric power of 15.51 W/m3 and 36.72 W/m3 was obtained in MFC-1 (feed pH 6.0) and MFC-2 (feed pH 8.0), respectively. These results indicate that higher feed pH (8.0) led to higher power production . Zhuang et al. discovered that an alkaline anode (pH 10) led to higher power production during wastewater treatment compared to a MFC which worked at neutral pH. Their assumption is that there methanogenesis is suppressed at higher pHs, and this contributes to a significant enhancement of coulombic efficiency . In contradistinction to the abovementioned studies, there are several studies which described that higher power production was obtained in natural or acidic environments. An air-cathode MFC which operated with a mixed bacterial culture at different pHs showed that the anodic microbial process preferred a neutral pH and that microbial activities decreased at higher or lower pHs . A MFC with a pure culture of
In conclusion, we assume that MFC power production is dependent on the pH environment and may correlate with the bacterial cell anode species.
8. Anode biofilm thickness, morphology, viability and conductivity
CSLM analyses revealed a correlation between current and increasing coverage of the anode surface with cells. The average height of the biofilm pillars at currents nearing the maximum current outputs in batch mode was 40 μm (±6 μm), and some cell clusters were as high as 50 μm. Protein measurements of the biofilm biomass at different points during current production indicated a direct linear increase in the amount of biomass on the anodes as the current increased and the biofilms developed. Viability staining indicated that during biofilm development, cells at a distance from the electrode surface remained viable, metabolically active and involved in electron transfer to the anode .
Three MFCs (plain graphite electrodes, air cathode, Nafion membrane) were operated separately with variable biofilm coverage [control; anode surface coverage (0%), partially developed biofilm (coverage ∼44%) and fully developed biofilm (coverage of ∼96%)] under acidophilic conditions (pH 6) at room temperature. Higher specific power production [29 mW/kg CODR (CW and DSW)], specific energy yield [100.46 J/kg VSS (CW)], specific power yield [0.245 W/kg VSS (DSW); 0.282 W/kg VSS (CW)] and substrate removal efficiency of 66.07% (substrate degradation rate, 0.903 kg COD/m3-day) were observed, especially with fully developed biofilm operation .
The behavior of MFCs during initial biofilm growth and characterization of anodic biofilm were studied using two-chamber MFCs with activated sludge as inoculum. When the biofilms were well developed, a maximum closed circuit potential of 0.41 V and 0.37 V (1000 Ω resistor) was achieved using acetate and glucose, respectively. SEM analysis revealed rod-shaped cells, 0.2-0.3 mm wide by 1.5-2.5 mm long, in the anode biofilm in the acetate-fed MFC, mainly arranged in a monolayer. The biofilm in the glucose-fed MFC was made of cocci-shaped cells in chains and a thick matrix [60. A MFC was operated with a pure culture of
Electron transfer between bacterial cells and the electrode is one of the major blockages to a desired power density. A direct electron transfer process between
Introducing a pH-sensitive fluoroprobe into the anode chamber revealed a strong pH gradient within the
Millo et al. employed surface-enhanced resonance Raman (SERR) spectroscopy in combination with CV analysis. This technique, performed under strict electrochemical control, reveals the redox, coordination and spin states of the heme iron in addition to the nature of its axial ligand. Furthermore, by applying
Liu et al. showed that graphene modification improved power density and energy conversion efficiency by almost 3 times that of a
In conclusion, the biofilm strain composition, components, morphology and thickness play a major role in the electrochemical process in MFCs, and show marked influence on bioelectricity production. Changes in biofilm parameters may influence the anode electrochemical characteristics and performance in a MFC. It is therefore important to understand the characteristics, structure and composition of the biofilm in order to ensure optimized operation of MFCs.
This chapter was supported in part by the Research Authority of Ariel University, the Israel Ministry of Environmental Protection and the Samaria and Jordan Rift Valley Regional R&D Center.
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