**The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control**

Oleksandr Bilyy, Oresta Vasyliv and Svitlana Hnatush

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58347

## **1. Introduction**

[63] Kalathil, S., et al., Enhanced Performance of a Microbial Fuel Cell Using CNT/MnO2 Nanocomposites as a Bioanode Materials. Journal of nanoscience and nanotechnolo‐

[64] Liang, P., et al., Carbon nanotube powders as electrode modifier to enhance the ac‐ tivity of anodic biofilm in microbial fuel cells. Biosensors and Bioelectronics, 2011.

[65] Mink, J.E., et al., Vertically Grown Multiwalled Carbon Nanotube Anode and Nickel Silicide Integrated High Performance Microsized (1.25 µL) Microbial Fuel Cell. Nano

[66] Yong, Y.-C., et al., Macroporous and Monolithic Anode Based on Polyaniline Hybri‐ dized Three-Dimensional Graphene for High-Performance Microbial Fuel Cells. ACS

[67] Wang, H., et al., High power density microbial fuel cell with flexible 3D graphene-

nickel foam as anode. Nanoscale, 2013. 5(21): p. 10283-10290.

gy, 2013. 13: p. 7712-7716.

32 Technology and Application of Microbial Fuel Cells

Letters, 2012. 12(2): p. 791-795.

Nano, 2012. 6(3): p. 2394-2400.

26(6): p. 3000-3004.

The need for reducing dependence on fossil fuels and the promoting the use of renewable fuels requires the development of alternative sources such as waste biomass for environmental benefits and alternative global energy supplies [1]. Microbial fuel cells (MFCs) provide new opportunities for the sustainable production of energy from biodegradable and reduced compounds, and thus, have attracted substantial research efforts to develop various devices for generating electricity and removing wastes [1, 2]. The development of processes that can use bacteria to produce electricity represents a highly effective method for bioenergy produc‐ tion as the bacteria are self-replicating, and thus the catalysts of organic matter oxidation are self-sustaining [2]. The substrates used in MFCs range from carbohydrates (e.g. glucose, sucrose, cellulose, starch), volatile fatty acids (e.g. formate, acetate, butyrate), alcohols (e.g. ethanol, methanol), amino acids, proteins and even inorganic components such as sulfides or acid mine drainages [2, 3-9]. The type of substrate fed to a MFC potentially has an impact on the structure and composition of the microbial community. Untill now, no clear image of the effect of the type of substrate on electricity generation by the microbial fuel cells is available.

Analysis of external resistances, electron donor concentrations, cell densities, rates of electron transfer to electrodes at various voltages, and anode potentials can aid in understanding the power production capabilities using microorganisms [10]. A simplified model for the conver‐ sion of complex organic fuels to electricity is shown in figure 1 [10].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

efficient mechanisms of antioxidant defense system, ion efflux transport enzyme complexes etc. Examples of these genuses are *Feroplasma*, *Streptomyces*, *Thiobacillus*, *Desulfuromonas* etc [12]. The specific metal-transport systems that were found in gram-negative bacteria which support transport of iron, zinc and manganese, copper, nickel and cobalt are schematically

The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control

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35

**Figure 2.** Schematic metal homeostasis models for iron, zinc and manganese, copper, nickel and cobalt in gram-nega‐

The variety of ion transport systems in gram-negative bacteria represents sophisticated mechanisms of bacterial cell metal homeostasis regulation. This is possible because of the formation of specific protein-metal coordination complexes used to effect uptake, efflux,

Previous research has shown that gram-negative bacterium *Desulfuromonas acetoxidans* possesses resistance to copper, cadmium, lead, zinc, manganese, iron, etc. [14, 15]. Bacteria of *Desulfuromonas* genus are also shown to be highly effective in term of electricity generation [2, 16, 17]. However the research on its application as the anode biocatalyst in MFC is inadequate. *Desulfuromonas* sp. can be very promising for MFC development because of inexpensive cultivation medium, high survival rate and resistance to toxic xenobiotics such as the various

*D. acetoxidans,* which belongs to the class ∂-*Proteobacteria,* are uncolored obligate anaerobes that inhabit sulfur containing aquatic environments [18]. *D. acetoxidans* supports reductive stage of sulfur cycle in the nature, but it can not possess an ability to reduce sulfate or other oxoanions of this element. Sulfur is a crucial component of various biological active substances, such as

intracellular trafficking within compartments, and storage [12].

presented in figure 2 [13].

tive bacteria [13]

metal ions [2].

**Figure 1.** Usage of organic substrates as electron donors in microbial fuel cell [10]

Complex organic matter is hydrolyzed to constituents that in the most cases are primarily fermented, but there are microorganisms that can completely oxidize such compounds with an electrode serving as the sole electron acceptor or incompletely oxidize these substrates with electron transfer to an electrode [10]. Acetate and some other minor fermentation acids can be completely oxidized to carbon dioxide and it is typically the primary source of electrons for current production [10].

MFC is considered to be used also for hydrogen production from the generated potential of the organic matter electrolysis by bacteria [2].

Microbial fuel cell technologies also are a promising and yet completely distinct approach to wastewater treatment as the treatment process can become a method of capturing energy in the form of electricity or hydrogen gas, rather than a drain on electrical energy [2]. Wastewater treatment processes currently employ the biological activities of complex microbial biofilms to remove organic pollutants [11]. The most significant energy savings associated with the use of MFCs for wastewater treatment, besides electricity generation, result from savings in expenses for aeration and solids handling, because the major operating costs for wastewater treatment are wastewater aeration, sludge treatment, and wastewater pumping. The MFC process is inherently an anaerobic process, although, oxygen can diffuse into the system resulting in some aerobic organic matter removal [10].

At the same time, wastewater contains high concentrations of xenobiotics, such as heavy metal ions that have an overwhelming harmful effect towards all living organisms. These substances even in small concentration in the environment cause the increasing inhibition of physiological and biochemical properties of the most bacteria. Despite that, some genera of bacteria possess high resistance according to toxic heavy metals influence because of functioning of highlyefficient mechanisms of antioxidant defense system, ion efflux transport enzyme complexes etc. Examples of these genuses are *Feroplasma*, *Streptomyces*, *Thiobacillus*, *Desulfuromonas* etc [12]. The specific metal-transport systems that were found in gram-negative bacteria which support transport of iron, zinc and manganese, copper, nickel and cobalt are schematically presented in figure 2 [13].

**Figure 1.** Usage of organic substrates as electron donors in microbial fuel cell [10]

current production [10].

34 Technology and Application of Microbial Fuel Cells

the organic matter electrolysis by bacteria [2].

resulting in some aerobic organic matter removal [10].

Complex organic matter is hydrolyzed to constituents that in the most cases are primarily fermented, but there are microorganisms that can completely oxidize such compounds with an electrode serving as the sole electron acceptor or incompletely oxidize these substrates with electron transfer to an electrode [10]. Acetate and some other minor fermentation acids can be completely oxidized to carbon dioxide and it is typically the primary source of electrons for

MFC is considered to be used also for hydrogen production from the generated potential of

Microbial fuel cell technologies also are a promising and yet completely distinct approach to wastewater treatment as the treatment process can become a method of capturing energy in the form of electricity or hydrogen gas, rather than a drain on electrical energy [2]. Wastewater treatment processes currently employ the biological activities of complex microbial biofilms to remove organic pollutants [11]. The most significant energy savings associated with the use of MFCs for wastewater treatment, besides electricity generation, result from savings in expenses for aeration and solids handling, because the major operating costs for wastewater treatment are wastewater aeration, sludge treatment, and wastewater pumping. The MFC process is inherently an anaerobic process, although, oxygen can diffuse into the system

At the same time, wastewater contains high concentrations of xenobiotics, such as heavy metal ions that have an overwhelming harmful effect towards all living organisms. These substances even in small concentration in the environment cause the increasing inhibition of physiological and biochemical properties of the most bacteria. Despite that, some genera of bacteria possess high resistance according to toxic heavy metals influence because of functioning of highly-

**Figure 2.** Schematic metal homeostasis models for iron, zinc and manganese, copper, nickel and cobalt in gram-nega‐ tive bacteria [13]

The variety of ion transport systems in gram-negative bacteria represents sophisticated mechanisms of bacterial cell metal homeostasis regulation. This is possible because of the formation of specific protein-metal coordination complexes used to effect uptake, efflux, intracellular trafficking within compartments, and storage [12].

Previous research has shown that gram-negative bacterium *Desulfuromonas acetoxidans* possesses resistance to copper, cadmium, lead, zinc, manganese, iron, etc. [14, 15]. Bacteria of *Desulfuromonas* genus are also shown to be highly effective in term of electricity generation [2, 16, 17]. However the research on its application as the anode biocatalyst in MFC is inadequate. *Desulfuromonas* sp. can be very promising for MFC development because of inexpensive cultivation medium, high survival rate and resistance to toxic xenobiotics such as the various metal ions [2].

*D. acetoxidans,* which belongs to the class ∂-*Proteobacteria,* are uncolored obligate anaerobes that inhabit sulfur containing aquatic environments [18]. *D. acetoxidans* supports reductive stage of sulfur cycle in the nature, but it can not possess an ability to reduce sulfate or other oxoanions of this element. Sulfur is a crucial component of various biological active substances, such as vitamins, coenzymes, several amino acids etc. This is an element with variable valence, which participates in the different chemical and biochemical redox-reactions [19]. Sulfur deposits formation is tightly bound with the process of sulfate-reduction, which is carried out exclu‐ sively by microorganisms. SO4 2-is the prevalent hydrogen acceptor in the processes of organic matter destructions under anaerobic conditions. Majority of strains can use unspecific electron acceptors, such as L-malate or fumarate, instead of sulfur [20]. S0 -reduction by *D. acetoxidans* causes hydrogen sulfide production. Using metal-resistant strains of these bacteria also helps overcome H2S toxicity since divalent cations will interact with sulfide ions, forming insoluble precipitates in form of metal sulfides. *D. acetoxidans* contains NiFe-hydrogenase [20], which catalyses hydrogen uptake and production; polysulfide reductase [19], which supports sulfur reduction with hydrogen sulphide formation, and specific metaloreductase that reduce Fe3+and Mn4+ with formation of magnetite (Fe3O4), siderite (FeCO3) and rhodochrosite (MnCO3) as final products of Fe (ІІІ)-and Mn (IV)-dissimilative reductions [21].

and 2CO2 as anaplerotic reaction while most organisms oxidizing acetate to CO2 by using the glyoxylate bypass for this function. Besides acetate, *D. acetoxidans* can completely oxidize Lmalate, fumarate, propionate, ethanol etc as electron donors. *D. acetoxidans* was one of the first electrogenic bacterium described, a microorganism performing complete oxidation of an organic substrate with electron transfer directly to the electrode. It was calculated that in the fuel cell that contains acetate as the sole electron donor up to 82% of acetate is oxidized by *D. acetoxidans* with an electrode as the terminal electron acceptor [29]. Therefore, *D. acetoxidans* accumulates energy for growth by electron transfer to the electrodes. Similar results were obtained with *Geobacter metallireducens*, oxidizing aromatic compounds, and the predominant‐

The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control

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37

Thus, *D. acetoxidans* has a crucial role in the biosphere and shows prospect of prosperous development of microbial fuel cell with simultaneous control of toxic metals environmental pollution because of formation of insoluble precipitates of metal sulfides. Although profound analyses of *D. acetoxidans* physiology and its linkage with power generation in MFC and organic matter consumption need to be established for its efficient application as the anode

Microbial strain *D. acetoxidans* IMV B-7384, which was applied in these investigations, belongs to the Ukrainian Collection of Microorganisms of D.K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine. Bacteria have been cultivated under the anaerobic conditions in the modified Postgaite C medium [20] in which sterile sulfur (10 g/l) and biotin (20 µg) were added before cultivation. Biotin served as a growth factor. Optimal pH for growth was 6.8-7.5

Bacterial growth commonly can be investigated by the registration of bacterial suspension turbidity or by the methods of dynamic or static light dispersion. The new method of rapid measurement of cell size distribution and their relative content, which is based on cell light scattering changes [31, 32] is proposed in this study. It includes the sounding of flow suspended bacterial cells by monochromatic coherent light, the registration of cooperative signals of sounding radiation and the explored microbiological objects by detecting of amplitudes and duration of scattered light impulses. The distribution of particles in size is determined on the basis of the measured functional dependence of the number of registered particles upon the amplitude and duration of corresponding electric impulses on the photoreceiver output by

C.

**2.2. Cell size distribution and relative content measurement**

solving integral equation of Fredholm of the first kind (1):

ly freshwater *G. sulfurreducens* [17, 30].

biocatalyst in microbial fuel cell.

and optimal temperature was 30 0

**2.1. Microbial strains, medium and cultivation**

**2. Methodology**

Several redox-proteins have been elucidated of the cells of *D. acetoxidans* [22]. This bacterium, similarly to *Desulfovibrio* sp*.*, contains huge amount of cytochromes *c*-type, which possibly are involved in the electron transport to the elemental sulfur. Multiheme cytochromes *c*-type are shown to possess metaloreductase activity, which possibly could have practical application in metal ions bioremediation from the environment. It was found that electric current production in MFCs operated with different organisms such as *Shewanella oneidensis*, *Pelobacter carbinoli‐ cum* or *Geobacter sulfurreducens* requires the presence of multiheme cytochromes *c* [23, 24]. Given the clear interest in *D. acetoxidans* for alternative processes of energy generation, the identification and understanding of the role of the macromolecular components responsible for these metabolic capabilities becomes a priority. Recently the final draft genome of *D. acetoxidans* was made available by the Joint Genome Institute [25] coding for "cytochromome" of 47 putative multiheme cytochromes *c*-type. Of those, up to now only the triheme cytochrome *c7* was characterized in detail. Its structure in the fully oxidized and fully reduced states, its thermodynamic and kinetic redox properties and its thermodynamic stability has been reported in the literature [22]. It shows high similarity to tetraheme cytochrome *c3*, extracted from sulfate-reducing bacteria [26]. Cytochrome synthesis by bacteria is observed under usage of insoluble extracellular electron acceptors, such as sulfur.

*D. acetoxidans* can obtain energy as a result of sulfur respiration and complete acetate oxidation via the citric acid cycle [27]:

Acetate- + 4S<sup>0</sup> + H<sup>+</sup> + 2H2O→2CO2+ 4H2S; *<sup>Δ</sup>*G =-39kJ×mol-1

This is the first investigated microorganism, which obtains energy by the complete acetate oxidation under the anaerobic conditions. It was shown that only 4% of consumed acetate by bacteria was assimilated into the cell material [28]. *D. acetoxidans* contains a succinyl-CoA: acetate CoA transferase instead of an acetyl-CoA synthetase and a succinyl-CoA synthetase. The succinyl-CoA: acetate-CoA transferase couples the formation of succinate from succinyl-CoA with the activation of acetate. The enzymes required for the assimilation of acetate and CO2 into pyruvate are acetyl-CoA synthetase and pyruvate synthase [27]. C14-labeling experi‐ ments shown that *D. acetoxidans* metabolism includes synthesis of oxaloacetate from acetate and 2CO2 as anaplerotic reaction while most organisms oxidizing acetate to CO2 by using the glyoxylate bypass for this function. Besides acetate, *D. acetoxidans* can completely oxidize Lmalate, fumarate, propionate, ethanol etc as electron donors. *D. acetoxidans* was one of the first electrogenic bacterium described, a microorganism performing complete oxidation of an organic substrate with electron transfer directly to the electrode. It was calculated that in the fuel cell that contains acetate as the sole electron donor up to 82% of acetate is oxidized by *D. acetoxidans* with an electrode as the terminal electron acceptor [29]. Therefore, *D. acetoxidans* accumulates energy for growth by electron transfer to the electrodes. Similar results were obtained with *Geobacter metallireducens*, oxidizing aromatic compounds, and the predominant‐ ly freshwater *G. sulfurreducens* [17, 30].

Thus, *D. acetoxidans* has a crucial role in the biosphere and shows prospect of prosperous development of microbial fuel cell with simultaneous control of toxic metals environmental pollution because of formation of insoluble precipitates of metal sulfides. Although profound analyses of *D. acetoxidans* physiology and its linkage with power generation in MFC and organic matter consumption need to be established for its efficient application as the anode biocatalyst in microbial fuel cell.

## **2. Methodology**

vitamins, coenzymes, several amino acids etc. This is an element with variable valence, which participates in the different chemical and biochemical redox-reactions [19]. Sulfur deposits formation is tightly bound with the process of sulfate-reduction, which is carried out exclu‐

matter destructions under anaerobic conditions. Majority of strains can use unspecific electron

causes hydrogen sulfide production. Using metal-resistant strains of these bacteria also helps overcome H2S toxicity since divalent cations will interact with sulfide ions, forming insoluble precipitates in form of metal sulfides. *D. acetoxidans* contains NiFe-hydrogenase [20], which catalyses hydrogen uptake and production; polysulfide reductase [19], which supports sulfur reduction with hydrogen sulphide formation, and specific metaloreductase that reduce Fe3+and Mn4+ with formation of magnetite (Fe3O4), siderite (FeCO3) and rhodochrosite

Several redox-proteins have been elucidated of the cells of *D. acetoxidans* [22]. This bacterium, similarly to *Desulfovibrio* sp*.*, contains huge amount of cytochromes *c*-type, which possibly are involved in the electron transport to the elemental sulfur. Multiheme cytochromes *c*-type are shown to possess metaloreductase activity, which possibly could have practical application in metal ions bioremediation from the environment. It was found that electric current production in MFCs operated with different organisms such as *Shewanella oneidensis*, *Pelobacter carbinoli‐ cum* or *Geobacter sulfurreducens* requires the presence of multiheme cytochromes *c* [23, 24]. Given the clear interest in *D. acetoxidans* for alternative processes of energy generation, the identification and understanding of the role of the macromolecular components responsible for these metabolic capabilities becomes a priority. Recently the final draft genome of *D. acetoxidans* was made available by the Joint Genome Institute [25] coding for "cytochromome" of 47 putative multiheme cytochromes *c*-type. Of those, up to now only the triheme cytochrome *c7* was characterized in detail. Its structure in the fully oxidized and fully reduced states, its thermodynamic and kinetic redox properties and its thermodynamic stability has been reported in the literature [22]. It shows high similarity to tetraheme cytochrome *c3*, extracted from sulfate-reducing bacteria [26]. Cytochrome synthesis by bacteria is observed under usage

*D. acetoxidans* can obtain energy as a result of sulfur respiration and complete acetate oxidation

This is the first investigated microorganism, which obtains energy by the complete acetate oxidation under the anaerobic conditions. It was shown that only 4% of consumed acetate by bacteria was assimilated into the cell material [28]. *D. acetoxidans* contains a succinyl-CoA: acetate CoA transferase instead of an acetyl-CoA synthetase and a succinyl-CoA synthetase. The succinyl-CoA: acetate-CoA transferase couples the formation of succinate from succinyl-CoA with the activation of acetate. The enzymes required for the assimilation of acetate and CO2 into pyruvate are acetyl-CoA synthetase and pyruvate synthase [27]. C14-labeling experi‐ ments shown that *D. acetoxidans* metabolism includes synthesis of oxaloacetate from acetate

(MnCO3) as final products of Fe (ІІІ)-and Mn (IV)-dissimilative reductions [21].

acceptors, such as L-malate or fumarate, instead of sulfur [20]. S0

of insoluble extracellular electron acceptors, such as sulfur.

+ 2H2O→2CO2+ 4H2S; *<sup>Δ</sup>*G =-39kJ×mol-1

via the citric acid cycle [27]:

+ 4S<sup>0</sup> + H<sup>+</sup>

Acetate-

2-is the prevalent hydrogen acceptor in the processes of organic


sively by microorganisms. SO4

36 Technology and Application of Microbial Fuel Cells

## **2.1. Microbial strains, medium and cultivation**

Microbial strain *D. acetoxidans* IMV B-7384, which was applied in these investigations, belongs to the Ukrainian Collection of Microorganisms of D.K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine. Bacteria have been cultivated under the anaerobic conditions in the modified Postgaite C medium [20] in which sterile sulfur (10 g/l) and biotin (20 µg) were added before cultivation. Biotin served as a growth factor. Optimal pH for growth was 6.8-7.5 and optimal temperature was 30 0 C.

## **2.2. Cell size distribution and relative content measurement**

Bacterial growth commonly can be investigated by the registration of bacterial suspension turbidity or by the methods of dynamic or static light dispersion. The new method of rapid measurement of cell size distribution and their relative content, which is based on cell light scattering changes [31, 32] is proposed in this study. It includes the sounding of flow suspended bacterial cells by monochromatic coherent light, the registration of cooperative signals of sounding radiation and the explored microbiological objects by detecting of amplitudes and duration of scattered light impulses. The distribution of particles in size is determined on the basis of the measured functional dependence of the number of registered particles upon the amplitude and duration of corresponding electric impulses on the photoreceiver output by solving integral equation of Fredholm of the first kind (1):

$$F\left(U,t\right) = \int\_{r\_{\min}}^{r\_{\max}} K\left(U,t,r\right) \mu\left(r\right) d\left(r\right),\tag{1}$$

**2.4. Measurement of catalase, superoxide dismutase activity and intracellular reduced**

Antioxidant defense system activity has been measured in the cell-free extract after the second, third and fourth day of bacterial growth. Cells were washed by 0.9% NaCl solution and disintegrated on the ultrasonic homogenizer at 22 kHz at 0ºC during five minutes. Cell debris were sedimented by centrifugation at 5640-8800g at 4 ºC during 30 minutes. Catalase activity was measured spectrophotometrically at λ=410 nm by the degree of breakdown of hydrogen peroxide in the cell-free extract [34]. Superoxide dismutase activity was measured by the level of inhibition of 2,3,5-triphenyltetrazolium chloride reduction that follows formazan formation (absorbance maximum λ=405 nm) [35, 36]. Reduced glutathione content has been measured by the degree of dithionitrobenzoic acid reduction in cell-free extract (absorbance maximum

The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control

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39

In this study two chamber microbial fuel cell has been constructed, in which *D. acetoxidans* IMV B-7384 was applied as the anode biocatalyst. Bacteria were cultivated in the modified Postgaite С medium [20] without sulfates under the anaerobic conditions and temperature

were applied as electrodes. Graphite is electrically conductive and conforms to the require‐ ments of electrodes in MFC: non-corrosive, highly conductive, large surface area, high porosity etc [2]. Bacteria were cultivated periodically in the anode chamber of constructed MFC under separate addition of such electron donors as acetic, lactic and fumaric acids in form of sodium salts in concentrations 6 and 9 g/l. Electrode material served as the sole electron acceptor. All

Electric current and voltage generation in constructed MFC were determined from the measured voltage drop across the resistor by multimeter DT-830C. The external load resistor with value 2.2 kΩ, which was shown to be the most optimal in constructed microbial fuel cell, was applied. The power output of an MFC was calculated from the measured voltage, EMFC,

Power generation by *D. acetoxidans* IMV B-7384 was investigated during twenty days of bacterial growth under the application of lactic, acetic and fumaric acids in form of their

C during twenty days in MFC. 0.1% potassium permanganate solution served as catholyte and bacterial suspension with 0.30±0.05 g/l dry cell weight/liter initial biomass served as anolyte respectively. 0.1% KMnO4 was replaced after 14 days of bacterial growth in MFC. Anode and cathode chambers with 0.3 l volume were separated by proton-exchange mem‐

. Graphite rods with the surface area of 130 cm<sup>2</sup>

*PIE* = ´ *MFC* (2)

**glutathione content**

λ=412 nm) [37].

25-28 0

**2.5. Microbial fuel cell construction and maintenance**

brane (Millipore) with surface area of 2.5 cm<sup>2</sup>

across the load and the current as (2):

sodium salts.

experiments were conducted under strictly sterile conditions.

**2.6. Power output measurements of the microbial fuel cell**

where rmin and rmax – upper and lower limits of particle size distribution, which is registered; *n(r)* – the function of particle size distribution;

*K(U,t,r)* – the function of distribution of normalized values of amplitude and duration of registered impulses of scattered light by the calibrating particles, which is a result of the previous probing of liquid flow by the monochromatic coherent light of the polymeric latex with set sizes and known refractive index.

However, presence of bacterial metabolism products in the growth medium could lead to errors in cell size distribution measurement because of additional light scattering. These errors were eliminated in the next way. Bacteria were cultivated in the liquid growth medium. Dependence between quantities of microbial cells and background particles in the growth medium had been determined during the time of bacterial cultivation. Liquid growth medium with and without bacterial cells was diluted in the same proportions by using highly purified liquid (deionized water). Then were registered separately the total quantity of cells and background particles in the highly purified liquid, which contains cells, and the total size distribution of background particles in the growth medium without cells. Then, relative content of bacterial cells was determined in the chosen interval of sizes, which equaled 0.3-1.9 µm. The cells relative content was measured by the calculation of quantity ratio of the set size cells to their total quantity. Specimens for determination of cell size distribution were prepared by dilution of 1 ml of bacterial suspension in 100 ml of deionized water [31, 32]. Measurements have been carried out by using the equipment PRM-6M, which was constructed at the Laboratory of Optical-Electronic Device of Faculty of Electronics of Ivan Franko National University of Lviv. The errors of cell size distribution measurement of constructed equipment are 5%.

#### **2.3. Electron microscopy**

After the third day of *D. acetoxidans* IMV B-7384 growth, the cells were harvested by centrifu‐ gation (2500 g, 30 min, 4°C) and washed three times in a buffer (50 mM potassium-phosphate buffer, pH 7.5). Intact cells were fixed at 1.5% KMnO4 solution during 20 min under the room temperature (20 0 C). Post-fixation was carried out with 1% OsO4 in cacodylic buffer during 90 min under 0 0 C. Fixed cells were washed and dehydrated in solutions with gradient concen‐ trations of ethanol and propylene oxide. Specimens were fixed in epoxy Epon 812. Ultrathin cross-sections were obtained with the ultramicrotome UMTP-6 and contrasted by lead citrate [33]. Electron photographs were obtained by transmission electron microscopes UEMB-100B and PEM-100 at acceleration voltage 75 kV. Final photographs magnification – 10000 times.

#### **2.4. Measurement of catalase, superoxide dismutase activity and intracellular reduced glutathione content**

Antioxidant defense system activity has been measured in the cell-free extract after the second, third and fourth day of bacterial growth. Cells were washed by 0.9% NaCl solution and disintegrated on the ultrasonic homogenizer at 22 kHz at 0ºC during five minutes. Cell debris were sedimented by centrifugation at 5640-8800g at 4 ºC during 30 minutes. Catalase activity was measured spectrophotometrically at λ=410 nm by the degree of breakdown of hydrogen peroxide in the cell-free extract [34]. Superoxide dismutase activity was measured by the level of inhibition of 2,3,5-triphenyltetrazolium chloride reduction that follows formazan formation (absorbance maximum λ=405 nm) [35, 36]. Reduced glutathione content has been measured by the degree of dithionitrobenzoic acid reduction in cell-free extract (absorbance maximum λ=412 nm) [37].

#### **2.5. Microbial fuel cell construction and maintenance**

( ) ( ) ( ) ( ) max

, ,, , <sup>=</sup> ò

where rmin and rmax – upper and lower limits of particle size distribution, which is registered;

*K(U,t,r)* – the function of distribution of normalized values of amplitude and duration of registered impulses of scattered light by the calibrating particles, which is a result of the previous probing of liquid flow by the monochromatic coherent light of the polymeric latex

However, presence of bacterial metabolism products in the growth medium could lead to errors in cell size distribution measurement because of additional light scattering. These errors were eliminated in the next way. Bacteria were cultivated in the liquid growth medium. Dependence between quantities of microbial cells and background particles in the growth medium had been determined during the time of bacterial cultivation. Liquid growth medium with and without bacterial cells was diluted in the same proportions by using highly purified liquid (deionized water). Then were registered separately the total quantity of cells and background particles in the highly purified liquid, which contains cells, and the total size distribution of background particles in the growth medium without cells. Then, relative content of bacterial cells was determined in the chosen interval of sizes, which equaled 0.3-1.9 µm. The cells relative content was measured by the calculation of quantity ratio of the set size cells to their total quantity. Specimens for determination of cell size distribution were prepared by dilution of 1 ml of bacterial suspension in 100 ml of deionized water [31, 32]. Measurements have been carried out by using the equipment PRM-6M, which was constructed at the Laboratory of Optical-Electronic Device of Faculty of Electronics of Ivan Franko National University of Lviv. The errors of cell size distribution measurement of constructed equipment

After the third day of *D. acetoxidans* IMV B-7384 growth, the cells were harvested by centrifu‐ gation (2500 g, 30 min, 4°C) and washed three times in a buffer (50 mM potassium-phosphate buffer, pH 7.5). Intact cells were fixed at 1.5% KMnO4 solution during 20 min under the room

trations of ethanol and propylene oxide. Specimens were fixed in epoxy Epon 812. Ultrathin cross-sections were obtained with the ultramicrotome UMTP-6 and contrasted by lead citrate [33]. Electron photographs were obtained by transmission electron microscopes UEMB-100B and PEM-100 at acceleration voltage 75 kV. Final photographs magnification – 10000 times.

C). Post-fixation was carried out with 1% OsO4 in cacodylic buffer during 90

C. Fixed cells were washed and dehydrated in solutions with gradient concen‐

*F Ut K Utrn r d r* (1)

min

*r*

*n(r)* – the function of particle size distribution;

38 Technology and Application of Microbial Fuel Cells

with set sizes and known refractive index.

are 5%.

**2.3. Electron microscopy**

temperature (20 0

min under 0 0

*r*

In this study two chamber microbial fuel cell has been constructed, in which *D. acetoxidans* IMV B-7384 was applied as the anode biocatalyst. Bacteria were cultivated in the modified Postgaite С medium [20] without sulfates under the anaerobic conditions and temperature 25-28 0 C during twenty days in MFC. 0.1% potassium permanganate solution served as catholyte and bacterial suspension with 0.30±0.05 g/l dry cell weight/liter initial biomass served as anolyte respectively. 0.1% KMnO4 was replaced after 14 days of bacterial growth in MFC. Anode and cathode chambers with 0.3 l volume were separated by proton-exchange mem‐ brane (Millipore) with surface area of 2.5 cm<sup>2</sup> . Graphite rods with the surface area of 130 cm<sup>2</sup> were applied as electrodes. Graphite is electrically conductive and conforms to the require‐ ments of electrodes in MFC: non-corrosive, highly conductive, large surface area, high porosity etc [2]. Bacteria were cultivated periodically in the anode chamber of constructed MFC under separate addition of such electron donors as acetic, lactic and fumaric acids in form of sodium salts in concentrations 6 and 9 g/l. Electrode material served as the sole electron acceptor. All experiments were conducted under strictly sterile conditions.

#### **2.6. Power output measurements of the microbial fuel cell**

Electric current and voltage generation in constructed MFC were determined from the measured voltage drop across the resistor by multimeter DT-830C. The external load resistor with value 2.2 kΩ, which was shown to be the most optimal in constructed microbial fuel cell, was applied. The power output of an MFC was calculated from the measured voltage, EMFC, across the load and the current as (2):

$$P = I \times E\_{MFC} \tag{2}$$

Power generation by *D. acetoxidans* IMV B-7384 was investigated during twenty days of bacterial growth under the application of lactic, acetic and fumaric acids in form of their sodium salts.

## **3. Results and discussion**

#### **3.1. Particularities of** *D. acetoxidans* **IMV B-7384 growth physiology**

Biomass accumulation of *D. acetoxidans* IMV B-7384, its cell size distribution and relative content changes have been carried out [14, 15]. Investigated bacterium has been shown to accumulate biomass the most intensively during third-fourth days of its growth. (fig. 3). Since bacterial growth processes were shown to be maximal during third-fourth days of cultivation, possibly, the highest power output in microbial fuel cell should be observed during this period. Bacterial growth usually is characterized by increasing of cell quantity or cell size. Thus, analyses of cell size distribution and their relative content allow to obtain more detailed data of cell growth and division processes under various cultivation conditions in comparison with standard turbidimetric method of biomass measurement. Proposed method of cell light scattering determination allows to calculate possible changes of cell division on the basis of cumulative analyses of three histogram parameters: cell size distribution maximum, cell relative content and half-width of cell size distribution. It can be the basis for inventing of new methodologies for obtaining of synchronous cell cultures and also for development of new

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Cell size distribution maximum equaled 0.55±0.01 µm on the third day of *D. acetoxidans* IMV B-7384 growth (period of maximal biomass accumulation). Cell relative content increased from 0.275 to 0.420 relative units under normal growth conditions on the third day of bacterial

0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8 2,0

Cell size, µm

**Figure 5.** Medial values of *D. acetoxidans* IMV B-7384 cell relative content and size distribution during the third day of

From the first to the fifth day of *D. acetoxidans* IMV B-7384 growth the maximum of cell size distribution was 0.49-0.55±0.01 µm and cell relative content with the maximum of size distribution changed from 0.275±0.011 to 0.398±0.011 relative units (table 1). Half-width of cell size distribution curves decreased from 0.23±0.01 to 0.14±0.01 µm with the increase of bacterial

It indicated the decrease of cell size variations with the increase of cultivation time. Obviously, it is caused by intensive bacterial division on the third-fourth days of their cultivation. As a result cell's relative content with lower size distribution maximum (0.49±0.01 µm) increased in comparison with its initial value with higher maximum (0.55±0.01 µm). Possible inhibition of cell division on the fifth day of bacterial growth caused increase of cell relative content with

effective cytometers with low self-cost and high productivity.

0 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40 0,45

higher size distribution maximum, which equaled 0.55±0.02 µm.

Cell content, relative units

cultivation time from the first to the third day.

cultivation (fig. 5).

growth (five repeats, *P* ≤ 0.05)

**Figure 3.** Changes of *D. acetoxidans* IMV B-7384 biomass during ten days under normal cultivation conditions (addi‐ tion of lactic acid (6 g/l) and elemental sulfur (10 g/l) respectively as electron donor and acceptor)

Electron micrographs of *D. acetoxidans* IMV B-7384 cross-sections are presented on the fig. 4. Cells were obtained on the third day of their growth when maximal bacterial biomass accumulation was observed. These are rod shaped or slightly curved cells.

**Figure 4.** Cross-sections of the cells of *D. acetoxidans* IMV B-7384, harvested under normal growth conditions (third day) (electron micrograph (TEM), ×10000 times; bar, 0.5 μm)

Bacterial growth usually is characterized by increasing of cell quantity or cell size. Thus, analyses of cell size distribution and their relative content allow to obtain more detailed data of cell growth and division processes under various cultivation conditions in comparison with standard turbidimetric method of biomass measurement. Proposed method of cell light scattering determination allows to calculate possible changes of cell division on the basis of cumulative analyses of three histogram parameters: cell size distribution maximum, cell relative content and half-width of cell size distribution. It can be the basis for inventing of new methodologies for obtaining of synchronous cell cultures and also for development of new effective cytometers with low self-cost and high productivity.

**3. Results and discussion**

40 Technology and Application of Microbial Fuel Cells

**3.1. Particularities of** *D. acetoxidans* **IMV B-7384 growth physiology**

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1 1,2 1,3

Biomass, g/l

day) (electron micrograph (TEM), ×10000 times; bar, 0.5 μm)

Biomass accumulation of *D. acetoxidans* IMV B-7384, its cell size distribution and relative content changes have been carried out [14, 15]. Investigated bacterium has been shown to accumulate biomass the most intensively during third-fourth days of its growth. (fig. 3). Since bacterial growth processes were shown to be maximal during third-fourth days of cultivation, possibly, the highest power output in microbial fuel cell should be observed during this period.

0123456789 10 11

Time of cultivation, days

**Figure 3.** Changes of *D. acetoxidans* IMV B-7384 biomass during ten days under normal cultivation conditions (addi‐

Electron micrographs of *D. acetoxidans* IMV B-7384 cross-sections are presented on the fig. 4. Cells were obtained on the third day of their growth when maximal bacterial biomass

**Figure 4.** Cross-sections of the cells of *D. acetoxidans* IMV B-7384, harvested under normal growth conditions (third

tion of lactic acid (6 g/l) and elemental sulfur (10 g/l) respectively as electron donor and acceptor)

accumulation was observed. These are rod shaped or slightly curved cells.

Cell size distribution maximum equaled 0.55±0.01 µm on the third day of *D. acetoxidans* IMV B-7384 growth (period of maximal biomass accumulation). Cell relative content increased from 0.275 to 0.420 relative units under normal growth conditions on the third day of bacterial cultivation (fig. 5).

**Figure 5.** Medial values of *D. acetoxidans* IMV B-7384 cell relative content and size distribution during the third day of growth (five repeats, *P* ≤ 0.05)

From the first to the fifth day of *D. acetoxidans* IMV B-7384 growth the maximum of cell size distribution was 0.49-0.55±0.01 µm and cell relative content with the maximum of size distribution changed from 0.275±0.011 to 0.398±0.011 relative units (table 1). Half-width of cell size distribution curves decreased from 0.23±0.01 to 0.14±0.01 µm with the increase of bacterial cultivation time from the first to the third day.

It indicated the decrease of cell size variations with the increase of cultivation time. Obviously, it is caused by intensive bacterial division on the third-fourth days of their cultivation. As a result cell's relative content with lower size distribution maximum (0.49±0.01 µm) increased in comparison with its initial value with higher maximum (0.55±0.01 µm). Possible inhibition of cell division on the fifth day of bacterial growth caused increase of cell relative content with higher size distribution maximum, which equaled 0.55±0.02 µm.


reaction [41]. Thus, aforementioned components of antioxidant defense system protect the cell

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It was revealed that under the influence of various concentrations of FeSO4, FeCl3, MnCl2, NiCl2, CoCl2 and CuCl2 on the cells of *D. acetoxidans* IMV B-7384 activity of antioxidant defense system appears in spite of their obligate anaerobic metabolism. At fig. 6 is presented the complete scheme of the dependences between the highest values of catalase and superoxide dismutase activity and glutathione content, which were observed in the cells of investigated

**Figure 6.** Maximal observed values of reduced glutathione content and superoxide dismutase, and catalase activity in the cell-free extract of *D. acetoxidans* IMV B-7384 under the influence of 0.5-2.5 mM of FeSO4, FeCl3, MnCl2, NiCl2,

Obtained results show that investigated bacteria possess specific mechanisms of rapid defense against toxic influence of external factors, such as high concentrations of various metal ions. It allows to assume their tolerance according to detrimental xenobiotics, which wastewaters are enriched with. This shows the prospect of *D. acetoxidans* IMV B-7384 application into wastewater treatment with simultaneous power generation in MFC. Also specific activity of antioxidant defense system enzymes, such as catalase and superoxide dismutase could help to prevent harmful influence of reactive oxygen species on the integrity of proton-exchange membranes in microbial fuel cells. Since catalase causes two-electron decomposition of H2O2

with O2 and H2O production, this enzyme could prevent Fenton reaction: Мn+(=Cu+

reaction could significantly increase longevity of expensive proton-exchange membranes in microbial fuel cell, such as Nafion, with the next increasing of reliability and durability of

, Fe2+, Ti3+, Co2+)+•ОН+ОН-

, Fe2+, Ti3+,

[42]. Neutralization of products of this

against oxidative stress, which may be caused by reactive oxygen species.

bacteria during four days of their cultivation.

CoCl2 and CuCl2 during four days of bacterial cultivation [36, 38, 39]

Co2+)+H2O2 → М(n+1)+(=Cu+

power generation in MFC.

**Table 1.** Changes of cell size distribution maximum, cell relative content with size distribution maximum and halfwidth of curves of cell size distribution of *D. acetoxidans* IMV B-7384 during five days of cultivation (five repeats, *P* ≤ 0.05)

Thus, it was shown that the maximal biomass accumulation is observed during third-fourth day of bacterial growth. High value of cell relative content with size distribution maximum and intensive decrease of half-width of cell size distribution indicates on significant increase of cell quantity with lower size distribution maximum. It is a possible result of intensive cell division during this time. Overall analysis of mentioned above parameters allow to assume that *D. acetoxidans* IMV B-7384 are in the middle of exponential phase of growth during the third day of their cultivation under normal growth conditions. Thus, the most intensive biosynthesis processes and power generation in microbial fuel cell possibly overlap this period of bacterial cultivation or immediately afterwards.

#### **3.2. Response of** *D. acetoxidans* **IMV B-7384 cells to the influence of heavy metals**

The activity of antioxidant defense system of *D. acetoxidans* IMV B-7384 has been revealed under the influence of external stress factors, such as Ferric iron, Ferrous iron, Nickel, Cobalt and Copper salts. It includes:


Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radical etc are produced as a result of prolonged influence of oxygen or toxic xenobiotics, such as heavy metal ions, on the living cell. Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the most abundant intracellular thiol-dependent antioxidant, which protects living cells against oxidative stress. It has low redox-potential (Е'0=-240 mV under pH=7.0) and it is constantly maintained in reduced state because of NADF-glutathione reductase functioning. Therefore it serves as cellular redox-buffer [40]. Superoxide dismutase catalyzes disproportionation of superoxides with oxygen and hydrogen peroxide formation. Catalase causes decomposition of produced H2O2 with neutralization of its toxicity. Н2О and О2 are final products of this reaction [41]. Thus, aforementioned components of antioxidant defense system protect the cell against oxidative stress, which may be caused by reactive oxygen species.

It was revealed that under the influence of various concentrations of FeSO4, FeCl3, MnCl2, NiCl2, CoCl2 and CuCl2 on the cells of *D. acetoxidans* IMV B-7384 activity of antioxidant defense system appears in spite of their obligate anaerobic metabolism. At fig. 6 is presented the complete scheme of the dependences between the highest values of catalase and superoxide dismutase activity and glutathione content, which were observed in the cells of investigated bacteria during four days of their cultivation.

Thus, it was shown that the maximal biomass accumulation is observed during third-fourth day of bacterial growth. High value of cell relative content with size distribution maximum and intensive decrease of half-width of cell size distribution indicates on significant increase of cell quantity with lower size distribution maximum. It is a possible result of intensive cell division during this time. Overall analysis of mentioned above parameters allow to assume that *D. acetoxidans* IMV B-7384 are in the middle of exponential phase of growth during the third day of their cultivation under normal growth conditions. Thus, the most intensive biosynthesis processes and power generation in microbial fuel cell possibly overlap this period

**Table 1.** Changes of cell size distribution maximum, cell relative content with size distribution maximum and halfwidth of curves of cell size distribution of *D. acetoxidans* IMV B-7384 during five days of cultivation (five repeats, *P* ≤

 0.55±0.01 0.275±0.011 0.23±0.01 0.55±0.02 0.268±0.009 0.23±0.03 0.55±0.01 0.420±0.022 0.14±0.01 0.49±0.01 0.383±0.14 0.14±0.02 0.55±0.02 0.398±0.011 0.16±0.01

**Cell content with size distribution maximum, relative units**

**Half-width of cell size distribution curves, μm**

**3.2. Response of** *D. acetoxidans* **IMV B-7384 cells to the influence of heavy metals**

The activity of antioxidant defense system of *D. acetoxidans* IMV B-7384 has been revealed under the influence of external stress factors, such as Ferric iron, Ferrous iron, Nickel, Cobalt

**•** biosynthesis of reduced glutathione (GSH), a tripeptide that serves as universal electron

**•** activity of catalase and superoxide dismutase, a basic enzymes of antioxidant defense

Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radical etc are produced as a result of prolonged influence of oxygen or toxic xenobiotics, such as heavy metal ions, on the living cell. Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the most abundant intracellular thiol-dependent antioxidant, which protects living cells against oxidative stress. It has low redox-potential (Е'0=-240 mV under pH=7.0) and it is constantly maintained in reduced state because of NADF-glutathione reductase functioning. Therefore it serves as cellular redox-buffer [40]. Superoxide dismutase catalyzes disproportionation of superoxides with oxygen and hydrogen peroxide formation. Catalase causes decomposition of produced H2O2 with neutralization of its toxicity. Н2О and О2 are final products of this

of bacterial cultivation or immediately afterwards.

**Cell size distribution maximum, μm**

donor detoxifying reactive oxygen species [38];

and Copper salts. It includes:

system [36, 39].

**Time of cultivation, day**

42 Technology and Application of Microbial Fuel Cells

0.05)

**Figure 6.** Maximal observed values of reduced glutathione content and superoxide dismutase, and catalase activity in the cell-free extract of *D. acetoxidans* IMV B-7384 under the influence of 0.5-2.5 mM of FeSO4, FeCl3, MnCl2, NiCl2, CoCl2 and CuCl2 during four days of bacterial cultivation [36, 38, 39]

Obtained results show that investigated bacteria possess specific mechanisms of rapid defense against toxic influence of external factors, such as high concentrations of various metal ions. It allows to assume their tolerance according to detrimental xenobiotics, which wastewaters are enriched with. This shows the prospect of *D. acetoxidans* IMV B-7384 application into wastewater treatment with simultaneous power generation in MFC. Also specific activity of antioxidant defense system enzymes, such as catalase and superoxide dismutase could help to prevent harmful influence of reactive oxygen species on the integrity of proton-exchange membranes in microbial fuel cells. Since catalase causes two-electron decomposition of H2O2 with O2 and H2O production, this enzyme could prevent Fenton reaction: Мn+(=Cu+ , Fe2+, Ti3+, Co2+)+H2O2 → М(n+1)+(=Cu+ , Fe2+, Ti3+, Co2+)+•ОН+ОН- [42]. Neutralization of products of this reaction could significantly increase longevity of expensive proton-exchange membranes in microbial fuel cell, such as Nafion, with the next increasing of reliability and durability of power generation in MFC.

#### **3.3.Power generation by** *D. acetoxidans* **IMV B-7384 in constructed microbial-anode fuel cell**

Therefore, all further experiments on MFC development were carried out with application of such external load resistance (2.2 kΩ), which was shown to be the most effective in term of

0123456789

 Current strength, mA Voltage, V

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0,1 0,2 0,3 0,4 0,5 0,6

Voltage, V

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45

under

External load resistance, kÎ hm

**Figure 8.** Volt-ampere characteristic of constructed MFC under the influence of various external load resistances on the third-beginning of the fourth days of *D. acetoxidans* IMV B-7384 growth under normal cultivation conditions

Accurate selection of external load resistance plays an important role in effective MFC development, because it significantly influences the value of generated power by bacteria,

*3.3.2. Power output in MFC under usage of various electron donors by D. acetoxidans IMV B-7384 in*

addition the external electron acceptors, such as sulfur and Ferric iron in concentrations, which are favorable for *D. acetoxidans* IMV B-7384 metabolism [43]. Excluding of these additional electron acceptors and external load resistance optimization caused increasing of power output

Lactic acid was applied as the sole organic electron donor in concentrations 6 and 9 g/l during *D. acetoxidans* IMV B-7384 cultivation in the anode chamber of constructed microbial fuel cell. The highest power output equaled 5.74±0.29 mW/m2 on the 64 hour (third day) of *D. acetoxi‐ dans* IMV B-7384 cultivation under addition of 6 g/l of C3H6O3 (fig. 9). Its value decreased by 41% in comparison with the maximal power, obtained in this investigation by 250 hour of bacterial cultivation (tenth day). Power output equaled 1.33±0.18 mW/m2 on the 480 hour of bacterial cultivation (twentieth day), which was lower by 77% in comparison with its maximal

In previous researches it was shown that maximal power output equaled 4.3 mW/m2

electric power generation by *D. acetoxidans* IMV B-7384 in constructed MFC.

0 0,05 0,10 0,15 0,20 0,25 0,30

which are cultivated under different conditions in constructed MFC.

Current strength, mA

*3.3.2.1. Lactic acid and power output in MFC*

*MFC*

value.

in constructed MFC.

*3.3.1. The influence of external load resistance on power generation by D. acetoxidans IMV B-7384 as the anode biocatalyst in constructed MFC*

External load resistance in microbial fuel cell is one of the crucial factors, which influence the electricity generation. Correlation between value of external load resistance and power generation in constructed MFC was determined. The influence of changes of external load resistance on volt-ampere characteristic of constructed MFC was investigated during *D. acetoxidans* IMV B-7384 cultivation in Postgaite C medium under normal growth conditions on the third-beginning of the fourth day, when the highest power generation was observed (fig. 7).

**Figure 7.** Power generation by *D. acetoxidans* IMV B-7384 during eight days under normal cultivation conditions in MFC, and application of 0.2 kΩ external load resistor

The maximal value of power density equaled 4.7 mW/m2 on 84 hour of bacterial cultivation with addition of lactate (6 g/l) as electron donor and elemental sulfur (10 g/l) as electron acceptor (normal growth conditions), and application of an external load resistor of 0.2 kΩ. With the increase of cultivation time till 192 hour (eighth day of growth) generated power decreased by 42% in comparison with its maximal value.

With the aim to determine the most optimal load in term of electricity generation by *D. acetoxidans* IMV B-7384 in constructed MFC, external load resistors with values of 0.45-8.15 kΩ were applied in this investigation. The highest current strength and voltage were observed in constructed MFC under application of external load resistor with value of 2.2 kΩ (fig. 8). The highest power density, which was obtained in MFC under these conditions, equaled 5.8 mW/m2 . Increasing of external load resistor value caused decrease of generated power.

Therefore, all further experiments on MFC development were carried out with application of such external load resistance (2.2 kΩ), which was shown to be the most effective in term of electric power generation by *D. acetoxidans* IMV B-7384 in constructed MFC.

**Figure 8.** Volt-ampere characteristic of constructed MFC under the influence of various external load resistances on the third-beginning of the fourth days of *D. acetoxidans* IMV B-7384 growth under normal cultivation conditions

Accurate selection of external load resistance plays an important role in effective MFC development, because it significantly influences the value of generated power by bacteria, which are cultivated under different conditions in constructed MFC.

#### *3.3.2. Power output in MFC under usage of various electron donors by D. acetoxidans IMV B-7384 in MFC*

#### *3.3.2.1. Lactic acid and power output in MFC*

**3.3.Power generation by** *D. acetoxidans* **IMV B-7384 in constructed microbial-anode fuel**

*3.3.1. The influence of external load resistance on power generation by D. acetoxidans IMV B-7384 as*

External load resistance in microbial fuel cell is one of the crucial factors, which influence the electricity generation. Correlation between value of external load resistance and power generation in constructed MFC was determined. The influence of changes of external load resistance on volt-ampere characteristic of constructed MFC was investigated during *D. acetoxidans* IMV B-7384 cultivation in Postgaite C medium under normal growth conditions on the third-beginning of the fourth day, when the highest power generation was observed

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

Time of cultivation, hours

on 84 hour of bacterial cultivation

**Figure 7.** Power generation by *D. acetoxidans* IMV B-7384 during eight days under normal cultivation conditions in

with addition of lactate (6 g/l) as electron donor and elemental sulfur (10 g/l) as electron acceptor (normal growth conditions), and application of an external load resistor of 0.2 kΩ. With the increase of cultivation time till 192 hour (eighth day of growth) generated power

With the aim to determine the most optimal load in term of electricity generation by *D. acetoxidans* IMV B-7384 in constructed MFC, external load resistors with values of 0.45-8.15 kΩ were applied in this investigation. The highest current strength and voltage were observed in constructed MFC under application of external load resistor with value of 2.2 kΩ (fig. 8). The highest power density, which was obtained in MFC under these conditions, equaled 5.8

. Increasing of external load resistor value caused decrease of generated power.

**cell**

(fig. 7).

0 0,02

MFC, and application of 0.2 kΩ external load resistor

The maximal value of power density equaled 4.7 mW/m2

decreased by 42% in comparison with its maximal value.

0,03

0,04

0,05

Power, mW/m

mW/m2

2

0,06

0,07

*the anode biocatalyst in constructed MFC*

44 Technology and Application of Microbial Fuel Cells

In previous researches it was shown that maximal power output equaled 4.3 mW/m2 under addition the external electron acceptors, such as sulfur and Ferric iron in concentrations, which are favorable for *D. acetoxidans* IMV B-7384 metabolism [43]. Excluding of these additional electron acceptors and external load resistance optimization caused increasing of power output in constructed MFC.

Lactic acid was applied as the sole organic electron donor in concentrations 6 and 9 g/l during *D. acetoxidans* IMV B-7384 cultivation in the anode chamber of constructed microbial fuel cell. The highest power output equaled 5.74±0.29 mW/m2 on the 64 hour (third day) of *D. acetoxi‐ dans* IMV B-7384 cultivation under addition of 6 g/l of C3H6O3 (fig. 9). Its value decreased by 41% in comparison with the maximal power, obtained in this investigation by 250 hour of bacterial cultivation (tenth day). Power output equaled 1.33±0.18 mW/m2 on the 480 hour of bacterial cultivation (twentieth day), which was lower by 77% in comparison with its maximal value.

Thus, gradual lactate oxidation and the following diminishing of its quantity in the anode chamber because of bacterial growth in MFC caused gradual decrease of produced power with the increase of duration of cultivation time. Thus, application of lactic acid as the single electron donor in the aforementioned concentration in constructed MFC can't be used for sustainable long-term electricity generation.

Concentration of lactic acid in the anode chamber has been increased up to 9 g/l. Under these cultivation conditions the highest power output equaled 5.90±0.21 mW/m2 on the 64 hour (third day) of *D. acetoxidans* IMV B-7384 cultivation (fig. 10). After ten days it decreased by 37% in comparison with its highest measured value. By the 480 hour (the twentieth day) of *D. acetoxidans* IMV B-7384 cultivation power production in constructed MFC decreased by 33% in comparison with its maximal measured value.

value decreased by 38% on the 250 hour (the tenth day) and by 43% on the 480 hour (the twentieth day) of *D. acetoxidans* IMV B-7384 cultivation. The medial power density, which was

**Figure 10.** Power density in MFC during twenty days of *D. acetoxidans* IMV B-7384 cultivation under application of 9

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

Time of cultivation, hours

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0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

**Figure 11.** Power density in MFC during twenty days under addition of 6 g/l of fumaric acid into the growth medium

Thus, application of fumaric acid (6 g/l) causes higher stability of power generation by investigated bacteria in constructed MFC in comparison with the lactic acid in the same concentration. Therefore, fumaric acid is more preferable electron donor in term of power

generation by *D. acetoxidans* IMV B-7384 in comparison with application of lactic acid.

Time of cultivation, hours

.

observed under these cultivation conditions equaled 3.58±0.20 mW/m2

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5

of *D. acetoxidans* IMV B-7384

Power density, mW/m

2

g/l of lactic acid

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5

Power density, mW/m

2

**Figure 9.** Power density in MFC during twenty days under addition of 6 g/l of lactic acid into the growth medium of *D. acetoxidans* IMV B-7384

Increase of lactic acid content in the anode chamber caused enhance of stability of power generation in constructed MFC, but such manipulation did not boost its value significantly in comparison with application of lower concentration of lactic acid.

Thus, lactic acid as the sole electron donor in high concentrations supports durability of constructed MFC with application of *D. acetoxidans* IMV B-7384 as the anode biocatalyst. However, its effectiveness does not change significantly under the application of lower and higher lactic acid concentrations.

#### *3.3.2.2. Fumaric acid and power output in MFC*

With the aim to determine the most optimal electron donor in term of electricity generation lactic acid has been substituted by fumaric acid in the anode chamber of constructed MFC. The highest value of generated power equaled 5.69±0.29 mW/m2 on the 56 hour (the third day) of bacterial cultivation under usage of 6 g/l of fumaric acid as the sole electron donor (fig. 11). It's

Thus, gradual lactate oxidation and the following diminishing of its quantity in the anode chamber because of bacterial growth in MFC caused gradual decrease of produced power with the increase of duration of cultivation time. Thus, application of lactic acid as the single electron donor in the aforementioned concentration in constructed MFC can't be used for sustainable

Concentration of lactic acid in the anode chamber has been increased up to 9 g/l. Under these

day) of *D. acetoxidans* IMV B-7384 cultivation (fig. 10). After ten days it decreased by 37% in comparison with its highest measured value. By the 480 hour (the twentieth day) of *D. acetoxidans* IMV B-7384 cultivation power production in constructed MFC decreased by 33%

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

**Figure 9.** Power density in MFC during twenty days under addition of 6 g/l of lactic acid into the growth medium of *D.*

Increase of lactic acid content in the anode chamber caused enhance of stability of power generation in constructed MFC, but such manipulation did not boost its value significantly in

Thus, lactic acid as the sole electron donor in high concentrations supports durability of constructed MFC with application of *D. acetoxidans* IMV B-7384 as the anode biocatalyst. However, its effectiveness does not change significantly under the application of lower and

With the aim to determine the most optimal electron donor in term of electricity generation lactic acid has been substituted by fumaric acid in the anode chamber of constructed MFC. The

bacterial cultivation under usage of 6 g/l of fumaric acid as the sole electron donor (fig. 11). It's

comparison with application of lower concentration of lactic acid.

highest value of generated power equaled 5.69±0.29 mW/m2

Time of cultivation, hours

on the 64 hour (third

on the 56 hour (the third day) of

cultivation conditions the highest power output equaled 5.90±0.21 mW/m2

long-term electricity generation.

46 Technology and Application of Microbial Fuel Cells

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5

*acetoxidans* IMV B-7384

higher lactic acid concentrations.

*3.3.2.2. Fumaric acid and power output in MFC*

Power density, mW/m

2

in comparison with its maximal measured value.

**Figure 10.** Power density in MFC during twenty days of *D. acetoxidans* IMV B-7384 cultivation under application of 9 g/l of lactic acid

value decreased by 38% on the 250 hour (the tenth day) and by 43% on the 480 hour (the twentieth day) of *D. acetoxidans* IMV B-7384 cultivation. The medial power density, which was observed under these cultivation conditions equaled 3.58±0.20 mW/m2 .

**Figure 11.** Power density in MFC during twenty days under addition of 6 g/l of fumaric acid into the growth medium of *D. acetoxidans* IMV B-7384

Thus, application of fumaric acid (6 g/l) causes higher stability of power generation by investigated bacteria in constructed MFC in comparison with the lactic acid in the same concentration. Therefore, fumaric acid is more preferable electron donor in term of power generation by *D. acetoxidans* IMV B-7384 in comparison with application of lactic acid.

It was shown that increase of fumaric acid concentration up to 9 g/l caused maximal power generation (2.38±0.10 mW/m2 ) at 56 hour (fig. 12).

**Figure 12.** Power density in MFC during twenty days under addition of 9 g/l of fumaric acid into the growth medium of *D. acetoxidans* IMV B-7384

usage of 6 g/l of lactic acid by bacteria in the anode chamber of MFC. Increase of cultivation

**Figure 13.** Power density in MFC during twenty days under addition of 6 g/l of acetic acid into the growth medium of

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

Time of cultivation, hours

The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control

http://dx.doi.org/10.5772/58347

49

Thus, increase of acetic acid concentration as electron donor in the anode chamber of MFC caused partial inhibition of electricity generation in comparison with application of its lower

It can be summarized that low concentrations of investigated organic acids caused higher stability of power generation in constructed microbial fuel cell apart from their higher

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

**Figure 14.** Power density in MFC during twenty days under addition of 9 g/l of acetic acid into the growth medium of

Time of cultivation, hours

.

time up to 480 hour caused decrease of power value till 1.60±0.11 mW/m2

content.

concentrations.

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5

*D. acetoxidans* IMV B-7384

Power density, mW/m

2

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5

*D. acetoxidans* IMV B-7384

Power density, mW/m

2

It was less by 2.4 times in comparison with the highest power density value, which was observed under bacterial cultivation with application of less concentration of fumaric acid (6 g/l). The minimal observed power density value equaled 1.95±0.047 mW/m2 on the 160 hour of bacterial cultivation under these conditions. Increasing of cultivation time caused insignif‐ icant enhance of power production. On the 480 hour it value equaled 1.97±0.07 mW/m2 , which was less by 17% in comparison with its maximal measured value in this investigation.

Thus, increase of fumaric acid concentration from 6 to 9 g/l in the anode chamber of constructed MFC reduced its productivity but enhanced its stability in comparison to the application of lower concentration of investigated electron donor.

#### *3.3.2.3. Acetic acid and power output in MFC*

Acetic acid was applied as the separate electron donor in constructed MFC. It was shown that the maximal power value equaled 5.78±0.24 mW/m2 on the 40 hour (second day) of bacterial cultivation under addition of 6 g/l of CH3COOH (fig. 13).

Its value decreased by 42% on the 232 hour (10 day) of *D. acetoxidans* IMV B-7384 growth. Generated power insignificantly enhanced with the increase of cultivation time. It equaled 3.1±0.12 mW/m2 on the 480 hour of bacterial cultivation. Thus, application of acetic acid as well as fumaric acid in low concentrations caused high stability of electricity generation in con‐ structed fuel cell apart from application of lactic acid.

*D. acetoxidans* IMV B-7384 has been cultivated in MFC under addition of 9 g/l of acetic acid as the sole electron donor into the growth medium (fig. 14). Under these cultivation conditions the maximal power value equaled 3.6±0.30 mW/m2 on the 64 hour (third day) of bacterial cultivation. It was lower by 61% in comparison with the maximal power value obtained under

It was shown that increase of fumaric acid concentration up to 9 g/l caused maximal power

0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

**Figure 12.** Power density in MFC during twenty days under addition of 9 g/l of fumaric acid into the growth medium

It was less by 2.4 times in comparison with the highest power density value, which was observed under bacterial cultivation with application of less concentration of fumaric acid (6 g/l). The minimal observed power density value equaled 1.95±0.047 mW/m2 on the 160 hour of bacterial cultivation under these conditions. Increasing of cultivation time caused insignif‐ icant enhance of power production. On the 480 hour it value equaled 1.97±0.07 mW/m2

Thus, increase of fumaric acid concentration from 6 to 9 g/l in the anode chamber of constructed MFC reduced its productivity but enhanced its stability in comparison to the application of

Acetic acid was applied as the separate electron donor in constructed MFC. It was shown that the maximal power value equaled 5.78±0.24 mW/m2 on the 40 hour (second day) of bacterial

Its value decreased by 42% on the 232 hour (10 day) of *D. acetoxidans* IMV B-7384 growth. Generated power insignificantly enhanced with the increase of cultivation time. It equaled

as fumaric acid in low concentrations caused high stability of electricity generation in con‐

*D. acetoxidans* IMV B-7384 has been cultivated in MFC under addition of 9 g/l of acetic acid as the sole electron donor into the growth medium (fig. 14). Under these cultivation conditions

cultivation. It was lower by 61% in comparison with the maximal power value obtained under

on the 480 hour of bacterial cultivation. Thus, application of acetic acid as well

on the 64 hour (third day) of bacterial

was less by 17% in comparison with its maximal measured value in this investigation.

lower concentration of investigated electron donor.

cultivation under addition of 6 g/l of CH3COOH (fig. 13).

structed fuel cell apart from application of lactic acid.

the maximal power value equaled 3.6±0.30 mW/m2

*3.3.2.3. Acetic acid and power output in MFC*

Time of cultivation, hours

, which

) at 56 hour (fig. 12).

generation (2.38±0.10 mW/m2

48 Technology and Application of Microbial Fuel Cells

0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 3,0

of *D. acetoxidans* IMV B-7384

3.1±0.12 mW/m2

Power density, mW/m

2

**Figure 13.** Power density in MFC during twenty days under addition of 6 g/l of acetic acid into the growth medium of *D. acetoxidans* IMV B-7384

usage of 6 g/l of lactic acid by bacteria in the anode chamber of MFC. Increase of cultivation time up to 480 hour caused decrease of power value till 1.60±0.11 mW/m2 .

Thus, increase of acetic acid concentration as electron donor in the anode chamber of MFC caused partial inhibition of electricity generation in comparison with application of its lower content.

It can be summarized that low concentrations of investigated organic acids caused higher stability of power generation in constructed microbial fuel cell apart from their higher concentrations.

**Figure 14.** Power density in MFC during twenty days under addition of 9 g/l of acetic acid into the growth medium of *D. acetoxidans* IMV B-7384

Possibly, it may be explained because of raising of by-products concentrations in the growth medium under increasing of organic source concentration. It may cause negative influence according to *D. acetoxidans* IMV B-7384 metabolism and their respective ability of exoelectro‐ genesis.

*dans* IMV B-7384 as the anode biocatalyst may include detailed analyses of its molecular biochemistry with the aim of profound understanding of interconnections between the processes of organic source consumption and electric current generation. Further analyses of interrelations between specific reactions of sulfur cycle (e.g. polysulfide reductase activity), reduction of transition metals, such as iron and manganese, and processes of electrogenesis, which are conducted by the cells of *D. acetoxidans* IMV B-7384 may substantially influence the microbial fuel cell study with the aim of increasing of its productivity, reliability and durability.

The Anode Biocatalyst with Simultaneous Transition Metals Pollution Control

http://dx.doi.org/10.5772/58347

51

We are highly grateful to Dr. Neelkanth G. Dhere, Jaroslav Ferensovych, Dr. Vasyl Getman, Dr. Dariya Fedorovych, Dr. Yurij Boretsky, Dr. Oleksandr Kulachkovskyj, and all other our coworkers for their support provided in carrying out of investigations and book chapter

and Svitlana Hnatush2

1 Ivan Franko National University of Lviv, Faculty of Electronics, Laboratory of Optical-

2 Ivan Franko National University of Lviv, Biological Faculty, Department of Microbiology,

[1] Zhuwei D., Haoran L., Tingyue G. A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy. Biotechnology Ad‐

[3] Cheng S., Dempsey BA., Logan BE. Electricity generation from synthetic acid-mine drainage (AMD) water using fuel cell technologies. Environmental Science & Tech‐

[2] Logan BE. Microbial Fuel Cells. John Wiley & Sons., New Jersey; 2007.

**Acknowledgements**

preparation.

Ukraine

**References**

**Author details**

Oleksandr Bilyy1\*, Oresta Vasyliv2

vances 2007; 25, 464-482.

nology 2007; 41, 8149-8153.

Electronic Device, Ukraine

\*Address all correspondence to: bily2011@yandex.ru

## **4. Conclusions**

*D. acetoxidans* IMV B-7384 is exoelectricigenic sulfur-reducing bacterium which influences environmental biogeochemistry by maintenance of reductive stage of sulfur cycle. Its metalresistant strains play significant role in heavy metal ions remediation from the aquatic environments because of interaction between the final product of bacterial dissimilative sulfurreduction – hydrogen sulfide and metal ions with their next combining in form of insoluble metal sulfide precipitates. It was shown that *D. acetoxidans* IMV B-7384 synthesizes such components of antioxidant defense system as catalase, superoxide dismutase and reduced glutathione under the influence of aggressive external factors, such as heavy metal ions. It causes its resistance against environmental pollution by these xenobiotics. Enzymatic and nonenzymatic components of antioxidant defense system found in the cells of *D. acetoxidans* IMV B-7384 are highly effective in neutralization of reactive oxygen species. Antioxidant system activity of investigated bacterial cells may be useful for increasing the durability of protonexchange membranes in MFCs because of the creation of defensive barrier against detrimental influence of these oxidants. It shows a prospect of efficient and economic application of *D. acetoxidans* IMV B-7384 as the anode biocatalyst in microbial fuel cell with simultaneous wastewater treatment.

The optimal external resistance in constructed MFC in term of power generation was deter‐ mined to be 2.2 kΩ. Separate application of lactic, fumaric, and acetic acids caused differences in power generation by investigated bacterium. It was shown that addition of fumaric and acetic acids in concentration 6 g/l improved stability of generated power in constructed microbial fuel cell in comparison with application of lactic acid in the same concentration. Increase of concentration of investigated organic electron donors up to 9 g/l reduced generated electric power .

Thus, *D. acetoxidans* IMV B-7384 may be applied for effective treatment of wastewater enriched with heavy metals, acetic and fumaric acids-containing refuses with simultaneous electricity generation in the scaled-up microbial fuel cells. Additionally it can be used for treatment of highly polluted sulfur-containing aquatic environments with alterations of sulfur cycle.

#### **4.1. Possible directions of further research on application of** *D. acetoxidans* **IMV B-7384 as the anode biocatalyst in MFC**

Exploration of the utility of *D. acetoxidans* IMV B-7384 for development of efficient MFC through determination of optimal cultivation and fuel cell construction parameters may be highly beneficial for the progress of microbial fuel cell study with simultaneous heavy metals pollution control for cost effective environmental remediation. Further research of *D. acetoxi‐* *dans* IMV B-7384 as the anode biocatalyst may include detailed analyses of its molecular biochemistry with the aim of profound understanding of interconnections between the processes of organic source consumption and electric current generation. Further analyses of interrelations between specific reactions of sulfur cycle (e.g. polysulfide reductase activity), reduction of transition metals, such as iron and manganese, and processes of electrogenesis, which are conducted by the cells of *D. acetoxidans* IMV B-7384 may substantially influence the microbial fuel cell study with the aim of increasing of its productivity, reliability and durability.

## **Acknowledgements**

Possibly, it may be explained because of raising of by-products concentrations in the growth medium under increasing of organic source concentration. It may cause negative influence according to *D. acetoxidans* IMV B-7384 metabolism and their respective ability of exoelectro‐

*D. acetoxidans* IMV B-7384 is exoelectricigenic sulfur-reducing bacterium which influences environmental biogeochemistry by maintenance of reductive stage of sulfur cycle. Its metalresistant strains play significant role in heavy metal ions remediation from the aquatic environments because of interaction between the final product of bacterial dissimilative sulfurreduction – hydrogen sulfide and metal ions with their next combining in form of insoluble metal sulfide precipitates. It was shown that *D. acetoxidans* IMV B-7384 synthesizes such components of antioxidant defense system as catalase, superoxide dismutase and reduced glutathione under the influence of aggressive external factors, such as heavy metal ions. It causes its resistance against environmental pollution by these xenobiotics. Enzymatic and nonenzymatic components of antioxidant defense system found in the cells of *D. acetoxidans* IMV B-7384 are highly effective in neutralization of reactive oxygen species. Antioxidant system activity of investigated bacterial cells may be useful for increasing the durability of protonexchange membranes in MFCs because of the creation of defensive barrier against detrimental influence of these oxidants. It shows a prospect of efficient and economic application of *D. acetoxidans* IMV B-7384 as the anode biocatalyst in microbial fuel cell with simultaneous

The optimal external resistance in constructed MFC in term of power generation was deter‐ mined to be 2.2 kΩ. Separate application of lactic, fumaric, and acetic acids caused differences in power generation by investigated bacterium. It was shown that addition of fumaric and acetic acids in concentration 6 g/l improved stability of generated power in constructed microbial fuel cell in comparison with application of lactic acid in the same concentration. Increase of concentration of investigated organic electron donors up to 9 g/l reduced generated

Thus, *D. acetoxidans* IMV B-7384 may be applied for effective treatment of wastewater enriched with heavy metals, acetic and fumaric acids-containing refuses with simultaneous electricity generation in the scaled-up microbial fuel cells. Additionally it can be used for treatment of highly polluted sulfur-containing aquatic environments with alterations of sulfur cycle.

**4.1. Possible directions of further research on application of** *D. acetoxidans* **IMV B-7384 as**

Exploration of the utility of *D. acetoxidans* IMV B-7384 for development of efficient MFC through determination of optimal cultivation and fuel cell construction parameters may be highly beneficial for the progress of microbial fuel cell study with simultaneous heavy metals pollution control for cost effective environmental remediation. Further research of *D. acetoxi‐*

genesis.

**4. Conclusions**

50 Technology and Application of Microbial Fuel Cells

wastewater treatment.

electric power .

**the anode biocatalyst in MFC**

We are highly grateful to Dr. Neelkanth G. Dhere, Jaroslav Ferensovych, Dr. Vasyl Getman, Dr. Dariya Fedorovych, Dr. Yurij Boretsky, Dr. Oleksandr Kulachkovskyj, and all other our coworkers for their support provided in carrying out of investigations and book chapter preparation.

## **Author details**

Oleksandr Bilyy1\*, Oresta Vasyliv2 and Svitlana Hnatush2

\*Address all correspondence to: bily2011@yandex.ru

1 Ivan Franko National University of Lviv, Faculty of Electronics, Laboratory of Optical-Electronic Device, Ukraine

2 Ivan Franko National University of Lviv, Biological Faculty, Department of Microbiology, Ukraine

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**Chapter 4**

**Anode Biofilm**

Michal Schechter, Alex Schechter,

http://dx.doi.org/10.5772/58432

pH conditions on power production.

matrix of a primarily polysaccharide material [3].

**2. Biofilm definition**

**1. Introduction**

Shmuel Rozenfeld, Emanuel Efrat and Rivka Cahan

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 [1]. Power production by MFCs varies with bacterial cell species, specific

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 [1]. Knowledge and understanding of the anode biofilm components, morphology formation steps and electron

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

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

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

substrate concentration, cathode catalysts and the MFC configuration [2].

transfer mechanism may lead to better biofilm conductivity in MFC.

Additional information is available at the end of the chapter

## **Anode Biofilm**

Michal Schechter, Alex Schechter, Shmuel Rozenfeld, Emanuel Efrat and Rivka Cahan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58432

**1. Introduction**

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 [1]. Power production by MFCs varies with bacterial cell species, specific substrate concentration, cathode catalysts and the MFC configuration [2].

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 [1]. 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].

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **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 [7].

Cell surface-associated proteins such as pili, flagella, curli and amyloid fibers are believed to be important factors for biofilm formation [20]. Cell-to-cell signaling (quorum sensing) has been demonstrated to play a role in biofilm formation. *P. aeruginosa* produces two different quorum sensing molecules, lasR-lasI and *rhlR-rhlI,* which were shown to be involved in biofilm formation [21]. The biofilm of a double mutant produced thinner biofilms than the wild type, its cells were more densely packed, and the typical biofilm architecture was absent. Addition of the quorum sensing molecules known as homoserine lactone to a medium that contained the mutant biofilms resulted in biofilms with a structure and thickness similar to that of the

Anode Biofilm

59

http://dx.doi.org/10.5772/58432

In conclusion, knowledge and understanding of the biofilm components, morphology and

**4. Spectroscopic methods for anode biofilm study, including advancing**

During the last decade, most efforts in microbial fuel cell (MFC) research focused on modifi‐ cations of MFC design and electrode materials, with little investigation of the properties of the anode microorganisms that are essential for maximal current production. However, under‐ standing the functions of microbial cell surfaces requires knowledge of their chemical struc‐

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

In this chapter we will discuss the principle, the advantages and the disadvantages of each of

Confocal laser scanning microscopy (CLSM) is often used in optical imaging of sliced microfluidic 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 *ex situ* method [23, 24].

formation steps may lead to better biofilm conductivity in microbial fuel cells (MFC).

wild type [17,22].

**real-time analysis**

tural, physical properties and biological processes.

biofilm formation, functionality and activity.

**4.1. Confocal laser scanning microscopy**

the methods and the application of these techniques in MFCs.

The EPS serves as a scaffold which holds the cell aggregates together [4]. 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 [8]. The amount and thickness of the EPS increase with the biofilm's age [9].

The EPS in thin biofilms is often rich in proteins, contrary to thicker biofilms [10]. The EPS is more abundant in the interior of the biofilm, whereas cell densities are highest in the top layer [11].

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 [6]. Nucleic acids that are found in the EPS are extracellular DNA (eDNA) that exhibit some similarities to genomic DNA but also distinct differences. In *Pseudomonas aeruginosa*, the release of eDNA is under the control of quorum sensing systems. The eDNA is necessary for the initial establishment of *P. aeruginosa* biofilms [12, 13]. Filamen‐ tous networks of eDNA were shown to stabilize the biofilm architecture. EPS lipids contribute to the hydrophobic properties of EPS [14].

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 [15]. Biofilm formation occurs in a sequen‐ tial 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 [16].

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 [17]. Furthermore, microbial colonization increases with the increase in surface roughness [18]. The biofilm architecture changes constantly, due to external and internal processes [16]. The biofilm thickness may be affected by the number and species of microorganisms. Biofilms of pure cultures of either *Klebsiella. pneumoniae* or *P. aeruginosa* in a laboratory reactor were thinner (15 µ and 30 µ, respectively), whereas a biofilm containing both species was thicker (40 µ) [19]. Mature bacterial biofilms can adopt various architectures depending on the characteristics of the surrounding environment, such as nutrients, pH, temperature, shear forces, osmolarity and composition of the microbial consortia. The common complex biofilm is a mushroom-like structure which is surrounded by highly permeable water channels that facilitate the transport of nutrients and oxygen to the interior of the biofilm [8].

Cell surface-associated proteins such as pili, flagella, curli and amyloid fibers are believed to be important factors for biofilm formation [20]. Cell-to-cell signaling (quorum sensing) has been demonstrated to play a role in biofilm formation. *P. aeruginosa* produces two different quorum sensing molecules, lasR-lasI and *rhlR-rhlI,* which were shown to be involved in biofilm formation [21]. The biofilm of a double mutant produced thinner biofilms than the wild type, its cells were more densely packed, and the typical biofilm architecture was absent. Addition of the quorum sensing molecules known as homoserine lactone to a medium that contained the mutant biofilms resulted in biofilms with a structure and thickness similar to that of the wild type [17,22].

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 modifi‐ cations of MFC design and electrode materials, with little investigation of the properties of the anode microorganisms that are essential for maximal current production. However, under‐ standing the functions of microbial cell surfaces requires knowledge of their chemical struc‐ tural, 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**

**3. Biofilm composition**

58 Technology and Application of Microbial Fuel Cells

[11].

1-10% nucleic acids and 1-40% lipids [7].

to the hydrophobic properties of EPS [14].

biofilm maturation; and (v) detachment [16].

of nutrients and oxygen to the interior of the biofilm [8].

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,

The EPS serves as a scaffold which holds the cell aggregates together [4]. 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

The EPS in thin biofilms is often rich in proteins, contrary to thicker biofilms [10]. 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 [6]. Nucleic acids that are found in the EPS are extracellular DNA (eDNA) that exhibit some similarities to genomic DNA but also distinct differences. In *Pseudomonas aeruginosa*, the release of eDNA is under the control of quorum sensing systems. The eDNA is necessary for the initial establishment of *P. aeruginosa* biofilms [12, 13]. Filamen‐ tous networks of eDNA were shown to stabilize the biofilm architecture. EPS lipids contribute

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 [15]. Biofilm formation occurs in a sequen‐ tial 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)

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 [17]. Furthermore, microbial colonization increases with the increase in surface roughness [18]. The biofilm architecture changes constantly, due to external and internal processes [16]. The biofilm thickness may be affected by the number and species of microorganisms. Biofilms of pure cultures of either *Klebsiella. pneumoniae* or *P. aeruginosa* in a laboratory reactor were thinner (15 µ and 30 µ, respectively), whereas a biofilm containing both species was thicker (40 µ) [19]. Mature bacterial biofilms can adopt various architectures depending on the characteristics of the surrounding environment, such as nutrients, pH, temperature, shear forces, osmolarity and composition of the microbial consortia. The common complex biofilm is a mushroom-like structure which is surrounded by highly permeable water channels that facilitate the transport

EPS [8]. The amount and thickness of the EPS increase with the biofilm's age [9].

Confocal laser scanning microscopy (CLSM) is often used in optical imaging of sliced microfluidic 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 *ex situ* method [23, 24].

#### **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.

recorded to give topographic, conductive, magnetic, mechanical (friction forces) or potential

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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 [25]. 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 hydropho‐ bicity, surface charges, mechanical properties and local electrochemical properties [28]. For example, AFM enables imagining of biofilms and the EPS distribution within the biofilm [29].

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

Power-producing bacterial species that have exoelectrogenic activity without exogenous mediators include: *Shewanella putrefaciens, Clostridium butyricum, Desulfuromonas acetoxidans, Geobacter metallireducens, Geobacter sulfurreducens, Rhodoferax ferrireducens, Pseudomonas aeruginosa, Desulfobulbus propionicus, Geothrix fermentans, Shewanella oneidensis, Escherichia coli, Rhodopseudomonas palustris, Ochrobactrum anthropi, Desulfovibrio desulfuricans, Acidiphilium sp. Klebsiella pneumonia, Thermincola sp*. [30] and *Cupriavidus basilensis* [31]. Microorganisms which were detected on the anode of microbial electrolysis cells (MECs) include: *Stenotrophomonas, Lactobacillus, Curtobacterium, Agrobacterium, Flavobacterium, Bradyrhizobium, Pseudomonas,*

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

Exoelectrogenic bacteria transfer electrons to anodes either directly or via self-produced mediators. In the direct mechanism, electron transfer occurs via membrane-associated Ccytochrome or through conductive pili or appendages [33]. 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 [34]. Well-known exoelectrogens include *Geobacter* sp. and *Shewanella* sp. [35] which are known to have outer-membrane cytochromes

*Desulfovibrio, Shewanella, Desulfonauticus, Xenohaliotis* and *Marinicola* [32].

images of the surface.

**5. Power-producing bacterial cells**

all other parameters are identical.

[25].

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 [25].

#### **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 [25].

#### **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 noncontact 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 [25]. 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 hydropho‐ bicity, surface charges, mechanical properties and local electrochemical properties [28]. For example, AFM enables imagining of biofilms and the EPS distribution within the biofilm [29].

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 [25].

## **5. Power-producing bacterial cells**

**4.2. Scanning electron microscopy**

60 Technology and Application of Microbial Fuel Cells

low pressure inside the SEM vacuum chamber [25].

in the study of solid, liquid and gaseous samples.

applications.

**4.3. Raman spectroscopy**

**4.4. Atomic force 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

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

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

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

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 noncontact 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

radiation can damage the sample and distort the Raman spectrum [25].

Power-producing bacterial species that have exoelectrogenic activity without exogenous mediators include: *Shewanella putrefaciens, Clostridium butyricum, Desulfuromonas acetoxidans, Geobacter metallireducens, Geobacter sulfurreducens, Rhodoferax ferrireducens, Pseudomonas aeruginosa, Desulfobulbus propionicus, Geothrix fermentans, Shewanella oneidensis, Escherichia coli, Rhodopseudomonas palustris, Ochrobactrum anthropi, Desulfovibrio desulfuricans, Acidiphilium sp. Klebsiella pneumonia, Thermincola sp*. [30] and *Cupriavidus basilensis* [31]. Microorganisms which were detected on the anode of microbial electrolysis cells (MECs) include: *Stenotrophomonas, Lactobacillus, Curtobacterium, Agrobacterium, Flavobacterium, Bradyrhizobium, Pseudomonas, Desulfovibrio, Shewanella, Desulfonauticus, Xenohaliotis* and *Marinicola* [32].

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 Ccytochrome or through conductive pili or appendages [33]. 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 [34]. Well-known exoelectrogens include *Geobacter* sp. and *Shewanella* sp. [35] which are known to have outer-membrane cytochromes and conductive pili, and *Pseudomonas* sp. that produce mediators that shuttle electrons from the bacteria through the biofilm matrix to the anode [36].

reductase and nitrate reductase. Bacterial two-hybrid showed pair-wise interactions among CymA, MtrA and some other periplasmic redox proteins, indicating that CymA is the major electron conduit to the periplasmic space and can interact directly with periplasmic redox

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Okamoto et al identified voltammetry signals of outer membrane C-cytochrome in monolayer biofilms of *S. oneidensis* [45]. Okamoto et al analyzed monolayer and multilayer biofilms of *S. oneidensis* on tin-doped indium oxide electrodes and compared the respective amounts of electroactive C-cytochrome using voltammetry techniques. The scan-rate dependence of cyclic voltammograms was used to investigate the role of C-cytochrome in the transfer of respiratory electrons to distant tin-doped indium oxide electrodes. Electron conduction in *S. oneidensis* MR-1 multilayer biofilms was demonstrated to be mediated by sequential redox cycling of outer membrane C-cytochrome under normal physiological conditions. It was also demon‐ strated that the electron transport outer membrane C-cytochrome conduit across the biofilms

Immunogold labeling of the outer-surface C-type cytochrome OmcZ showed that when *G. sulfurreducens* grew as a biofilm on a graphite electrode that served as an anode and sole electron acceptor for growth, OmcZ was highly concentrated at the biofilm–electrode interface. Control biofilms which were grown on the same graphite material but with fumarate as the electron acceptor did not have OmcZ accumulations at the biofilm–electrode interface. The researchers suggested that OmcZ may serve as an electrochemical gate facilitating electron transfer from *G. sulfurreducens* biofilms to the anode surface [46]. Direct current surfaceenhanced infrared (IR) absorption spectroscopy and FTIR demonstrated a linear correlation between the increasing presence of *Geobacter sulfurreduces* protein and current production. This result may confirm that the extracellular cytochromes [47] are responsible for the electron

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 electro‐ chemical 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 [49]. *In vitro* and *in vivo* experiments led to the proposal of several models based on the redox properties of bacterial C-cytochromes. A 4-step mechanism for electron transfer in *Geobacter* biofilms was proposed: step 1, acetate uptake and electron

proteins by forming a transient protein complex [44].

contributed to the anodic current generation [40].

transfer to the gold electrode [48].

## **6. Mechanisms of electron transfer from the biofilm to the anode**

#### **6.1. C-cytochrome**

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. *S. oneidensis* MR-1 has 42 putative C-cyto‐ chromes and 80% of them are located in the outer membrane, covering 8-34% of the cell surface. It was reported that in *Geobacter*, C-cytochromes are bound to the matrices of extracellular polymeric substrates [37]. Appendages termed bacterial nanowires that were identified in both *Shewanella* and *Geobacter* also contain surface-located C-cytochromes which are postulated to transport electrons from distant cells to electrodes [38, 39]. However, these studies are based on *in vitro* experiments and more research needs to be done to understand this mechanism [40]. *Geobacter* biofilm respiration was found to continue after the interruption of electrode polarization, since these bacteria can store electrons in the heme of the exocytoplasmic cytochromes. When the electrode was connected again, stored electrons were recovered as a current superimposed on the basal steady-state current [41].

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: *Geobacter, Desulfovibrio, Rhodopseudomonas* and *Shewanella*, all of which increased over three months of the MEC reactor's performance [32]. Microarray analysis of *Geobacter sulfurredu‐ cens* thick anode biofilms (>50 µm) in MFCs revealed 13 genes in current-harvesting biofilms. Up-regulated genes included two outer C-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane C-cytochromes OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface C-type cytochrome OmcE were more abundant and OmcS was less abundant in current-harvesting cells. The role of outer-surface proteins whose genes were expressed in the current-producing biofilm versus the control biofilm which reduced fumarate were evaluated by gene deletion and its impact on current production was examined. It was found that deletion of OmcS, OmcB, or OmcE had no impact on maximum current production. Deletion of OmcZ significantly reduced power production. These results suggest that OmcZ is a key component in electron transfer in MFCs with a *G. sulfurreducens* anode [42]. In thin (ca. 10 mm) wild type biofilms, genes for OmcS and OmcE are more highly expressed than in planktonic cells grown with a soluble electron acceptor such as Fe(III) citrate [43]. CymA, which participates in many *Shewanella* anaerobic respiration processes, is a tetraheme C-cytochrome whose C-terminal is exposed to the periplasm and its N-terminal is anchored in the inner membrane. In the case of electrode reduction, a deletion of the CymA gene caused >80% decrease in current generation. CymA can interact directly with many terminal reductases in the periplasm, such as fumarate reductase and nitrate reductase. Bacterial two-hybrid showed pair-wise interactions among CymA, MtrA and some other periplasmic redox proteins, indicating that CymA is the major electron conduit to the periplasmic space and can interact directly with periplasmic redox proteins by forming a transient protein complex [44].

and conductive pili, and *Pseudomonas* sp. that produce mediators that shuttle electrons from

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. *S. oneidensis* MR-1 has 42 putative C-cyto‐ chromes and 80% of them are located in the outer membrane, covering 8-34% of the cell surface. It was reported that in *Geobacter*, C-cytochromes are bound to the matrices of extracellular polymeric substrates [37]. Appendages termed bacterial nanowires that were identified in both *Shewanella* and *Geobacter* also contain surface-located C-cytochromes which are postulated to transport electrons from distant cells to electrodes [38, 39]. However, these studies are based on *in vitro* experiments and more research needs to be done to understand this mechanism [40]. *Geobacter* biofilm respiration was found to continue after the interruption of electrode polarization, since these bacteria can store electrons in the heme of the exocytoplasmic cytochromes. When the electrode was connected again, stored electrons were recovered as a

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: *Geobacter, Desulfovibrio, Rhodopseudomonas* and *Shewanella*, all of which increased over three months of the MEC reactor's performance [32]. Microarray analysis of *Geobacter sulfurredu‐ cens* thick anode biofilms (>50 µm) in MFCs revealed 13 genes in current-harvesting biofilms. Up-regulated genes included two outer C-type membrane cytochromes, OmcB and OmcZ. Down-regulated genes included the genes for the outer-membrane C-cytochromes OmcS and OmcT. Results of quantitative RT-PCR of gene transcript levels during biofilm growth were consistent with microarray results. OmcZ and the outer-surface C-type cytochrome OmcE were more abundant and OmcS was less abundant in current-harvesting cells. The role of outer-surface proteins whose genes were expressed in the current-producing biofilm versus the control biofilm which reduced fumarate were evaluated by gene deletion and its impact on current production was examined. It was found that deletion of OmcS, OmcB, or OmcE had no impact on maximum current production. Deletion of OmcZ significantly reduced power production. These results suggest that OmcZ is a key component in electron transfer in MFCs with a *G. sulfurreducens* anode [42]. In thin (ca. 10 mm) wild type biofilms, genes for OmcS and OmcE are more highly expressed than in planktonic cells grown with a soluble electron acceptor such as Fe(III) citrate [43]. CymA, which participates in many *Shewanella* anaerobic respiration processes, is a tetraheme C-cytochrome whose C-terminal is exposed to the periplasm and its N-terminal is anchored in the inner membrane. In the case of electrode reduction, a deletion of the CymA gene caused >80% decrease in current generation. CymA can interact directly with many terminal reductases in the periplasm, such as fumarate

**6. Mechanisms of electron transfer from the biofilm to the anode**

the bacteria through the biofilm matrix to the anode [36].

62 Technology and Application of Microbial Fuel Cells

current superimposed on the basal steady-state current [41].

**6.1. C-cytochrome**

Okamoto et al identified voltammetry signals of outer membrane C-cytochrome in monolayer biofilms of *S. oneidensis* [45]. Okamoto et al analyzed monolayer and multilayer biofilms of *S. oneidensis* on tin-doped indium oxide electrodes and compared the respective amounts of electroactive C-cytochrome using voltammetry techniques. The scan-rate dependence of cyclic voltammograms was used to investigate the role of C-cytochrome in the transfer of respiratory electrons to distant tin-doped indium oxide electrodes. Electron conduction in *S. oneidensis* MR-1 multilayer biofilms was demonstrated to be mediated by sequential redox cycling of outer membrane C-cytochrome under normal physiological conditions. It was also demon‐ strated that the electron transport outer membrane C-cytochrome conduit across the biofilms contributed to the anodic current generation [40].

Immunogold labeling of the outer-surface C-type cytochrome OmcZ showed that when *G. sulfurreducens* grew as a biofilm on a graphite electrode that served as an anode and sole electron acceptor for growth, OmcZ was highly concentrated at the biofilm–electrode interface. Control biofilms which were grown on the same graphite material but with fumarate as the electron acceptor did not have OmcZ accumulations at the biofilm–electrode interface. The researchers suggested that OmcZ may serve as an electrochemical gate facilitating electron transfer from *G. sulfurreducens* biofilms to the anode surface [46]. Direct current surfaceenhanced infrared (IR) absorption spectroscopy and FTIR demonstrated a linear correlation between the increasing presence of *Geobacter sulfurreduces* protein and current production. This result may confirm that the extracellular cytochromes [47] are responsible for the electron transfer to the gold electrode [48].

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 electro‐ chemical 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 [49]. *In vitro* and *in vivo* experiments led to the proposal of several models based on the redox properties of bacterial C-cytochromes. A 4-step mechanism for electron transfer in *Geobacter* biofilms was proposed: step 1, acetate uptake and electron transfer to periplasmic cytochromes; step 2, subsequent electron transfer to the exocytoplasmic cytochromes; step 3, electron transport along the biofilm matrix cytochromes; and step 4, transfer between the interfacial cytochromes and the electrode [41].

### **6.2. Pili**

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 [50]. Multilayer biofilms of *Geobacter sulfurreducens* on the anode surface of a MFC remained viable even at a distance from the anode. There was no decrease in the efficiency of current production with an increase in the thickness of the biofilm. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili which represent an electronic network promoting long-range electrical transfer in an energy-efficient manner in the MFC [38].

**Figure 1.** Measuring electrical transport along a bacterial nanowire. (A) Tapping-mode atomic force microscopy (AFM) amplitude image detailing the contact area with the bacterial nanowire from Fig. 1. (B) Contact-mode AFM deflection image of the junction after cutting the nanowire with FIB milling. The arrow marks the cut location. (C) Current-volt‐ age curve of the bacterial nanowire (ramp-up and ramp-down) both before (red) and after (black) cutting the nano‐

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**Figure 2.** CP-AFM of a bacterial nanowire. (A) Topographic AFM image showing air-dried S. oneidensis MR-1 cells and extracellular appendages deposited randomly on a SiO2/Si substrate patterned with Au microgrids. (B) Contact mode AFM image showing a nanowire reaching out from a bacterial cell to the Au electrode. (C) An I-V curve obtained by probing the nanowire at a length of 600 nm away from the Au electrode (at the position marked by the black dot in B). (Inset) The I-V curves obtained on bare Au and SiO2, respectively. (D) A plot of total resistance as a function of

distance between AFM tip and Au electrode [52].

wire [52].

*G. sulfurreducens* KN400 bacterial cells which were selected after 5 months of incubation at-400 mV produced higher current and power densities than the original inoculum (strain DL1). The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. The changes in outer surface components were associated with a greater propensity of strain KN400 to stick to surfaces than strain DL1. KN400 cells grown with fumarate as the electron acceptor were clumpy and strongly adhered to the glass surface of the culture tubes. This research showed that microor‐ ganisms' ability to electrochemically interact with electrodes can be enhanced with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide better microbe– electrode interactions [51].

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 *Geobacter* species. The existence of such pili raises the possibility of long-range direct electron transfer from each microbe to the electrode [36, 48]. El-Naggar et al. evaluated transport along the bacterial nanowires by conducting probe AFM at several points alongside a single nanowire using a metallic electrode and a conductive AFM tip. *S. oneidensis* MR-1 nanowires were electrically conductive along micrometer-length scales, yielding a corresponding electron transport rate, at 100 mV, of about 109 electrons per second, with an estimated resistance of 1 Ω (Fig 1). It was also found that mutants deficient in genes for the C-type decaheme-cytochromes MtrC and OmcA produced supplements that are morphologically consistent with bacterial nanowires, but were found to be nonconductive [52].

transfer to periplasmic cytochromes; step 2, subsequent electron transfer to the exocytoplasmic cytochromes; step 3, electron transport along the biofilm matrix cytochromes; and step 4,

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 [50]. Multilayer biofilms of *Geobacter sulfurreducens* on the anode surface of a MFC remained viable even at a distance from the anode. There was no decrease in the efficiency of current production with an increase in the thickness of the biofilm. Genetic studies demonstrated that efficient electron transfer through the biofilm required the presence of electrically conductive pili which represent an electronic network promoting long-range electrical transfer in an energy-efficient

*G. sulfurreducens* KN400 bacterial cells which were selected after 5 months of incubation at-400 mV produced higher current and power densities than the original inoculum (strain DL1). The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. The changes in outer surface components were associated with a greater propensity of strain KN400 to stick to surfaces than strain DL1. KN400 cells grown with fumarate as the electron acceptor were clumpy and strongly adhered to the glass surface of the culture tubes. This research showed that microor‐ ganisms' ability to electrochemically interact with electrodes can be enhanced with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide better microbe–

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 *Geobacter* species. The existence of such pili raises the possibility of long-range direct electron transfer from each microbe to the electrode [36, 48]. El-Naggar et al. evaluated transport along the bacterial nanowires by conducting probe AFM at several points alongside a single nanowire using a metallic electrode and a conductive AFM tip. *S. oneidensis* MR-1 nanowires were electrically conductive along micrometer-length scales, yielding a corresponding electron transport rate, at 100 mV, of about 109 electrons per second, with an estimated resistance of 1 Ω (Fig 1). It was also found that mutants deficient in genes for the C-type decaheme-cytochromes MtrC and OmcA produced supplements that are morphologically consistent with bacterial nanowires,

transfer between the interfacial cytochromes and the electrode [41].

**6.2. Pili**

64 Technology and Application of Microbial Fuel Cells

manner in the MFC [38].

electrode interactions [51].

but were found to be nonconductive [52].

**Figure 1.** Measuring electrical transport along a bacterial nanowire. (A) Tapping-mode atomic force microscopy (AFM) amplitude image detailing the contact area with the bacterial nanowire from Fig. 1. (B) Contact-mode AFM deflection image of the junction after cutting the nanowire with FIB milling. The arrow marks the cut location. (C) Current-volt‐ age curve of the bacterial nanowire (ramp-up and ramp-down) both before (red) and after (black) cutting the nano‐ wire [52].

**Figure 2.** CP-AFM of a bacterial nanowire. (A) Topographic AFM image showing air-dried S. oneidensis MR-1 cells and extracellular appendages deposited randomly on a SiO2/Si substrate patterned with Au microgrids. (B) Contact mode AFM image showing a nanowire reaching out from a bacterial cell to the Au electrode. (C) An I-V curve obtained by probing the nanowire at a length of 600 nm away from the Au electrode (at the position marked by the black dot in B). (Inset) The I-V curves obtained on bare Au and SiO2, respectively. (D) A plot of total resistance as a function of distance between AFM tip and Au electrode [52].

## **7. The effect of medium and pH conditions on power production**

**8. Anode biofilm thickness, morphology, viability and conductivity**

and involved in electron transfer to the anode [38].

current densities than any other species [58].

(substrate degradation rate, 0.903 kg COD/m<sup>3</sup>

developed biofilm operation [59].

CSLM (Fig 3) [31].

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 produc‐ tion 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 devel‐ opment, cells at a distance from the electrode surface remained viable, metabolically active

*Geobacter* can form multi-microbe thick, more than 20-cell length, persistent biofilms when the anode serves as their terminal electron acceptor. This phenomenon was not observed in the presence of insoluble oxidants for respiration. The combination of robust direct electron transfer and high cell surface density enables *Geobacter* biofilms to achieve higher anodic

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%

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 *Cupriavidus basilensis* bacterial cells grown in a defined medium containing acetate or phenol. Operating this mediator-less MFC under a constant external resistor of 1 kΩ with acetate or phenol led to a current generation of 902 and 310 mA m-2, respectively. SEM and confocal microscopy analyses demonstrated a developed biofilm with pili/appendages and a live anode *C. basilen‐ sis* population which was stained with a LIVE/DEAD viability kit and further analyzed by

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 *P. aeruginosa* bacterial cells and the electrode was investigated using cationic reagents which are known to "perforate" the bacterial membrane. Three reagents, chitosan, ethylenediaminetetraacetic acid (EDTA) and polyethyleneimine (PEI), were explored. The surface morphologies of chemically treated


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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 demonstrat‐ ed 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 electroca‐ talytic 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 [53]. 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 [53].

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 [54]. 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 [55]. 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 [56]. A MFC with a pure culture of *Enterobacter cloacae* was evaluated as a function of variations in the pH microenvironment. Operation under pH 6.5 and 7.4 led to maximum current generation of 0.40 mA and 0.42 mA, respectively. However, MFC operation under higher pH environments of 8.5 and 9.5 led to a maximum current generation of only 0.38 mA and 0.27 mA, respectively [57].

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**

**7. The effect of medium and pH conditions on power production**

anodes under different pH conditions [53].

66 Technology and Application of Microbial Fuel Cells

36.72 W/m3

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 demonstrat‐ ed 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 electroca‐ talytic 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 [53]. 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

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

results indicate that higher feed pH (8.0) led to higher power production [54]. 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 [55]. 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 [56]. A MFC with a pure culture of *Enterobacter cloacae* was evaluated as a function of variations in the pH microenvironment. Operation under pH 6.5 and 7.4 led to maximum current generation of 0.40 mA and 0.42 mA, respectively. However, MFC operation under higher pH environments of 8.5 and 9.5 led to a maximum

In conclusion, we assume that MFC power production is dependent on the pH environment

current generation of only 0.38 mA and 0.27 mA, respectively [57].

and may correlate with the bacterial cell anode species.

was obtained in MFC-1 (feed pH 6.0) and MFC-2 (feed pH 8.0), respectively. These

and

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 produc‐ tion 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 devel‐ opment, cells at a distance from the electrode surface remained viable, metabolically active and involved in electron transfer to the anode [38].

*Geobacter* can form multi-microbe thick, more than 20-cell length, persistent biofilms when the anode serves as their terminal electron acceptor. This phenomenon was not observed in the presence of insoluble oxidants for respiration. The combination of robust direct electron transfer and high cell surface density enables *Geobacter* biofilms to achieve higher anodic current densities than any other species [58].

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/m<sup>3</sup> -day) were observed, especially with fully developed biofilm operation [59].

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 *Cupriavidus basilensis* bacterial cells grown in a defined medium containing acetate or phenol. Operating this mediator-less MFC under a constant external resistor of 1 kΩ with acetate or phenol led to a current generation of 902 and 310 mA m-2, respectively. SEM and confocal microscopy analyses demonstrated a developed biofilm with pili/appendages and a live anode *C. basilen‐ sis* population which was stained with a LIVE/DEAD viability kit and further analyzed by CSLM (Fig 3) [31].

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 *P. aeruginosa* bacterial cells and the electrode was investigated using cationic reagents which are known to "perforate" the bacterial membrane. Three reagents, chitosan, ethylenediaminetetraacetic acid (EDTA) and polyethyleneimine (PEI), were explored. The surface morphologies of chemically treated

real time. It was also suggested that the accumulation of protons that are released from organic

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 *in situ* SERR spectroscopy electrochemical analysis on a catalytically active biofilm grown on silver electrodes, it was revealed that two bis (histidine) coordinated heme cytochrome redox couples are involved in the mediated electron transfer between the

Liu et al. showed that graphene modification improved power density and energy conversion efficiency by almost 3 times that of a *P. aeruginosa* mediator-less MFC. Raman analysis revealed that the improvement was credited to the high biocompatibility of graphene which promotes bacterial growth on the electrode surface and results in the creation of more direct electron transfer activation centers and stimulates excretion of mediating molecules for higher electron transfer [65]. Raman spectroscopy analysis at different growth stages revealed changes in the vibrational properties of *Geobacter* Cyt *c* resulting from shifts in the anodic potential between

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 charac‐ teristics 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

, Shmuel Rozenfeld1

, Emanuel Efrat1

and Rivka Cahan1\*

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69

http://dx.doi.org/10.5772/58432

matter oxidation within anode biofilms can limit current production [62].

bacteria and the electrode [63].

different redox conditions [65].

**Acknowledgements**

Center.

**Author details**

Michal Schechter1

, Alex Schechter2

1 Department of Chemical Engineering, Ariel University, Ariel, Israel

2 Department of Biological Chemistry, Ariel University, Ariel, Israel

\*Address all correspondence to: rivkac@ariel.ac.il

**Figure 3.** *Cupriavidus basilensis* biofilm grown on a graphite anode in MFC. CSLM micrograph, magnification of 40 (A). SEM micrograph, magnification of 1,000 (B).

*P. aeruginosa* cells were examined with SEM and AFM images. The results showed that the chemical treatment caused changes in the cell shape and perforated bacterial cells, contrary to the original *P. aeruginosa* cells that were plump and smooth. The cells were flattened with pores and clusters on the surface. The chemical treatment disorganized the outer mem‐ brane of *P. aeruginosa* which resulted in higher permeability, like "drilling", which significant‐ ly improved electron transfer for high power density MFCs. Cyclic voltammograms (CV) of the anaerobically activated control and perforated *P. aeruginosa* cells were measured in glucose-free medium and showed two pairs of redox peaks at-0.59 and-0.47 V. The two redox waves represented different electron transfer mechanisms of this bio-anode. The output current profiles at a constant load of chitosan, EDTA-and PEI-treated cells were much higher than in control cells, and the PEI-treated cell catalysts produced the highest discharge (oxidation) current density, which parallels the result of the CV experiment. The polariza‐ tion curves revealed that the PEI-treated cell has much lower polarization for better energy conversion efficiency than the control cells. It was explained that the increased permeabili‐ ty of the bacterial outer membrane by the large pores and channels facilitates the transport of redox mediators and catabolic enzymes through the cell membrane and thus provides faster electron transfer [61].

Introducing a pH-sensitive fluoroprobe into the anode chamber revealed a strong pH gradient within the *G. sulfurreducens* anode biofilm. The pH decreased with increased proximity to the anode surface and from the exterior to the interior of the biofilm pillars. pH levels near the anode surface were as low as 6.1 compared to pH 7 near the cathode. Various controls demonstrated that proton accumulation was associated with current production. Decreasing the pH of the culture medium from 7 to 6 limited the growth of *G. sulfurreducens*. The results demonstrated that it is feasible to non-destructively monitor the activity of anode biofilms in real time. It was also suggested that the accumulation of protons that are released from organic matter oxidation within anode biofilms can limit current production [62].

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 *in situ* SERR spectroscopy electrochemical analysis on a catalytically active biofilm grown on silver electrodes, it was revealed that two bis (histidine) coordinated heme cytochrome redox couples are involved in the mediated electron transfer between the bacteria and the electrode [63].

Liu et al. showed that graphene modification improved power density and energy conversion efficiency by almost 3 times that of a *P. aeruginosa* mediator-less MFC. Raman analysis revealed that the improvement was credited to the high biocompatibility of graphene which promotes bacterial growth on the electrode surface and results in the creation of more direct electron transfer activation centers and stimulates excretion of mediating molecules for higher electron transfer [65]. Raman spectroscopy analysis at different growth stages revealed changes in the vibrational properties of *Geobacter* Cyt *c* resulting from shifts in the anodic potential between different redox conditions [65].

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 charac‐ teristics 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.

## **Acknowledgements**

*P. aeruginosa* cells were examined with SEM and AFM images. The results showed that the chemical treatment caused changes in the cell shape and perforated bacterial cells, contrary to the original *P. aeruginosa* cells that were plump and smooth. The cells were flattened with pores and clusters on the surface. The chemical treatment disorganized the outer mem‐ brane of *P. aeruginosa* which resulted in higher permeability, like "drilling", which significant‐ ly improved electron transfer for high power density MFCs. Cyclic voltammograms (CV) of the anaerobically activated control and perforated *P. aeruginosa* cells were measured in glucose-free medium and showed two pairs of redox peaks at-0.59 and-0.47 V. The two redox waves represented different electron transfer mechanisms of this bio-anode. The output current profiles at a constant load of chitosan, EDTA-and PEI-treated cells were much higher than in control cells, and the PEI-treated cell catalysts produced the highest discharge (oxidation) current density, which parallels the result of the CV experiment. The polariza‐ tion curves revealed that the PEI-treated cell has much lower polarization for better energy conversion efficiency than the control cells. It was explained that the increased permeabili‐ ty of the bacterial outer membrane by the large pores and channels facilitates the transport of redox mediators and catabolic enzymes through the cell membrane and thus provides

**Figure 3.** *Cupriavidus basilensis* biofilm grown on a graphite anode in MFC. CSLM micrograph, magnification of 40 (A).

Introducing a pH-sensitive fluoroprobe into the anode chamber revealed a strong pH gradient within the *G. sulfurreducens* anode biofilm. The pH decreased with increased proximity to the anode surface and from the exterior to the interior of the biofilm pillars. pH levels near the anode surface were as low as 6.1 compared to pH 7 near the cathode. Various controls demonstrated that proton accumulation was associated with current production. Decreasing the pH of the culture medium from 7 to 6 limited the growth of *G. sulfurreducens*. The results demonstrated that it is feasible to non-destructively monitor the activity of anode biofilms in

faster electron transfer [61].

SEM micrograph, magnification of 1,000 (B).

68 Technology and Application of Microbial Fuel Cells

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.

## **Author details**

Michal Schechter1 , Alex Schechter2 , Shmuel Rozenfeld1 , Emanuel Efrat1 and Rivka Cahan1\*


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**Chapter 5**

**Removal of COD by Two-Chamber Microbial Fuel Cells**

Microbial fuel cells (MFC) are regarded as special bio-electrochemical systems which seem to have a high potential in the future technology for energy production in the section of renewable

A classical MFC in general [4-8] consist of an anodic and cathodic chamber divided by a proton (cation) selective membrane (Figure 1). Microbes in the anodic cell – mostly attached to the electrode surface forming a biofilm – oxidize the substrates and generate electrons and protons in the process. Then electrons are transported from the anode to the cathode through an external circuit (wire) resulting measurable electric current. Meanwhile protons are passing through the membrane and enter the cathode cell where they combine with oxygen to form

MFC-s can be operated by either monoculture (e.g. *Geobacter sulfurreducens* [5], *Shewanella oneidensis, Lactococcus lactis* [9], *Lysinibacillus sphaericus* [10]) – where the electrogenic species release electron to the anode electrode directly or with the use of electroactive metabolites –, or multiculture (microbe consortia) system, which can be found in e.g. anaerobic sewage sludge acting in a similar way. The substrates of the MFCs operated by sewage sludge can be provided from various wastewaters, thus electricity generation in the MFCs can be coupled with the degradation of organic matters even e.g. wastewater treatment processes [11-20]. Many types of wastewaters were investigated (e.g. beer brewery wastewater [22], food industrial wastewaters [22, 23]) where the degradation can be followed by COD removal determination [14]. In MFCs applied for treatment of organic matters, the feed can be enriched with various substrates. It has been noted that non-fermentable substrates are superior to fermentable substrate as the electron donor for power output and electron recovery. E.g. it was reported that an acetate enriched MFC produced higher power output than a glucose enriched

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Katalin Belafi-Bako, Balazs Vajda, Peter Bakonyi and

Additional information is available at the end of the chapter

Nandor Nemestothy

**1. Introduction**

water.

green energy sources [1-5].

http://dx.doi.org/10.5772/58373
