Bioremediation, Nutrients, and Other Valuable Product Recovery: Using Bioelectrochemical Systems.

Bioremediation and Nutrients and Other Valuable Products Recovery: Using Bio-electrochemical Systems reviews key applications in transforming fuel waste substrates into simple low impact and easily assimilative compounds that are environmentally non-labile and tolerant. The book emphasizes waste treatment and nutrient removal and recovery from a diverse array of waste substrates, utilizing Bioelectrochemical Systems (BES) approaches. Throughout, the work emphasizes the utilization of electrode and/or electrolyte components in building self-sustaining fuel cell systems that target the removal of both conventional and emerging pollutants, along with the production of energy.

Bioremediation strategies with potential scale-up options for wastewater treatment, metal removal and soil remediation drug derivates and emerging contaminants are discussed with particular emphasis. Chapters explore applications for these varied pollutants, together with prospects in waste minimization, nutrient recycling, water purification and bioremediation of natural resources.

• Explores a detailed panorama of potential known pollutants with detailed reviews on their removal and recovery
• Discusses bioproduct recovery application frontiers across wastewater treatment and bioremediation, metal removal and soil remediation, extraction of drug derivates and emerging contaminants
• Emphasizes pilot scale-up and commercialization potential for each recovery application discussed

• Language: English
• Publisher: Elsevier Science
• Release date: Nov 17, 2020
• ISBN: 9780128227527

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Korea

Chapter 1

Fundamentals of bioelectroactive fuel cells

Bhim Sen Thapa¹, Sang-Eun Oh¹, Shaik Gouse Peera² and Lakhveer Singh³,    ¹Department of Biological Environment, Kangwon National University, Chuncheon, South Korea,    ²Department of Environmental Science and Engineering, Keimyung University, Daegu, South Korea,    ³Department of Environmental Science, SRM University-AP, Amaravati, India

Abstract

The bioelectroactive fuel cells (BFC) or bioelectrochemical systems (BES) are regarded as a potent source for producing green and sustainable energy. The two major BFCs studied extensively are the enzymatic fuel cells and the microbial fuel cells. The main difference between them is the bioactive source that catalyzes the electrochemical reaction in the system. In recent times BES has gained tremendous publicity due to its unique technique for producing fuel, bioremediation, and many other applications. The BES differs from chemical fuel cells (CFC) in terms of catalysts, reaction mechanism, operational conditions, yield, and quantity of the product, etc. Although the BES has some limitations, it is considered to be advantageous over conventional CFC in several ways. However, the losses and the limitations present in BES are the main drawbacks. The present chapter highlights the working mechanism, operational condition, and other fundamental concepts of the BFC.

Keywords

Bioelectrochemical fuel cell; microorganisms; cell potential; microbial metabolism; exoelectron transfer; electrochemical analysis

1.1 Introduction

The depletion of available resources and increase in demand for alternative and sustainable energy has been a most warranted concept to be focused on. The urgency of producing fuel and energy through alternate sources has been a trending topic of research in current times [1]. In the fuel cell, electric energy is produced from chemical energy present in organic compounds catalyzed in an electrochemical system. Biologically active fuel cells are a type of electrochemical fuel cell that catalyzes reactions through a biotic means, such as a microorganism. The biocatalysts can be either a secretions of a cell, such as proteins, enzymes, or any other molecules, or due to the action of whole cell. The two major bioelectroactive fuel cells are the enzymatic fuel cell (EFC) and the microbial fuel cell (MFC). The MFC has garnered much attention because of its numerous applications and easy-to-operate features whereas the EFC has a role in biosensors applications [2]. Both the MFC and EFC are known to operate with near-similar principles but differ in terms of the biocatalyst performing the reactions. In MFCs, microorganisms, predominantly bacteria, convert organic molecules to produce fuel whereas in EFCs redox enzymes such as glucose oxidase catalyze the reaction supported by cofactors such as NAD+ and FAD. Another major difference between the EFC and MFC is specificity. Due to the enzyme biocatalyst, EFCs are highly specific toward the substrate, product, and the reaction they catalyze. In contrast, products obtained from the MFC are through a nonspecific manner, irrespective of substrate and the source of inocula. Hence a wide range of compounds are degraded for the generation of fuel in MFC. The commonly used enzymes in EFC are glucose oxidase, lactate dehydrogenase, NAD-dependent glucose dehydrogenase (GDH), NAD(P)+-dependent gluconate-5-dehydrogenase (Ga5DH), etc. [3]. In addition to these, bilirubin oxidase and laccase are also used for the cathode oxygen reduction catalyst in EFCs and MFCs. Other than biocatalyst and reaction specificity, operational conditions and product yield are some of the factors differentiating EFCs and MFCs. The basic differences between EFC, MFC, and other bioelectrochemical systems (BES) are illustrated in Fig. 1.1.

Figure 1.1 Schematic representation of different types of bioelectrochemical fuel cell and their applications [4].

The electric potential generation from microorganisms was first reported by Potter, 100 years ago with the bacterium Bacillus coli (E. coli) and yeast Saccharomyces cerevisiae [5]. This observation went unnoticed for a long time because of a lack of strong evidence at that time. In recent times, BES has become one of the trending research concepts across many countries. Beside generating bioelectricity, this technique is applied widely for treating wastewater and bioremediation by many researchers due to the reduction of organic chemicals, harmful dyes, metals, and other toxic compounds which are not easily treated by conventional and other chemical methods. The BES works with the same principle as that in chemical fuel cells, differing in its catalyst source and operational conditions. Microbe-catalyzed reactions are found to be advantageous over chemical reactions where the catalytic conditions are stringent and selective. However, further understanding and improvements are required to overcome the limitations and losses of the current system to construct an efficient bioelectroactive fuel cell to achieve higher performance and make it practically applicable.

1.1.1 The biotic factor

The interaction between two different molecules or atoms results in the generation of an electric charge, which is due to the free protons and electrons liberated from the reaction. When the free electrons from the reaction are collected and passed through the circuit in an electrochemical system, electricity is produced. The electrochemical systems are commonly employed for producing electric energy from chemical energy by catalyzing the reactions under stringent conditions in a closed reactor known as a fuel cell. Such biochemical reactions between molecules also takes place inside the cells of living systems during metabolism and reproduction required for their growth and survival. The electrochemical gradient and proton motive force generated from these reactions inside the cell provides the energy to maintain the thermal, pH, and other favorable metabolic conditions in the organism.

Microorganisms are a common and preferred source of inocula in BESs. Their availability and ability to metabolize a wide range of chemical compounds, reduce metals, faster growth rates, and survival in any environmental conditions are some of the main reasons for this. The energy source for carrying out cellular metabolic activities can originate from chemical compounds (chemotrophs), while some microorganisms obtain energy from sunlight (phototrophs) depending on their habitats and physiology. The organic compounds are metabolized through different pathways depending on the nature of the molecule. However, the prime source for producing energy inside the cell is carbohydrates. When a carbohydrate molecule is catabolized, a set of free protons and electrons are released at each reaction step. Under aerobic conditions the protons and electrons are accepted by oxygen to produce a water molecule whereas in an anoxic environments they are reduced by inorganic molecules and metals depending on their habitat. Irrespective of the environmental conditions of the cell the transfer of free protons and electrons to the terminal electron acceptor requires an electron mediator. The two major intracellular electron mediators in cells are NAD+ and FAD which shuttle the electrons between the cytoplasm and oxygen. During the oxidation of organic molecules, the free electrons and protons liberated are collected by NAD+ and FAD which are then reduced to NADH and FADH2. Upon transferring the electrons to oxygen, NADH and FADH2 are oxidized to their native states NAD+ and FAD, respectively. This process, a series of metabolic pathways, generates ATP, the energy currency of the living cell, and occurs continuously inside the cell allowing its growth and metabolism. The regeneration of NAD+ and FAD occurs only when a terminal electron acceptor such as oxygen accepts the electrons and protons (Fig. 1.2). The electrochemical balance inside the cell is maintained by sets of proteins and molecules allowing the favorable conditions for cellular activities to take place inside the cell.

Figure 1.2 Pictorial representations showing cellular metabolism occurring inside the bacterial cells under (A) aerobic conditions and (B) anaerobic conditions in an MFC.

In BESs, physiological conditions are anaerobic. When bacteria are inoculated in the anode chamber, they tend to find an alternate electron acceptor to oxygen for regenerating NAD+ and FAD. The electrode is the sole electron acceptor in the system and having a high redox potential easily accepts the electrons from NADH and FADH2 to oxidize them back to participate in another set of reactions (Fig. 1.4). These electrons are then passed through the circuit to complete the half-cell reaction at the cathode. Hence the obligate anaerobe and facultative anaerobic bacteria dominates the microbial inocula in bioelectroactive fuel cells.

Figure 1.4 Pictorial representation of exoelectron transfer mechanisms in Geobacter and Shewanella. (I) The OMC-based direct electron transfer in Geobacter; (II) electron conduction through bacterial nanowire; (III) electron transfer network of Shewanella including flavins and c-type cytochromes; and (IV) electrode respiration-coupled proton motive force and energy (ATP) generation [13].

1.1.1.1 Bacteria in microbial fuel cell

Bacteria are the most predominant microorganisms on Earth. They range in size from 0.2 to 0.5 µm in diameter and 0.5–3 µm in length. They are single celled microorganisms, hence the cellular metabolic activities take place within the cytoplasm of the cell. The role of bacteria in wine preparation, fermenting alcohol, antibiotics synthesis, probiotics, etc. has been studied for centuries. The generation of bioelectricity in a microbial fuel cell is the latest application of bacteria being studied over the past two decades, though the observation was made 100 yearsago by Potter [5]. The bacteria Geobacter and Shewanella are known for their property of dissimulatory metal reduction. When inoculated in MFCs, they actively transfer electrons toward extracellular space and directly to the electrode by forming a biofilm or attachment through pili. Due to their electroactive properties, they are the common choice for inocula in bioelectroactive systems. The predominant electroactive bacteria in MFC belong to the class Proteobacteria (mostly Gammaproteobacteria and Deltaproteobacteria), as revealed by many ecological and metagenomic studies [6]. This could be due to their physiological condition related to oxygen. However, the electrical output produced depends on the choice of growth condition, source for the electron donors, type of electrode material, reactor size, and other operational conditions. Mixed microbial consortia are found to be advantageous and the common choice of inocula because of the wide and diverse microbial population enabling them to form synergistic relations among themselves. Over time, several notable contributions have been made in the field of bioelectrochemical systems, ranging from the discovery of novel electroactive bacteria to exploring the exoelectron transfer mechanism in them, improvements in bioelectricity generation to some practical applications, and others [1].

1.1.1.2 Fungi (yeasts) in microbial fuel cell

Bacteria have been the preferred choice of inocula in bioelectrochemical systems. However, use of yeasts have also been reported as being used as biocatalysts in BESs. Yeasts are unicellular eukaryotic fungi, often placed in between bacteria and fungi in taxonomy classification. Though the initial observation of their electroactive properties was made by Porter in Saccharomyces cerevisiae using a galvanic cell, not many studies have since been reported in yeasts. The electrochemical activity in Hansenula anomala was observed by Prasad et al., and it is due to the presence of cell-bound redox enzymes, lactate dehydrogenase and ferricyanide reductase [7]. S. cerevisiae inocula with methylene blue as an electron mediator has been reported to produce a maximum power density of 146.71±7.7 mW/m³. Other than these, the other major electroactive yeasts reported in bioelectroactive fuel cells in the literature are Arxula adeninivorans and Candida melibiosica [7,8]. However, extracellular electron transfer mechanism in yeasts has not been explored as much as that of in bacteria. This could be due to its complex cell structure compared to bacteria. Yeasts are commonly considered as electrogens which require the aid of electron mediators (thionin, methylene blue, neutral red, AQDS, flavins, etc.) for shuttling electrons between the cell and electrode. The extracts from yeasts have also shown to act as an excellent electron mediator producing a potential of 1 V in an open air cathode MFC.

1.1.2 Type of exoelectron transfer mechanism

Transfer of free electrons from the bacterial cell to the electrode in BES can be acomplished in different ways [9]. They are broadly classified into two types: direct transfer and indirect transfer of electrons. In a direct transfer systems, electrons are transferred directly from the bacterium to the electrode by forming a contact between them. Hence the electrons do not require any external support to facilitate transfer of electrons. On the other hand, in indirect transfer systems, electron transfer requires an external factors such as redox compounds or chemical molecules to aid in the transfer of electrons to the electrode. These mediators temporarily accept the electrons and shuttle them between the electrode and bacterial cell, as shown in Fig. 1.3.

Figure 1.3 The different types of exoelectron transfer mechanisms used by electroactive microorganisms and their interactions with electrodes in the bioelectrochemical systems [9].

1.1.2.1 Indirect transfer mechanism

In BES some bacteria are weak electrogens. They are unable to transfer the electrons to the electrode actively. Oxidation of chemical compounds at the anode continuously generates free electrons and protons. Due to the lack of contact between the bacterium and the electrode, the free electrons start accumulating in the medium. Upon addition of an external electron mediators in the media, the free electrons are continuously transferred to the electrode [10]. These electron shuttlers are cyclic or heterocyclic chemical compounds that can temporarily accept the electrons due to the presence of conjugate double bonds in them. They also have a high redox potential to participate in redox reactions, thereby enhancing the electron transfer rate in BESs. Chemicals such as AQDS, DMSO, flavins, neutral red, thionine, and methylene blue are some of the commonly employed electron mediators. For example, when a neutral red was added to the media, a 10-fold higher current density was produced with an inoculum of a weak electrogen, E. coliof neutral red in the media [11]. Some bacteria can produce electrochemically active compounds that aid in exoelectron transfer of electrons to the electrode. One such self-synthesized electron mediator produced by Pseudomonas aeruginosa was first reported by Rabaey and coworkers [12]. Pyocyanin is a phenazine derivative compound produced by P. aeruginosa that has the ability to shuttle the electrons in MFCs, thereby increasing bioelectricity production. Apart from pyocyanin, riboflavin, menaquinone, and flavins are some of the self-synthesized chemical compounds that shuttle electrons between the bacterium and the electrode in MFCs.

1.1.2.2 Direct transfer mechanism

In the direct mode of electron transfer, the bacterium interacts with the electrode either by forming a biofilm or through extracellular cell appendage pili, thereby passing electrons directly without the aid of any external support. This kind of exoelectron transfer mechanism is commonly observed in Geobacter sulfurreducens, Shewanella oneidensis, and other electroactive bacteria. This is believed to be due to the presence of electric conductive pili and a set of dedicated exoelectron transfer proteins in the cell membrane (Fig. 1.4) [14,15]. The direct transfer of electrons is also sometimes observed in mixed culture inocula and is made possible by other electroactive bacteria that can form biofilm on the surface of the electrode. The factors governing the transfer of electrons from the cell to the electrode are discussed below.

1.1.2.2.1 Electroconductive pili

Pili are small, thin, hair-like structures present on the cell walls of bacteria. Pili are present in Gram-negative bacteria and aid in horizontal gene transfer during conjugation [16]. They are also reported for their antigenic property in some bacteria. In some electroactive bacteria they are found to be electric conductive. Reguera et al. have shown that the bacterium G. sulfurreducens can transfer electron through pili [17]. Due to their electric conductive properties, these special pili are called nanowires. When analyzed with an atomic force microscope, these nanowires were found to be highly electrically conductive in nature. A similar study on conductive pili was also reported for the bacterium S. oneidensis by Gorby and coworkers [18]. A current of 10 pA was observed in the nanowire when measured using a conductive probe atomic force microscope. The bacteria deficient in pili synthesis were incapable of conducting electrons and the output produced was comparatively less compared to the wild-type strains.

1.1.2.2.2 Biofilm

A common phenomenon observed in bacteria is colonization on the surface of any material through secretion a signaling molecule to form a biofilm. Biofilm formation is also observed in electroactive bacteria for electron transfer. This is observed in pure culture inocula as well as in mixed culture inocula in MFC. Biofilm formation is the most commonly observed mode of microbe–electrode interaction. The extracellular polysaccharide substances present in biofilm are believed to play the role of electron mediators for transferring electrons to the electrode. Formation of biofilm depends on the nature of the electrode material used. Carbon cloth provides an excellent source for biofilm formation due to its rough surface, ultimately resulting in higher current densities compared to graphite and other carbon materials. However, there are reports on biofilm formation on metal electrodes [19]. Baudler et al. reported that the use of metals like steel, silver, gold, and copper providing a rough surface on which electroactive bacterial cells can colonize and form a biofilm. The antibacterial properties of these metals does not affect electroactive bacteria. The current densities produced by them were comparable to those produced using graphite electrodes.

1.1.2.2.3 Cytochrome

Cytochromes are iron-binding proteins that play an important role in intracellular electron transfer and oxidative phosphorylation. In S. oneidensis MR 1, c-type cytochromes present on the cell wall facilitate the transfer of electrons from the bacterium to the electrode. The cytochromes are multiheme-binding cell-bound proteins. The c-type cytochrome forms a multilayer biofilm that produces a higher potential than a monolayered biofilm. The cyclic voltammetry (CV) analysis of biofilm showed active redox peaks, confirming the role of cytochromes in electron shuttling. The mutants deficient in cytochrome synthesis were incapable of electron mediation. Inoue et al. showed that c-type cytochrome omcZ is essential for electron transfer in G. sulfurreducens. Mutants lacking the gene for omcZ showed poor electron conduction and current production [20]. Flavins such as FAD and riboflavins in S. oneidensis have been shown to be electron mediators. The redox property and ability to transfer electrons between the bacterial cell and electrode were analyzed using CV and showed the role of riboflavin in electron shuttling and metal reduction in S. oneidensis.

The electroactive bacteria in MFCs may use more than one mechanism of electron transfer. More often the direct electron transfer occurs with the cumulative effects of a biofilm, pili, and cytochromes for exoelectron transfer in MFC. Several studies have reported on the role of pili in biofilm formation, electron mediators present in the extracellular polysaccharide matrix of biofilm, and the outer membrane cytochromes for electrode and metal reduction.

1.1.3 The cell potential

The potential of an electroactive cell is the measure of difference in charge (negative) between its two ends, the anode and cathode, or in simple terms it is the potential difference between the cathode and anode. The performance of a fuel cell is measured as the potential generated from the system. The maximum voltage generated by the system is called the open circuit voltage, which is the voltage measured at infinite resistance and zero current. The voltage (V) is a function of the current (I) and resistance (R) (internal and external), calculated by using the Ohm’s equation.

(1.1)

The maximum voltage produced thermodynamically in a fuel cell from a particular compound is calculated using the Nernst equation,

(1.2)

E0 is the standard cell potential, R is the universal gas constant (8.31447 J/mol K), T is the temperature in K, n is the number of electrons transferred, and F is the Faraday’s constant (96,485 C/mol).

In BES, the current produced from the system is very small hence it is calculated from the potential measured by varying external resistance or using a potentiostat. Similarly, power is the product of voltage and current, calculated by applying the formula. It is difficult to predict the performance and efficiency of the MFC from the power and current produced by it. Thus it is a good practice to report the output produced in unit scale, normalizing the current and power to the surface area of the electrode and present them as current and power densities,