Comparative study on the metabolic behaviour of anode biofilm in microbial fuel cell under different external resistance

Abstract In this study the influence of the external circuit resistance on the metabolic behaviour of anodic biofilm in Microbial fuel cell (MFC) was studied. The results obtained demonstrated that extremely low and extremely high circuit loads could deteriorate the bio-electrochemistry of anodic respiration by shifting the microbial metabolism towards typical fermentation of the substrates. The best conditions for respiration and efficient substrate mineralisation of nearly 70% were found in the MFC reactor with 0.1 kΩ resistance in the anode and cathode connecting circuit. Two species of electrochemically active bacteria were isolated from the anodic community and were taxonomically affiliated to Pseudomonas and Bacillus genera based on 16 s rRNA genes amplification and sequencing.


Introduction
Bio-electrochemical systems (BES) have recently attracted many researchers for their ability to convert the chemical energy of organic compounds directly into electricity. This is a result of the specific metabolic activity of electrochemically active microorganisms known as electrogenes. They can use the electrode of the bio-electrochemical reactor as a terminal electron acceptor during biological oxidation of substrates, and this characteristic creates diverse applications [1]. The most known BES is the microbial fuel cell (MFC) in which the electron flow is generated via microbial decomposition of organic matter at anode connected to cathode by an external circuit to deliver the electrons for the reduction of a soluble chemical acceptor at higher potential. The process has specific biochemical and molecular factors such as terminal reductases, redox-active proteins, soluble metabolic mediators and even conductive cellular structures (often called nanowires in the literature) that perform direct extracellular transfer of electrons from the cell respiratory chains to the electrode surface [2]. An important detail is that the anodic processes are usually anaerobic and driven by anaerobic (obligate and facultative) bacterial communities. Biochemically, however, the anaerobic bio-electrochemical processes are fundamentally different from conventional anaerobic metabolism. While anaerobic digestion involves a fermentative type of metabolism without any intricate electron transport mechanisms for ATP synthesis, anodic processes support a respiratory type of metabolism with all the typical energy benefits for the microbial cell. In addition, anaerobic respiration is part of the biochemical mechanisms underlying the global nitrogen, iron, sulphur and carbon cycles and is quite common in a range of environments [3].
The anaerobic cellular respiration in the MFC depends on the ability of the anode to actively intercept electrons and thus to drive the cellular respiration and ATP synthesis. Studies have already described how simulating different potentials could affect the coupling of the anodic culture with the electrode surface, which results in significant variations in the biomass yield due to activation or inhibition of the respiration activity [4][5][6]. However, these suggestions are mainly based on controlled maintaining of the desired potential values in test systems and not in an actual MFC.
Microbial fuel cell; external resistance; electrochemically active biofilm; anodic respiration One of the factors which is often a subject of design and operation optimisation in the MFCs is the external resistance of the circuit connecting the anode and cathode. Most studies explore its influence on the power performance and very few of them focus on the relation between external resistance, the anode potential and the resulting effects on the microbial metabolism and diversity [7][8][9][10]. The typical anode culture in MFCs is mixed consortia of species, and recent studies have suggested that the metabolic behaviour of the microorganisms depends on the ability of the anode to attract the electrons harvested during the biological oxidation of a substrate [11]. A high-potential anode stimulates the expression of respiratory genes associated with the extracellular electron transport cytochromes and, contrarily, a lower anode potential triggers fermentative metabolism [12,13].
For the power performance of the MFC, fermentative processes are considered undesirable, as they consume electrons and involve reduction of cellular metabolites. In addition, fermentation is not an effective catabolic pathway in terms of cells' own energy economy. In the present work, we explored how the external resistance influences the metabolic behaviour of the microbial consortium present on the anode surface of a MFC.

MFC configuration and operation
The MFC used in this study was designed as a cylindrical plastic reactor consisting of two chambers separated by a Nafion® 424 perfluorinted proton exchange membrane. The cell segments were equipped with the respective sampling and gas/liquid transport ports. The electrodes were 30 mm in diameter made of carbon cloth with stainless steel current collectors. For the different experimental setups, five MFC reactors loaded with resistors ranging from 0 to 10 KΩ were assembled ( Figure 1). The volumes of cathode and anode chambers were 45 dm 3 . The anode compartment is fed by 10 g.dm −3 luria-Bertani (lB) nutrient medium with 2 g.dm −3 glucose (initial pH 7.5 and organic load COD equivalent to 9558 mgO 2 .dm −3 ) and mixed culture of electrochemically active bacteria isolated from lake bottom sediments [14] with initial cell density of 10 6 CFu per millilitre. A 2% solution of potassium ferricyanide was used as catholyte and terminal electron acceptor by permanent recirculation to maintain the concentration and the state of the cathode reactions. The MFC experiments were carried out in controlled ambient temperature of 18 C.

16s rRNA gene sequencing and taxonomic affiliation
16s rRNA gene amplification and sequencing were performed for identification of the bacterial species populating the anode biofilm. Strains were isolated from the anode surface after disassembly of the operating MFC. The isolates were grown anaerobically on rich agar medium (lB with 10 g.dm −3 glucose). genomic DNA from the studied isolated bacterial culture was isolated by Sigma Aldrich -genElute bacterial DNA isolation kit (cat. No.: NA2100) following the procedure described by the manufacturer. Polymerase chain reaction (PCR) was performed by universal prokaryotic primers 27 F and 1492 R [15] using an ESCO Swift™ MiniPro® Thermal Cycler and PuReTaq™Ready-To-go™PCR beads (Amersham Bioscience, uSA) under the following conditions: Reaction volume − 25 µl; Primers concentration in the final reaction volume − 10 pmol and 25 ng to 50 ng genomic DNA template. PCR program: Initial denaturation 95 °C − 5 min; 35 cycles as follows: 95 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min and 5 min final extension at 72 °C. The obtained PCR products were purified by the gFX™PCR DNA and gel band purification kit (gEHealthcare) and then sent to Macrogene Europe (Netherlands) for sequencing by 785 F and 907 R primers [16]. The sequences obtained were analysed by the BlAST tool of the NCBI gene Bank database for taxonomic affiliation of the studied strains.

Analytical methods
The COD and the organic acids content of the substrate in the anode compartment was measured by HACH lange lCK314 and lCK365 cuvette tests on a lange DR 3900 spectrophotometer.
All analyses and measurements were performed in three replicates and the mean values with standard deviation (±SD) are presented in the corresponding graphs and tables.

Influence of external circuit resistance
This study was based on the initial suggestion that the external resistance could play a significant role beyond its influence on the powers performance of the MFC. The initial hypothesis to be tested was that it is also a factor that can 'switch' the catabolic biochemistry of the anode biofilm from anoxic respiration (using the electrode as the terminal electron acceptor) to fermentation regime. Thus, the experimental design applied a functional approach in which the amount of organic acids (measured as acetate equivalents) in the anode compartment served as an indicator for the biochemical state of the microbial culture in terms of the substrate catabolism. It is common knowledge that acetate, lactate etc. are the typical product of fermentation and impor tant inter mediates of methanogenesis.
This assumption was made based on well known and previously described electrochemical mechanisms involved in the MFC processes. Previous studies described how power performance and biological activity balance between the potential difference, which polarises the cell to develop electromotive force and the electron flux through the circuit -both depending on the resistance between anode and cathode [17].
Four MFCs were started in a parallel experiment under the same conditions. Within the first week of operation all of them were configured at external circuit resistance of 1 kΩ for anode biofilm formation and stabilisation according to the procedure developed earlier [17]. After this initial stage, the operation conditions of the reactors were maintained at the same levels for all the experimental variants except for the external circuit load, which varied from 0 to 10 kΩ. A control sample including the same growth medium, substrate and microbial culture was incubated under anaerobic conditions in order to provide data for comparison of the MFC driven process and typical fermentation. Within 6 weeks of cultivation, the COD removal dynamics and the organic acids content of the medium were monitored (Figures 2 and 3).
The results showed that the mechanism and dynamics of organic substrates degradation differed significantly under different external resistance values. The organic acids content indicated the transition from fermentation to respiration when the electrochemical conditions on the anode are suitable for extracellular export of electrons. At 0 Ω (short-circuited anode and cathode) and 10 kΩ, the high organic acids concentrations measured indicate fermentation like substrate degradation. Although with the same effect on the microbial metabolism, the presumed mechanisms of these two cases significantly differ. In the first case (0 Ω), the process is characterised by low potential difference and lack of polarization, which results in insufficient electromotive force and low anode electron affinity. In the second scenario (10 kΩ), the extremely high values of the external resistance create high potential difference and polarisation (560 mV measured during the experiment), however the electron flux is significantly hindered. In both cases, the anode biofilm could not effectively export electrons towards the electrode surface, which   Bacillus cereus mn956536 p4 Pseudomonas protegens mn956537 p5 Pseudomonas syringae mn956538 p6 Aeromonas fluvialis mn956539 suppresses respiration and forces cells to ferment the available substrates. The most efficient anoxic respiration was observed under 100 Ω of external resistance, and the data obtained demonstrated the lowest levels of organic acids and fastest COD removal rate in this particular MFC reactor. The substrate mineralisation rate was nearly 70% (Figure 4), which also supports the potential use of bio-electrochemical processes as an alternative approach in biological wastewater treatment, especially when waste streams with high organic load are subject of treatment [18].

Isolation and characterisation of bacterial species with electrochemical activity
The secondary objective of this study was to isolate and characterise bacterial species with electrochemical activity and the metabolic ability to oxidise substrates via extracellular electron transport towards insoluble terminal acceptors (such as the anode in MFC). The results showed that the initial mixed culture used as inoculum in this study predominantly consisted of five strains (designated as P2, P3, P4, P5, P6) differentiated by cultural and morphological characteristics. Then, genomic DNA was extracted, PCR amplification and sequencing of 16 s rRNA gene fragments was performed. The resulting sequences were used for comparative analysis and taxonomical identification. All original sequences were registered in the NCBI geneBank database under the corresponding accession numbers ( Table 1).
The mixed culture profile was modified on the anode surface of the best performing 0.1 kΩ MFC after 6 weeks of operation; it was dominated by the P2 and P3 stains, namely Pseudomonas lutea and Bacillus cereus. Taking the anode respiration as a selective factor, these two strains could be considered as most electrogenic among the species presented in the initial consortia. Previous studies have reported representatives of different Bacillus and Pseudomonas genera in anode biofilms [19,20]. P. lutea (as many other species from the family Pseudomonadaceae) are usually considered as strict aerobes and their isolation from anaerobic cultures could be unexpected. However, it was recently reported that they could adapt and grow in anaerobic environments if alternative terminal electron acceptors are present. Furthermore, in this condition the metabolic profile of the culture is modulated by the external electron acceptor by regulating the gene expression involved in this process [21,22]. Our finding is in support of this understanding and beyond that, both P. lutea and B. cereus were isolated in all MFC variants in this study.

Conclusions
The results obtained demonstrated that the resistance in the MFC external circuit is a factor that significantly affected the metabolic behaviour of the anode biofilm and the overall performance of the reactor. In optimal electrochemical conditions the substrate mineralization is significantly improved due to effective respiration and suppressed fermentation processes. Current evidence indicates that the microbial species involved in the process seem to be adaptable to the electrochemical conditions on the anode surface and perform both respiration and fermentation depending on the anode potential, external resistance, MFC polarisation and electron flux through the circuit. Further research on the genetic and regulatory factors which determine this behaviour remains a question of interest.

Data availability statement
The data that support the findings of this study are available on request from the corresponding author.