Comparison of exoelectrogenic bacteria detected using two different methods: U-tube microbial fuel cell and plating method.

Microbial fuel cells (MFCs) have been considered to be a promising technology in the field of sustainable and renewable energy. An MFC is a system for generating electricity from organic compounds using microorganisms as a bio-catalyst. In the anode chamber of MFCs, microorganisms degrade organic compounds, such as glucose, acetate and ethanol, etc., and the electrons produced from this degradation are transferred to the anode as an electron acceptor. The microorganisms capable of transferring electrons to the anode are called “exoelectrogens”. Exoelectrogens function as electrochemically active bacteria, capable of transferring electrons from their cell body to outside of the cell, and play an important role in electricity generation (7, 8). In general, there are direct and indirect ways for exoelectrogens to transfer electrons. Microorganisms transfer electrons directly by developing a biofilm on the anode surface or indirectly through electron shuttles that exist in the anodic suspension. Current can be generated from differences in the potential due to the movement of electrons; they contribute to the production of electricity in both ways; however, such information on electron transfer mechanisms is still insufficient to understand the physiology of the exoelectrogen, the ecology of anodic microbial communities on the electrodes, and the relationship between the exoelectrogen and other bacteria. Therefore, the identification and characterization of exoelectrogens are the most significant factors for increasing the efficiency of transfer electrons and producing higher power via MFCs (7, 8). Methods for the isolation of exoelectrogens from the anode of MFCs can be categorized as follows: dilution to extinction and plating methods. The plating method is known as a generally convenient method to isolate exoelectrogens from MFC anodes. So far, a great number of exoelectrogens isolated from MFC anodes have been reported. There have also been many investigations on these bacteria, such as Clostridium butyricum(10), Aeromonas hydrophila(13), Rhodoseudomonas palustris(17), Aeromonas sp. (2) and Acrobacter butzleri(3). The main advantage of the plating method for their isolation is the conventional and relatively convenient experimental process. With plating, however, it is possible that it will discover not only exoelectrogens but also other bacteria that are not able to transfer electrons extracellularly on an MFC anode. The exoelectrogens identified by the plating method are also known as dissimilatory Fe (III) reducing bacteria, which are able to reduce insoluble iron (7). One reason is that the media for metal-reducing bacteria have been used to isolate exoelectrogens, even though not all of the microorganisms growing in the media are exoelectrogens. Therefore, microorganisms were generally identified by their electrochemical activity after isolation (by plating). It was impracticable to observe the electrochemically activity of bacteria discovered in the microbial community in MFCs. Moreover, the cultivation-dependent method, the plating method, is well known for significant limiting the numbers and populations of bacteria that represent the entire microbial community. The dilution to extinction method, a different and cultivation-independent method for the isolation of exoelectrogens devised in previous studies, is an alternative method that enables exoelectrogens to be isolated by continuous monitoring of the electricity produced in MFCs. With this method, dominant strains of anode respiring bacteria or the electrochemically active microbial community can be isolated. Ochrobactrum anthropi(20) and Comamonas denitrificans(16) have been reported as microorganisms isolated via the dilution to extinction method. O. anthrophi YZ-1 isolated from U-tube MFCs using the dilution to extinction method showed lower maximum current density of 708 mA/m2, but higher coulombic efficiency of 93% than the mixed culture (1730 mA/m2). They produced current using a wide range of substrates (acetate, lactate, propionate, butyrate, glucose, sucrose, cellobiose, glycerol, and ethanol) (20). Most of the defined strains isolated by the plating method have revealed the capability of reducing metals. It is unclear whether electrochemically active metal-reducing bacteria can significantly affect electricity generation; thus it should be considered that metal-reducing bacteria are really major exoelectrogens in MFC. However, previous studies have generally been carried out using a single method, or with different studies using different inocula, which have made it difficult to collectively compare studies. Thus, in this study, we used two methods, the dilution to extinction and plating methods, to identify and to isolate exoelectrogen. Through dilution to extinction experiments using a U-tube MFC, we tried to reduce the diversity of the exoelectrogenic community and to identify exoelectrogen. Then we isolated iron-reducing bacteria from the anodic microbial community of U-tube MFC by the plating method, and they were compared to the exoelectrogen identified using U-tube MFC.