Influence of substrate on electricity generation of Shewanella loihica PV-4 in microbial fuel cells
© Wu et al.; licensee BioMed Central Ltd. 2014
Received: 23 November 2013
Accepted: 7 May 2014
Published: 16 May 2014
The substrate, serving as carbon and energy source, is one of the major factors affecting the performance of microbial fuel cells (MFCs). We utilized BIOLOG system to rapidly screen substrates for electricigens, and further evaluated influence of these substrates on electricity generation of Shewanella loihica PV-4 in MFCs.
Three of most favorable substrates (lactate acid, formic acid and cyclodextrin) with OD590/750 of 0.952, 0.880 and 0.849 as well as three of most unfavorable substrates (galactose, arabinose and glucose) with OD590/750 of 0.248, 0.137 and 0.119 were selected by BIOLOG system under aerobic conditions. The chronoamperometry results showed that MFCs fed with these substrates exhibited different current behaviors. Cyclic voltammograms results showed that arabinose, galactose and glucose promoted electron transfer from outer membrane c-Cyts of cells to the electrode surface. Lactic acid, formic acid and cyclodextrin produced lower quantity of electric charge of 10.13 C, 9.83 C and 10.10 C, the corresponding OD600 value was 0.180, 0.286 and 0.152 in BES; while galactose, arabinose and glucose generated higher quantity of electric charge of 12.34 C, 13.42 C and 17.45 C, and increased OD600 values were 0.338, 0.558 and 0.409 in BES. SEMs results showed that plenty of plump and stretched cells as well as appendages were observed when lactic acid, formic acid, and cyclodextrin were utilized as substrates, while sparse cells in short shape were obtained when galactose, arabinose and glucose were used as substrates.
These results suggest that substrate not only has important role in electrochemical performances of MFCs but also in biological properties of electricigens. Lactic acid, formic acid, and cyclodextrin beneficial for cell growth under aerobic conditions are unfavourable for planktonic cell growth and current generation under anaerobic conditions, while consumptions of galactose, arabinose and glucose adverse to cell growth under aerobic conditions are favourable for planktonic cell growth and current generation under anaerobic conditions due to the increase of cell numbers with more outer membrane c-Cyts transferring electrons between the electrode surface and cells.
Microbial fuel cells (MFCs), harvesting electricity from renewable biomass, have attracted great interest in the area of wastewater treatment, bioremediation, biosensors and so on [1–4]. The microbial fuel cell consists of an anode, which accepts electrons released from the microbial metabolism and passes electrons to a cathode, where they are accepted by molecular oxygen. The knowledge that bacteria can generate electric current was first reported by Potter . However, the low power density is still one of the main limiting factors restricting the practical application of MFC [6, 7]. To overcome this problem, many researchers devote research to the optimization of MFC construction and operation condition [8, 9], screening of active electricigens , as well as the modification of electrode with nanostructures [11–14]. The substrate serving as carbon and energy source is also considered as a major factor which affects the performance of MFC . However, most of these studies are focused on the limited sorts of single substrate or complex substrates in MFCs with pure culture or activated sludge, respectively.
Shewanella loihica PV-4, a dissimilatory metal reducing bacterium isolated from the Loihi Seamount in Hawaii, has received attention because it generates higher current density than other Shewanella strains . In contrast to most Shewanella species, S. loihica PV-4 was able to utilize fumarate, galactose, glucose, citrate, lactate, malate, maltose, N-acetylglucosamine, succinate and alanine as substrates but unable to utilize acetate, propionate or Tween 40 . Although the effect of lactate  and a mix of volatile fatty acids  as substrates on the performance of MFCs have been evaluated, there is rare report about influence of other single substrates on electricity generation performance and cell growth as well as the interaction between them.
BIOLOG system, taking advantage of microbe’s ability to use particular carbon sources to produce a unique “fingerprint” of 95 single carbon sources in a MicroPlate, is widely used for the assessment of bacterial functional diversity in environmental samples [18–20]. The ability of a microbe to use a particular carbon source produces respiration, which reduces a tetrazolium redox dye and causes a color change in that well. The end result is a pattern of colored wells that is characteristic for that organism. Herein, we utilize BIOLOG system to rapidly screen favorable and unfavorable substrates for the growth of S. loihica PV-4, and further evaluate influence of these substrates on electricity generation of S. loihica PV-4 in MFCs.
Results and discussion
Substrate screening results
Quantity of electric charge and cell growth
Lactic acid, formic acid, cyclodextrin, galactose, arabinose and glucose serving as electron donors for S. loihica PV-4 in MFCs were selected by BIOLOG system. Lactic acid, formic acid and cyclodextrin beneficial for cell growth under aerobic conditions were unfavourable for planktonic cell growth under anaerobic conditions and produced lower quantity of electric charge, while galactose, arabinose and glucose adverse to cell growth were favourable for planktonic cell growth under anaerobic conditions and generated higher quantity of electric charge. The electron donor played an important role not only in electrochemical performances of cells but also in cellular morphologies, especially the formation of appendages. Further researches including underlying formation mechanism and electrical properties of appendages are still necessary.
Shewanella loihica PV-4 strain (ATCC BAA-1088) was aerobically cultured in 10 mL of Marine Broth (20 g L−1) at 30°C for 24 h. After centrifugation, the Marine Broth was replaced with 10 mL of defined media (DM)  at 30°C for 48 h with different substrates. The suspension was centrifuged for 10 min and the resultant cell suspension was washed with DM three times prior to being used for electrochemical experiments. As substrates, lactate acid, formic acid, cyclodextrin, galactose, arabinose and glucose (10 mM) were used respectively.
Screening of substrates by BIOLOG system
S. loihica PV-4 was inoculated and aerobically cultured in Marine Agar (20 g L−1) at 30°C for 16–24 h. A uniform suspension within the specified turbidity range was prepared by dipping the swab picking up cells into the inoculating fluid. Inoculate cells (150 μl per well) into GN2 MicroPlate (Biolog Catalog #1011), incubate aerobically at 30°C for 25 h and then read by BIOLOG MicroLog System (Release 4.2, Biolog Inc., USA) with two appropriate filters (590 and 750 nm).
A single-chamber, three-electrode system was used for the electrochemical measurements, where ITO electrode was used as the working electrode on the bottom of the cell , a platinum wire as the counter, and an Ag/AgCl (saturated KCl) electrode as the reference. The reactor filled with 4 mL DM containing 10 mM different substrates mentioned above was deaerated by purging with N2 gas (30 min) and subsequently injected with bacterial culture (OD600 2.0) as described above at a constant poised potential of 0.2 V using a CHI 660D potentiostat (CH Instruments, Chenhua Co. Shanghai, China) at 25°C, pH 7.8. After these electrochemical measurements, the final optical density (OD600) of the bacterial culture in the reactor after 25 h was measured using a spectrophotometer (Mapada, China). All these experimental tests were repeated for one time.
S. loihica PV-4 attached on the electrodes were imaged using an SEM (HITACHI, S-300 N). Samples were fixed in 2.5% glutaraldehyde for 2 h, rinsed three times in phosphate buffer (pH 7.0, 50 mM), dehydrated by alcoholic series (60, 70, 80, 90, 95, and 100%), and then air-dried.
This work was supported by National Natural Science Foundation of China (NSFC 81301290), Quanzhou Science and Technology Plan Project (2013Z24) and Research Foundation for High-level Talents of Huaqiao University (12BS207).
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