Development of Microbial Fuel Cells using Microorganisms NotEliminated by Indigenous Bacteria
Kengo INOUE
Department of Biochemistry and Applied Biosciences, Faculty ofAgriculture, University of Miyazaki
Research objective
Microbial fuel cells are energy conversion technologies that utilize microorganisms
capable of transferring electrons to electrodes, allowing power generation while treatingorganic compounds in organic wastewater (Fig. 1). In our previous study, microbial fuel cells
using livestock waste, such as cattle manure, has been established 1). By utilizing microbial
fuel cells, electricity can be generated while maintaining the treatment efficiency of the mostcommon wastewater treatment technology, aerobic treatment (activated sludge method).
In this study, the long-term operation of microbial fuel cells using swine wastewater
was conducted. Microorganisms were isolated from the anode biofilm of microbial fuel cells
to develop high-performance microbial fuel cells using the isolate, which was not eliminatedby indigenous bacteria in swine wastewater.

Methodsi) Long-term operation of swine wastewater microbial fuel cells: Microbial fuel cells using
swine wastewater were operated for 183 d. External resistances ranging from 5.1 to 100 Ω
were installed, and the voltage was recorded every minute using a data logger. Swine
wastewater was subjected to solid-liquid separation using only the liquid portion. The swine
wastewater was replaced approximately once a week. During the operation period, aeration
was performed in the swine wastewater while processing the reactor, including the microbial
fuel cells. The treatment efficiency was evaluated by measuring the chemical oxygen demand(COD) of swine wastewater in the reactor. Additionally, samples of swine wastewater fromthe reactor were collected at intervals of 3–4 d, and the pH, nitrate ion concentration, andelectrical conductivity were measured.
ii) Microbial community structure analysis: Swine wastewater samples before introduction
into the microbial fuel cells, anode biofilms on day 28 of operation, and anode biofilms on day
137 of operation were collected, and DNA was extracted. The extracted DNA was used as atemplate to amplify the 16S rDNA using PCR. Nucleic acid sequences were determined using
next-generation sequencing.
iii) Isolation of microorganisms from anode biofilm samples and identification of isolated
strains: To isolate anaerobic electricity-generating bacteria, media containing lactate-sulfate
for nitrate-reducing bacteria, acetate-carbonate for methanogens, R2A, and modified GAM
were prepared under anaerobic conditions. Noble agar or gelatin gum was added to the solidmedium. Biofilm samples scraped from the anode surface were diluted in phosphate buffer to10-4–10-8 under anaerobic conditions and spread onto solid media. The plates were incubatedat 30 °C in an anaerobic chamber for several months. Colonies that appeared during the culture
period were subjected to colony PCR using universal primers for bacteria and archaea toamplify the 16S rDNA. The obtained DNA fragments were sequenced to identify the genus
and species.
Results
During the 183-d operation of microbial fuel cells using swine wastewater, theaverage power density was 40.4 mW/m2 (per electrode projection area), and the maximum
power density was 83.9 mW/m2. The organic matter processing rate averaged 432.2 mgCOD/L·day, and the processing efficiency (average processing over 8.7 d) after each swine
wastewater replacement was 67.8%.
As considerable output and processing efficiency were achieved, anode biofilm
samples were collected from the microbial fuel cells on days 28 (power density 51.4 mW/m2)
and 137 (60 mW/m2) after the start of operation. Microbial community analysis revealed thatthe dominant microbial species changed significantly. Before the operation, Sphirochaetaceae

(19%), Clostridiaceae (14%), Prevotellaceae (6.8%), Ruminococcaceae (5.2%), and otherfamilies were relatively abundant in swine wastewater at the family level. In the anode biofilm
on day 28, Bacteroidales (11.6%), Porphyromonadaceae (8.9%), and Pelobacteraceae (8.7%)
were predominant, indicating a significant shift in the dominant microorganisms. On day 137
of operation, Clostridiaceae (16%), Methanosaetaceae (7.4%), and Bacteroidales (6.5%)
were predominant. Microorganisms belonging to the Pelobacteraceae and Methanosaetaceae
families, which were almost undetectable in the original swine wastewater before treatment,
have been suggested to contribute to the decomposition of organic matter and electricity
generation in the microbial fuel cells using swine wastewater. Additionally, microorganisms
with a high potential for sulfate reduction, such as those from the Desulfubulbaceae andDesulfobacteraceae families, significantly increased after microbial fuel cell operation.
Interestingly, Geobacteraceae, which are often dominant in microbial fuel cells near neutral
pH, were not detected before or after operation.
After collecting the anode biofilm samples from the microbial fuel cells after
operation, the samples were diluted and spread onto various solid media (as mentioned above)
under anaerobic conditions. Although numerous colonies were observed in each medium,diversity was not clearly observed in colony morphology. Analysis of the 16S rDNA sequencesof the 11 isolates revealed that they belonged to the genera Bacillus, Brachymonas,
Corynebacterium, Parabacteroides, and Rhodococcus. These microorganisms did not matchthe 16S rDNA sequences of the dominant microorganisms in the anode biofilm after microbial
fuel cell operation and were not closely related to them.
Conclusion
In the anode biofilm of microbial fuel cells using swine wastewater, no dominantmicroorganism species were found after the operation. Therefore, it is difficult to predict themicroorganisms with a high likelihood of contributing to electricity generation by comparing
microbial community structures. Consequently, despite attempts to isolate microorganisms
from various solid media, the isolated strains did not match the target microorganisms. In thefuture, a narrower focus will be placed on microbial species that are speculated to be highly
important, such as methanogens and sulfate-reducing bacteria, and isolation efforts will beconducted using specialized media for each species.
Reference1) Inoue, K., Ito, T., Kawano, Y., Iguchi, A., Miyahara, M., Suzuki, Y., and Watanabe, K.
(2013) Electricity generation from cattle manure slurry by cassette-electrode microbial
fuel cells. J. Biosci. Bioeng. 116: 610–615.