29

Noda Institute for Scientific Research GRANT2014 Young Investigator Research GrantOutline of Research Result

Mechanism of electricity generation based on the stressresponse of metal-reducing bacteriaYoshihiro OjimaDepartment of Materials Engineering Science, Graduate School of Engineering Science, Osaka University

Research aimsShewanella oneidensis is a gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration.1) It plays an importantrole in many environmental and biotechnological processes, including a microbial fuel cell (MFC) system and inreduction of toxic metals, such as uranium and chromium.
Our study aims at clarifying the mechanism of electricitygeneration of S. oneidensis in terms of stress response. S.
oneidensis and other gram-negative bacteria (Escherichiacoli etc.) have the rpoS gene, a global regulon gene, whichregulates the transcription of structural genes responsiblefor adaptation to a variety of stresses, including oxidativeand osmotic pressures, heat, starvation, acids. Additionally,S. oneidensis and Escherichia coli share many characteristics. For example, the plasmid used for gene recombinationof E. coli can be used in the S. oneidensis cells.
In this study, I investigated the effects of deleting thestress response gene rpoS on electricity generation by S.
oneidensis cells. Genetic engineering techniques using E.
coli cells were applied to construct the rpoS-deficient S.
oneidensis mutant.
MethodsIn-frame deletion of rpoS from the S. oneidensis genomerpoS (SO3442) was deleted in-frame from the S. oneidensis genome. Following multiple rounds of fusion PCR,the amplicon was cloned into the suicide plasmid pRE112,and then transformed into E. coli SM10 λpir via the calcium chloride method. The resulting plasmid was transferred into recipient S. oneidensis MR-1 cells by conjugation, and the transformants were selected on the basis ofchloramphenicol resistance. Integration of the mutagenesisconstruct into the chromosome was induced to generate thefinal deletion strain. The remaining plasmids were removedvia sucrose-induced lethality. The deletion was verified byPCR amplification of rpoS and realtime-PCR analysis.
Microbial fuel cellA two-chambered MFC was fabricated using two 300-mlPyrex glass bottles that were manually modified. A Nafion®
perfluorinated membrane was clamped between the adapters. The cathode and anode were graphite unrolled by 0.20

g of untreated carbon cloth. Copper wires were used to connect the circuit, and the connections were secured with alligator clips. The anode chamber was filled with 300 ml oflysogeny broth (LB) medium (10 g/l polypeptone, 5 g/lbacto-yeast extract, and 10 g/l NaCl). It also contained 2 g/llactate as substrate. The initial pH of the medium was 7.0.
The cathodic chamber was filled with 300 ml of 100mmol/l phosphate buffer (pH 7.0) containing 320 mmol/lpotassium ferricyanide. S. oneidensis cells were pre-cultured in LB medium and harvested by centrifugation. Theconcentrated E. coli cell suspension was added into the anodic chamber to obtain an initial optical density at 660 nm(OD660) of ~1. The MFC was operated at room temperature(ca. 25°C) and the voltage was measured with a voltagelogger by connecting to a 10-Ω resistor.
ResultsIn-frame deletionTo confirm the in-frame deletion of rpoS, genomic DNAwas extracted from wild type S. oneidensis and the ΔrpoSmutant. The rpoS gene (981 bp) plus 250 bp upstream wasamplified by PCR. The electrophoresis results are shown inFig. 1. An approximately 1.2-kbp fragment was amplifiedfrom the wild type cells, corresponding to the combinedsize of rpoS plus surrounding regions. In contrast, a 250-bp

Fig. 1. Electrophoresis of rpoS fragment amplified from genomic DNAisolated from wild type (WT) and ΔrpoS strains.

30Fig. 2. Time profile of viable cells, lactate concentration, and voltage ofthe constructed MFC with wild type cells.

band was amplified from the putative ΔrpoS mutant cells,demonstrating that the rpoS region was successfully deletedfrom genomic DNA of S. oneidensis. Furthermore, realtime PCR analysis did not detect rpoS mRNA expression inthe mutant cells. These results indicate the successful deletion of rpoS from the chromosome of S. oneidensis.
Microbial fuel cellTime profile of viable cells, lactate concentration, andvoltage of the constructed MFC with wild type cells areshown in Fig. 2. The voltage gradually increased andreached about 150 mV at 50 h, indicating that S. oneidensiswild type cells convert lactate to electric energy. Since viable cell concentration did not increase during the reactionperiod, it can be considered that the gradual increase involtage was due to the increase of the efficiency of electrontransfer from S. oneidensis cells to electrode. Lactate concentration slightly decreased from 2 g/l, but it was not completely consumed during the culture period, suggesting thatthe substrate supply does not limit the output of this system.
Next, Figure 2 shows the time profiles of the constructedMFC with ΔrpoS cells. The voltage rapidly increased andreached 90 mV at 5 h with no appreciable increase afterward. The value remained constant at around 90 mV. Meanwhile, time profiles of viable cells and lactate concentration

Fig. 3. Time profile of viable cells, lactate concentration, and voltage ofthe constructed MFC with ΔrpoS cells.

were not significantly different between wild type andΔrpoS cells, suggesting that the deletion of rpoS negativelyaffects the electron transfer from cells to the electrode.
Thus, it was clarified that the global stress response generpoS plays an important role in electricity generation of S.
oneidensis.
ConclusionThe rpoS gene of S. oneidensis was successfully deletedin-frame to examine the effect of oxidative stress on electricity production. Evaluation of the constructed MFC system showed that the deletion of rpoS gene decreased thevoltage of MFC without affecting the cell and lactate concentration. These results suggest that the rpoS gene is important for electricity generation by S. oneidensis cells.
References1)2)

Myers, C. R., and Nealson, K. H. (1988). Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor. Science 240: 1319–1321.
Ojima, Y., Kawata, T., Matsuo, N., Nishinoue, Y., and Taya, M.
(2014) Recovery of electric energy from formate by using a recombinant strain of Escherichia coli. Bioprocess and Biosystems Engineering 37: 2005–2008.