Activation of Ethanol Production by Combination of Recombinant Ralstonia eutropha and Electrochemical Reducing Power

Ralstonia eutropha was genetically modified to induce ethanol production from glucose. An electrochemical bioreactor was prepared to generate electrochemical reducing power coupled to regeneration of NADH. Growing cells of recombinant R. eutropha produced about 29 mM of ethanol in conventional conditions and 56 mM of ethanol in electrochemically reduced conditions from 100 mM glucose. Grown cells of the recombinant produced about 52 mM of ethanol in conventional conditions and 142 mM of ethanol in electrochemically reduced condition from 100 mM glucose. These results are a clue that electrochemical reducing power can induce the recombinant R. eutropha to produce more ethanol coupled to increase of NADH/NAD ratio.


Introduction
Electrochemical reduction reaction can be separated from the electrochemical oxidation reaction by a specially designed electrochemical bioreactor [1,2].The electrochemically reduced neutral red catalyzes regeneration of biochemical reducing power (NADH) without enzyme catalysis [3,4].Increase of the NADH/NAD + ratio induces an increase of metabolites that are produced coupled to NADH oxidation to NAD + in fermentative metabolism [5,6].The electrochemical reducing power can be obtained from electricity generated from solar energy without combustion of fossil fuels.An electrochemical bioreactor is a useful device to accomplish conversion of solar energy to biochemical energy and carbon dioxide to organic compounds [1,7].
The nutritionally versatile Ralstonia eutropha is a facultative autotroph that can assimilate CO 2 in coupling with metabolic oxidation of H 2 via Calvin-Benson-Sassham reductive pentose-phosphate cycle [8] and can also heterotrophically grow with glucose [9].Growth of R. eutropha was known to be less than 1.0 on an optical density basis in the autotrophic condition with H 2 and CO 2 as a sole energy and carbon source but maximally 6 times increased in the mixotrophic condition with H 2 , CO 2 , and amino acids [10][11][12].R. eutropha autotrophically grows with CO 2 and electrochemical reducing power as a sole carbon and energy source, but doesn't produce any metabolite that is alcohol and organic acid in both autotrophic and heterotrophic condition [13].Practically, ethanol production of R. eutropha may be induced by recombination of specific genes encoding pyruvate decarboxylase and alcohol dehydrogenase genes that were obtained from Zymomonas mobilis [14,15].Ethanol production was proportionally increased to the ratio of NADH/NAD + in fermentative metabolism [6].Accordingly, combination of intrinsically autotrophic metabolism, extrinsically genetic recombination, and electrochemically regenerated reducing power may be the best way to induce R. eutropha to produce ethanol from CO 2 .This study is performed to test the possibility that the metabolic function of the recombinant R. eutropha for ethanol production may be activated by the electrochemical reducing power under heterotrophic condition.
ing pyruvate decarboxylase and alcohol dehydrogenase II were kindly offered by Ingram [14,16].The plasmid was inserted into R. eutropha by using an electroporator (Mi-croPulser, BioRad Laboratories, Richmond, CA, USA) at 2.5 KV for 2.5 msec according to the user's manual.All of procedures for preparation of electrocompetent cells of R. eutropha, culture medium, plasmid DNA, buffers, and selection medium were performed according to methods used by Taghavi et al. [17] and transformant capable of producing ethanol was selectively isolated from agar plate medium according to the method used by Conway et al. [18].

Ethanol Production of Transformant
An electrochemical bioreactor that was designed for continuous culture in previous research was partially modified for batch culture of recombinant R. eutropha, shown in Figure 1.Graphite felt (thickness, 10 mm; height, 200 mm; length, 500 mm; Electrosynthesis, USA) modified with neutral red was used as a cathode and platinum wire (0.3 mm thickness, 400 mm length) was used as a counter electrode [13].Two thousand ml of the modified M9 medium was prepared in the electrochemical bioreactor (Figure 1) to which O 2 -free CO 2 (50 ml•min −1 ) was continuously supplied during cultivation.Wild type or recombinant R. eutropha previously cultivated for 24 h was inoculated at the ratio of 5% (v/v).DC −3 V of electricity was charged to the cathode for culture test in the electrochemical reduction condition but not during culture test in the conventional condition.One hundred mM of K 2 HPO 4 was used as anolyte.

Analysis
Ethanol production was analyzed by GLC (Acme 6000, Younglin Instrument, Seoul, Korea) equipped with a flame-ionized detector and DB-Wax column (30 m × 0.535 mm, Agilent Technology, CA, USA).Oven temperature was initially maintained at 50˚C for 5 min and then gradually increased to 150˚C at the rate of 20˚C a minute.Split ratio of sample and split flow of mobile phase (99.999% helium) was adjusted to 20:1 and 16.1 ml/min, respectively.Flow rate of mobile phase and injection volume was adjusted to 0.8 ml/min and 1 μl, respectively.20 mM of valeric acid was used as an internal standard.Glucose consumption was analyzed by HPLC equipped with a refractive index detector (Younglin Instrument, Seoul, Korea) and HPX-87H ion exchange column (BioRad Laboratories, Richmond, CA, USA).Temperature of column and detector was adjusted to 35˚C, and flow rate of mobile phase and injection volume of sample was adjusted to 0.6 ml/min and 20 μL, respectively.

Results and Discussion
In the time profile of batch culture for recombinant R. eutropha aimed at the production of ethanol by the introduction of genes encoding pyruvate decarboxylase and alcohol dehydrogenase II, glucose consumption and ethanol production was evaluated as shown in Figure 2. The recombinant R. eutropha produced approximate two times of ethanol in the electrochemically reduced condition than conventional condition; however, glucose was mostly consumed and not influenced by the cultivation conditions.Generally, the ethanol production by fermentative microorganisms that are Zymomonas mobilis and Saccharomyces cerevisiae is proportional to glucose consumption, with a ratio of ethanol to glucose of 2 to 1 [19,20].However, the ratio of ethanol production to glucose consumption by recombinant R. eutropha was about 0.28 to 1.0 in the conventional condition and 0.58 to 1.0 in the electrochemically reduced condition.The metabolic intermediates and reducing power (NADH) generated in coupling with glucose oxidation may be partially consumed for ethanol production and biosynthesis of building blocks in heterotrophically growing cells [21,22].The ratio of ethanol production to glucose consumption by grown cells of recombinant R. eutropha that was previously cultivated for 48 h and harvested and then suspended in 0.5 volume of fresh medium was increased to be 0.52 to 1.0 in conventional condition and 1.42 to 1.0 in electrochemically reduced condition as shown in Table 1.Theoretically, grown cells of fermentative bacteria may less consume reducing power for biosynthesis of building blocks but more consume reducing power for metabolite production than growing cells.The higher ethanol production by the grown cells compared to the growing cells may be caused by higher production of reducing power in proportion to biomass and higher consumption of reducing power coupled to ethanol production.The higher ethanol production in the electrochemically reduced condition may be caused by increased reducing power (ratio of NADH/NAD + balance) that is electrochemically reduced coupled to redox reaction of neutral red immobilized in cathode [6,23].Conversion of electrochemical reducing power to biochemical reducing power (high balance of NADH/NAD + ) has been employed to induce increase of specific metabolite production from glucose by fermentative metabolism [4].Ethanol produced from 1 M glucose by ethanol-fermentation bacteria or yeast has to be less than 2 M because all of NADH can't be oxidized coupled to reduction of acetaldehyde to ethanol; however, 2 M ethanol can be produced from 1 M glucose by the coupling with the electrochemical redox reaction of neutral red.A system for ethanol production from glucose may not be required to improve on the technical basis but may be required to develop on the material basis.
R. eutropha can autotrophically grow with electrochemical reducing power and CO 2 but doesn't produce any metabolite from CO 2 except biomass [1,13].In this research, R. eutropha was genetically modified with pyruvate decarboxylase and alcohol dehydrogenase to obtain a recombinant strain capable of producing ethanol from glucose.The recombinant R. eutropha produced ethanol from glucose, about two times increased in the electrochemically reduced culture condition.The recombinant R. eutropha, the autotrophic CO 2 fixation, and the electrochemical bioreactor will be combined to produce ethanol from CO 2 in near future.

Conclusion
R. eutropha autotrophically grew with the electrochemical reducing power and CO 2 , and ethanol production of the recombinant from glucose was activated by the electrochemical reducing power.On the basis of this result, it is possible that the recombinant R. eutropha may produce ethanol from CO 2 with electrochemical reducing power in medium without glucose by application of a specific signal for induction of pyruvate decarboxylase and alcohol dehydrogenase genes.

Figure 1 .Figure 2 .
Figure 1.Electrochemical bioreactor to induce biochemical reducing power for bacterial metabolism in coupling with redox reaction of neutral red immobilized in graphite felt.