J. Microbiol. Biotechnol. (2011), 21(6), 590–598 doi: 10.4014/jmb.1101.01032 First published online 20 April 2011 Enrichment of CO2-Fixing Bacteria in Cylinder-Type Electrochemical Bioreactor with Built-In Anode Compartment Jeon, Bo Young1, Il Lae Jung2, and Doo Hyun Park1* 1Department of Biological Engineering, Seokyeong University, Seoul 136-704, Korea 2Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon 305-353, Korea Received: January 21, 2011 / Revised: March 26, 2011 / Accepted: March 27, 2011 Bacterial assimilation of CO2 into stable biomolecules or NADH) with reduction energy generated by the + o using electrochemical reducing power may be an effective metabolic oxidation of NH4 , S , or H2, which functions as method to reduce atmospheric CO2 without fossil fuel an electron donor for the respiration and reduction of CO2 combustion. For the enrichment of the CO2-fixing bacteria [10, 22, 26, 28]. In a variety of metabolic carboxylation using electrochemical reducing power as an energy source, reactions, CO2 is assimilated into the biomolecules via the a cylinder-type electrochemical bioreactor with a built-in biochemical reducing power regenerated in tandem with anode compartment was developed. A graphite felt cathode the oxidation of organic compounds [29, 31, 32]. Meanwhile, modified with neutral red (NR-graphite cathode) was the biochemical reducing power can be regenerated in used as a solid electron mediator to induce bacterial cells combination with H2O decomposition by the electromagnetic to fix CO2 using electrochemical reducing power. Bacterial power in the photoautotrophic metabolism, along with O2 CO2 consumption was calculated based on the variation in generation [7]. the ratio of CO2 to N2 in the gas reservoir. CO2 consumed Biochemical reducing power also can be electrochemically by the bacteria grown in the electrochemical bioreactor regenerated via the catalysis of neutral red, without (2,000 ml) reached a maximum of approximately 1,500 ml per resorting to enzyme catalysis [23, 25]. Neutral red can be week. Time-coursed variations in the bacterial community electrochemically reduced by the cathodic reaction, which grown with the electrochemical reducing power and CO2 is coupled to the anodic reaction to generate O2 from H2O. in the mineral-based medium were analyzed via temperature Electrochemically reduced neutral red induces bacterial gradient gel electrophoresis (TGGE) of the 16S rDNA cells to regenerate biochemical reducing power; this is an variable region. Some of the bacterial community constituents effective reaction that is capable of inducing the noted at the initial time disappeared completely, but some autotrophic or mixotrophic CO2-fixing bacteria to fix CO2 of them observed as DNA signs at the initial time were with the electrochemical energy derived from solar energy. clearly enriched in the electrochemical bioreactor during Experimentally, NR-graphite felt activated ethanol production 24 weeks of incubation. Finally, Alcaligenes sp. and in the fermentation metabolism of Zymomonas mobilis, Achromobacter sp., which are capable of autotrophically denitrification reaction in Ochromobactrum sp., ammonium fixing CO2, were enriched to major constituents of the oxidation in Nitrosomonas sp., and the enrichment of bacterial community in the electrochemical bioreactor. hydrogenotrophic methanogens [1, 13-15, 17]. All of these reactions were activated by the electrochemically regenerated Keywords: Electrochemical reducing power, CO2-fixing bacteria, electrochemical bioreactor, built-in anode compartment, neutral biochemical reducing power in the intact bacterial cells. red-modified cathode The balance between the CO2 generated by heterotrophs and that fixed by autotrophs is unlikely to be profoundly altered in natural ecosystems; however, the concentration of CO2 in the atmosphere has been increased continuously Bacterial species capable of chemoautotrophically fixing via fossil fuel combustion. Atmospheric CO2 is naturally CO2 regenerate the biochemical reducing power (NADPH assimilated into biomolecules via autotrophic microorganisms and plants in the natural ecosystem; however, the extra *Corresponding author Phone: +82-2-940-7190; Fax: +82-2-919-0345; CO2 generated from the combustion of fossil fuels may E-mail: [email protected] not be biologically fixed. Accordingly, the bioreactor for 591 Jeon et al. bacterial CO2 fixation using solar energy may constitute a production of valuable organic products that are foods and method to reduce the atmospheric CO2 generated from fodders [34]. Some cyanobacteria were reported to produce fossil fuel combustion. Practically, a very small percentage secondary metabolites that have therapeutic effects [2]. Deng of solar energy is used for photosynthetic reactions; most and Coleman [8] transformed the cyanobacteria Synechococcus solar energy is converted to radiant heat, which can be sp. in order to produce a novel photoautotroph capable of saved by converting it to electric energy with solar panels. producing ethanol. Other useful products that are amino The electricity obtained by the solar panels can be converted acids and PHB were reported to be produced by Spirulina to electrochemical reducing power using neutral red. The sp. [5, 35]. According to the research data, the photoautotrophs bacterial cells cultivated using the electrochemical reducing may be the best organisms to fix the atmospheric carbon power and atmospheric carbon dioxide may be not useful dioxide without combustion of fossil fuel but may occupy for industrial, nutritional, and pharmacological purpose, but the ecological habitats for plants. Generally, cyanobacteria may be effective for conversion of carbon dioxide to the are cultivated in the water pool-type reactor under light of chemically stable biopolymers in the conditions without the sun like the agricultural system, by which the plants combustion of fossil fuel and occupation of ecological (trees and grasses) may be replaced by the cyanobacteria. habitats. The capacity of trees and grasses for carbon dioxide fixation Cyanobacteria that perform a similar oxygenic photosynthesis is greatly higher than the cyanobacteria. The purpose of to the higher plants [33] were studied with the object of agriculture is not to fix atmospheric carbon dioxide but to produce crops that are oxidized to carbon dioxide by consumption as the nutrients. Meanwhile, high cost may be required to cultivate the cyanobacteria in the tank-type bioreactor owing to the employment of an awful lot of electric lamps. In this study, a cylinder-type electrochemical bioreactor with a built-in anode compartment was designed and employed to enrich the CO2-fixing bacteria under CO2-N2 atmosphere. In order to convert electric energy to biochemical reducing power, neutral red was immobilized in the graphite felt electrode by a covalent bond. The time-coursed consumption of CO2 by the bacterial community grown in the electrochemical bioreactor was analyzed at intervals of 1 week for more than 30 weeks. The time-coursed variation of the bacterial community that was enriched in the electrochemical bioreactor was analyzed on the basis of the 16S rDNA variable region via the TGGE technique during 24 weeks of cultivation. MATERIALS AND METHODS Electrochemical Bioreactor A cylinder-type electrochemical bioreactor (diameter, 120 mm; height, 200 mm; working volume, 2,000 ml; total volume, 3,000 ml; Pyrex, USA) with a built-in anode compartment was designed in the scale- up procedure for a pilot reactor or industrial reactor, as shown in Fig. 1A. A sintered glass filter (diameter, 50 mm; thickness, 5 mm; pore, 1~1.6 µm; Duran, Germany) was fixed at the bottom end of the anode compartment (diameter, 36 mm; height, 200 mm; working volume, 200 ml). The anolyte consumed by H2O electrolysis was automatically refilled from the catholyte through the glass filter, as Fig. 1. Schematic structure of the electrochemical bioreactor shown in Fig. 1B. O2 generated by electrolysis in the anode compartment with a built-in anode compartment for the enrichment of CO2- is removed through the chimney. fixing bacteria. The glass filter septum was equipped at the bottom end of the anode Operation of Electrochemical Bioreactor compartment (A). Protons, electrons, and oxygen generated from water by the electrolysis may be transferred separately to the catholyte, the NR- The mixed culture of bacteria obtained from different sources was cathode, and the atmosphere (B). incubated in the electrochemical bioreactor under 50% (v/v) of CO2 ENRICHMENT OF CO2-FIXING BACTERIA IN ELECTROCHEMICAL BIOREACTOR 592 to N2 without fresh medium feeding for 2 weeks. Approximate 3 V mixture was filtered with filter paper (Whatman No.1) and the filtrate of electric power was charged directly to electrodes from a DC was used as the bacterial source for the enrichment of CO2-fixing power supply to induce 1.5 mA of current. The electric voltage and bacteria. current were measured precisely with a multimeter (Keithely 2700, USA). Approximate 50% (v/v) of CO2 was sparged circularly into the 16S rDNA Amplification medium at a rate of 200 ml/min to activate the dissolution of CO2 The bacterial cells were collected from the electrochemical bioreactor into carbonic acid in the medium, by which carbon dioxide consumed and NR-graphite felt using a long-needled syringe immediately after in the electrochemical bioreactor may be replenished spontaneously. inoculation,