**5. Enrichment and cultivation of carbon dioxide-fixing bacteria**

A cylinder-type electrochemical bioreactor composed of the built-in anode compartment and NR-graphite cathode was employed to enrich the facultative anaerobic mixotrophs capable of fixing carbon dioxide with electrochemical reducing power as shown in Fig 9. The NR-cathode was separated electrochemically from anode compartment by the glass filter (Fig 5). Mixture of the bacterial community obtained from aerobic wastewater treatment reactor, forest soil, and anaerobic wastewater was cultivated in the cylinder-type electrochemical bioreactor to enrich selectively carbon dioxide-fixing bacteria with the electrochemical reducing power generated from NR-graphite cathode. DC -3 volt of electricity that was generated by a solar panel was charged to NR-graphite cathode to induce generation of electrochemical reducing power. Electricity is the easiest energy to

wastewater containing other reduced organic and inorganic compounds and exhaust containing carbon dioxide, and the electrochemical reducing power as the electron donor

mL/L of reactant/day

0

mL/L of reactant/day

Fig. 8. Carbon dioxide consumption () and methane production () by anaerobic digestive sludge cultivated in conventional bioreactor (reactors A and B) and

**5. Enrichment and cultivation of carbon dioxide-fixing bacteria** 

electrochemical bioreactor (reactors C and D). Duplicate reactors were operated to enhance the comparability between the conventional bioreactor and the electrochemical bioreactor.

A cylinder-type electrochemical bioreactor composed of the built-in anode compartment and NR-graphite cathode was employed to enrich the facultative anaerobic mixotrophs capable of fixing carbon dioxide with electrochemical reducing power as shown in Fig 9. The NR-cathode was separated electrochemically from anode compartment by the glass filter (Fig 5). Mixture of the bacterial community obtained from aerobic wastewater treatment reactor, forest soil, and anaerobic wastewater was cultivated in the cylinder-type electrochemical bioreactor to enrich selectively carbon dioxide-fixing bacteria with the electrochemical reducing power generated from NR-graphite cathode. DC -3 volt of electricity that was generated by a solar panel was charged to NR-graphite cathode to induce generation of electrochemical reducing power. Electricity is the easiest energy to

0

20

40

60

80

D

100

20

40

60

80

B

100

Incubation time (day) 0 50 100 150 200

Incubation time (day) 0 50 100 150 200

(Skirnisdottir et al., 2001).

mL/L of reactant/day

mL/L of reactant/day

0

20

40

60

80

C

100

0

20

40

60

80

A

100

Incubation time (day) 0 50 100 150 200

Incubation time (day) 0 50 100 150 200 transfer and supply to any electronic device. The electrochemical bioreactor is also the simplest electronic device to convert electric energy to biochemical reducing power. The wastewater and exhausted gas can be used directly without purification or separation as the nutrient source for bacterial metabolism. Experimentally, the electricity generated from the 25 cm2 of the solar panel is very enough for operation of the 10 L of electrochemical bioreactor.

Fig. 9. Schematic diagram of the cylinder-type electrochemical bioreactor equipped with a built-in anode compartment for the cultivation of CO2-fixing bacteria. The glass filter septum equipped at the bottom end of the anode compartment functions as redox separator between anode and cathode compartment and micropore for transfer of catholyte to anode compartment.

During enrichment of the carbon dioxide-fixing bacteria using the cylinder-type electrochemical bioreactor, bacterial community was changed significantly as show in Fig 10. Some of bacteria community was increased or enriched as shown in the box A and C but decreased or died out as shown in the box B and D. These phenomena are a clue that the

Bioelectrochemical Fixation of Carbon Dioxide with Electric Energy Generated by Solar Cell 199

Lane Band Genus or Species Homology (%) Accession No.

1 Uncultured *Burkholderia* sp. 98 FJ393136 2 Groundwater biofilm bacterium 98 FJ204452 3 *Hydrogenophaga* sp. 98 FM998722 4 Uncultured bacterium sp. 97 HM481230 5 *Aquamicrobium* sp. 98 GQ254286 6 Uncultured *Actinobacterium* sp. 99 FM253013

1 Uncultured bacterium sp. 97 AF234127 2 Uncultured bacterium sp. 97 EU532796 3 Uncultured *Clostridum* sp. 99 FJ930072 4 Uncultured *Polaromonas* sp. 99 HM486175 5 Uncultured *Rhizobium* sp. 100 FM877981 6 *Raoultella planticola* 98 EF551363 7 Unidentified bacterium 98 AV669107 8 Uncultured bacterium 99 HM920740 9 Uncultured bacterium 97 GQ158957 10 Uncultured *Klebsiella* sp. 98 GQ416299

1 Uncultured bacterium sp. 97 AF234127 2 Uncultured bacterium sp. 97 EU532796 3 *Enterococcus* sp. 98 DQ305313 4 Uncultured bacterium 98 HM820223 5 *Aerosphaera taera* 99 EF111256 6 *Alcaligenes* sp. 98 GQ383898 7 Uncultured bacterium 98 HM231340 8 Uncultured bacterium sp. 97 FJ675330 9 *Stenotrophomonas* sp. 98 EU635492 10 Uncultured *Klebsiella* sp. 98 GQ416299

1 Uncultured bacterium sp. 97 AF234127 2 Uncultured bacterium sp. 97 EU532796 3 Uncultured bacterium sp. 98 HM575088 4 *Alcaligenes* sp. 98 GQ200556 5 *Alcaligenes* sp. 98 GQ383898 6 Uncultured bacterium 98 HM231340 7 *Achromobacter* sp. 96 GQ214399 8 Uncultured *Lactobacillales* bacterium sp. 96 HM231341 9 Uncultured *Ochrombacterum* sp. 97 EU882419 10 *Stenotrophomonas* sp. 98 EU635492 11 *Tissierella* sp. 96 GQ461822

1 Uncultured bacterium sp. 97 AF234127 2 Uncultured bacterium sp. 97 EU532796 3 Uncultured bacterium sp. 98 HM820116 4 *Alcaligenes* sp. 98 GQ200556 5 *Alcaligenes* sp. 97 GQ383898 6 *Enterococcus sp.* 99 FJ513901 7 Uncultured bacterium 98 HM231340 8 *Achromobacter* sp. 96 GQ214399

Table 1. The homologous bacterial species with the sequences of DNA extracted from TGGE

bands (Fig 10), which were identified based on the GenBank database.

1 (initial)

2 (2nd week)

3 (8th week)

4 (16th week)

5 (24th week)

bacterial species that can fix carbon dioxide with electrochemical reducing power are adapted selectively to the reactor condition but other bacteria that can't generate biochemical reducing power from the electrochemical reducing power are not. The DNA bands were extracted from TGGE gel and sequenced. Identity of the bacteria was determined based on the 16S-rDNA sequence homology.

Fig. 10. TGGE patterns of 16S-rDNA variable regions amplified with chromosomal DNA extracted from bacterial communities enriched in the cylinder-type electrochemical bioreactors. 50 ml of bacterial culture was isolated from the electrochemical bioreactor at the initial time immediately after inoculation (lane1), 2nd week (lane 2), 8th week (lane 3), 16th week (lane 4), and 24th week of incubation time (lane 5).

bacterial species that can fix carbon dioxide with electrochemical reducing power are adapted selectively to the reactor condition but other bacteria that can't generate biochemical reducing power from the electrochemical reducing power are not. The DNA bands were extracted from TGGE gel and sequenced. Identity of the bacteria was

Fig. 10. TGGE patterns of 16S-rDNA variable regions amplified with chromosomal DNA extracted from bacterial communities enriched in the cylinder-type electrochemical

week (lane 4), and 24th week of incubation time (lane 5).

bioreactors. 50 ml of bacterial culture was isolated from the electrochemical bioreactor at the initial time immediately after inoculation (lane1), 2nd week (lane 2), 8th week (lane 3), 16th

determined based on the 16S-rDNA sequence homology.


Table 1. The homologous bacterial species with the sequences of DNA extracted from TGGE bands (Fig 10), which were identified based on the GenBank database.

Bioelectrochemical Fixation of Carbon Dioxide with Electric Energy Generated by Solar Cell 201

of sequences identified with *Alcaligenes* sp. and *Achromobcter* sp. was 0.98 and 0.12%, respectively. Meanwhile, the most abundant sequences (43.83%) obtained from the bacterial culture after enrichment was identified as *Achromobacter* sp., and the most classifiable sequences were also identified as *Achromobacter* sp. and *Alcaligenes* sp. as shown in Table 2.

**Before enrichment After enrichment**

**Classifiable** 

876 17.96 *Brevundimonas* 100 2248 43.83 *Achromobacter* 100 153 3.14 *Pseudomonas* 100 748 14.58 *Achromobacter* 100 111 2.28 *Hydrogenophaga* 100 595 5.87 *Stenotrophomonas* 100 99 2.03 *Delftia* 100 301 2.28 *Achromobacter* 100 86 1.76 *Stenotrophomonas* 100 263 1.77 *Achromobacter* 100 70 1.44 *Pseudomonas* 100 219 1.23 *Achromobacter* 100 53 1.09 *Parvibaculum* 100 117 0.90 *Achromobacter* 100 52 1.07 *Brevundimonas* 100 91 0.66 *Achromobacter* 100 48 0.98 *Alcaligenes* 100 63 0.57 *Alcaligenes* 100 32 0.66 *Comamonas* 100 46 0.53 *Achromobacter* 100 31 0.64 *Bacillus* 100 34 0.49 *Achromobacter* 100 26 0.53 *Bosea* 100 29 0.49 *Castellaniella* 100 21 0.43 *Devosia* 100 27 0.45 *Achromobacter* 100 17 0.35 *Acidovorax* 100 25 0.45 *Achromobacter* 100 12 0.25 *Brevundimonas* 100 25 0.39 *Stenotrophomonas* 100 12 0.25 *Sphaerobacter* 100 23 0.16 *Achromobacter* 100 11 0.23 *Brevundimonas* 100 23 0.14 *Alcaligenes* 100 9 0.18 *Acinetobacter* 100 20 0.12 *Achromobacter* 100 9 0.18 *Sphaerobacter* 100 14 0.10 *Alcaligenes* 100 8 0.16 *Brevundimonas* 100 14 0.10 *Pseudomonas* 100 7 0.14 *Hyphomicrobium* 100 11 0.08 *Achromobacter* 100 7 0.14 *Thermomonas* 100 10 0.08 *Achromobacter* 100 6 0.12 *Achromobacter* 100 8 0.06 *Achromobacter* 100 6 0.12 *Brevundimonas* 100 7 0.06 *Achromobacter* 100 4 0.10 *Devosia* 100 7 0.06 *Achromobacter* 100 3 0.08 *Pseudoxanthomonas* 100 6 0.04 *Alcaligenes* 100 3 0.06 *Castellaniella* 100 6 0.04 *Achromobacter* 100 3 0.06 *Gordonia* 100 6 0.04 *Achromobacter* 100

Table 2. Relative abundances of dominant bacterial taxa in the bacterial culture before and after enrichment. The relative abundances were estimated from the proportion of classifiable sequences, excluding those sequences that could not be classified below the genus level and

100% homology with the specific bacterial genus.

**sequences** 

**Abundance** 

**(%)** 

**Bacterial** 

**genus** 

**Homology** 

**(%)** 

**Homology** 

**(%)** 

**Classifiable** 

**sequences** 

**Abundance** 

**(%)** 

**Bacterial** 

**genus** 

Some anaerobic bacteria (*Hydrogenophaga* sp. and *Clostridium* sp.) that may be originated from the anaerobic wastewater treatment reactor are detected at the initial cultivation time but disappeared after 8th week of incubation time (Kang and Kim, 1999; Willems et al., 1989; Lamed et al., 1988). On the other hand, the bacteria that are capable of fixing carbon dioxide by autotrophic or mixotrophic metabolism were enriched as shown in Table 1. All of the enriched bacteria may not be the carbon dioxide-fixing bacteria but *Achromobacter* sp. and *Alcaligenes* sp. are known to fix carbon dioxide autotrophically or mixotrophically (Freter and Bowien, 1994: Friedrich, 1982; Hamilton et al., 1965; Leadbeater and Bowien, 1984; Ohmura et al.). During the enrichment of the carbon dioxide-fixing bacteria, carbon dioxide consumption was increased and reached to stationary phase after 15th week of incubation time as shown in Fig 11. Various organic compounds contained in the bacterial cultures that were originated from anaerobic wastewater treatment reactor, aerobic wastewater treatment reactor, and forest soil might be consumed completely and then carbon dioxide-fixing bacteria might grow selectively. The carbon dioxide consumption was increased initially and then reached to stationary phase after 15th week of incubation time, which is proportional to the enrichment time of the *Achromobacter* sp. and *Alcaligenes* sp.

Fig. 11. Weekly consumption of CO2 in the electrochemical bioreactor from the initial incubation time to 30 weeks. CO2 consumption was analyzed weekly and the gas reservoir was refilled with 50±1% of CO2 to N2 at 4-week intervals.

Before and after enrichment, the bacterial community grown in the cylinder-type electrochemical bioreactor was analyzed using the pyrosequencing technique (Van der Bogert et al., 2011). The classifiable sequences obtained by the pyrosequencing were identified based on the Ribosomal Database Project (RDP), and defined at the 100 % sequence homologous level. The most abundant sequences (17.96%) obtained from the bacterial culture before enrichment was identified as *Brevundimonas* sp., and the abundance

Some anaerobic bacteria (*Hydrogenophaga* sp. and *Clostridium* sp.) that may be originated from the anaerobic wastewater treatment reactor are detected at the initial cultivation time but disappeared after 8th week of incubation time (Kang and Kim, 1999; Willems et al., 1989; Lamed et al., 1988). On the other hand, the bacteria that are capable of fixing carbon dioxide by autotrophic or mixotrophic metabolism were enriched as shown in Table 1. All of the enriched bacteria may not be the carbon dioxide-fixing bacteria but *Achromobacter* sp. and *Alcaligenes* sp. are known to fix carbon dioxide autotrophically or mixotrophically (Freter and Bowien, 1994: Friedrich, 1982; Hamilton et al., 1965; Leadbeater and Bowien, 1984; Ohmura et al.). During the enrichment of the carbon dioxide-fixing bacteria, carbon dioxide consumption was increased and reached to stationary phase after 15th week of incubation time as shown in Fig 11. Various organic compounds contained in the bacterial cultures that were originated from anaerobic wastewater treatment reactor, aerobic wastewater treatment reactor, and forest soil might be consumed completely and then carbon dioxide-fixing bacteria might grow selectively. The carbon dioxide consumption was increased initially and then reached to stationary phase after 15th week of incubation time, which is

proportional to the enrichment time of the *Achromobacter* sp. and *Alcaligenes* sp.

CO2 cosumption (ml)

**CO2 consumption (ml)** 

200

400

600

800

1000

1200

1400

1600

1800

Incubation time (week) 0 5 10 15 20 25 30

Fig. 11. Weekly consumption of CO2 in the electrochemical bioreactor from the initial incubation time to 30 weeks. CO2 consumption was analyzed weekly and the gas reservoir

Before and after enrichment, the bacterial community grown in the cylinder-type electrochemical bioreactor was analyzed using the pyrosequencing technique (Van der Bogert et al., 2011). The classifiable sequences obtained by the pyrosequencing were identified based on the Ribosomal Database Project (RDP), and defined at the 100 % sequence homologous level. The most abundant sequences (17.96%) obtained from the bacterial culture before enrichment was identified as *Brevundimonas* sp., and the abundance

was refilled with 50±1% of CO2 to N2 at 4-week intervals.

of sequences identified with *Alcaligenes* sp. and *Achromobcter* sp. was 0.98 and 0.12%, respectively. Meanwhile, the most abundant sequences (43.83%) obtained from the bacterial culture after enrichment was identified as *Achromobacter* sp., and the most classifiable sequences were also identified as *Achromobacter* sp. and *Alcaligenes* sp. as shown in Table 2.


Table 2. Relative abundances of dominant bacterial taxa in the bacterial culture before and after enrichment. The relative abundances were estimated from the proportion of classifiable sequences, excluding those sequences that could not be classified below the genus level and 100% homology with the specific bacterial genus.

Bioelectrochemical Fixation of Carbon Dioxide with Electric Energy Generated by Solar Cell 203

different metabolisms. The photoautotrophs, especially cyanobacteria that fix carbon dioxide by completely same metabolism (Calvin cycle) with plants, appear as if they are ideal organism to fix biologically carbon dioxide without chemical energy; however, they are unfavorable to be cultivated in the tank-type bioreactor owing to the limitation of reachable distance of solar radiation in aquatic condition. The chemoautotrophs may be useful to produce methane and acetic acid from carbon dioxide; however, they can grow only in the limit condition of the lower redox potential than -300 mV (vs. NHE) and with hydrogen. The mixotrophs can grow in the condition with electron donors, which are regardless of organic or inorganic compounds, for regeneration of reducing power under aerobic and anaerobic condition. This is the reason why the facultative anaerobic mixotrophs may be more effective than others to fix the atmospheric carbon dioxide directly by simple process. Especially, the cylinder-type electrochemical bioreactor equipped with the built-in anode compartment (Fig 9) is an optimal system for the cultivation or enrichment of facultative anaerobic mixotrophs. Basements of buildings or villages are used generally for maintenances or facilities for wastewater collection, electricity distribution, tap water distribution, and garage. The basements can't be the habitats for cultivation of plants with the natural sun light but can be utilized for cultivation of the carbon dioxide-fixing bacteria with electric energy generated from the

solar cells that can be installed on the rooftop as shown in Fig 12.

Fig. 12. Schematic structure of the electrochemical bioreactors installed in the building basement. The carbon dioxide-fixing bacteria can be cultivated using the electric energy

generated by the solar cells.

The *Achromobacter* sp. described in previous research was a facultative chemoautotroph (Hamilton *et al*., 1965; Romanov *et al*., 1977); however, it grew autotrophically with electrochemical reducing power under a CO2 atmosphere and consumed CO2 in this study. This result demonstrates that *Achromobacter* sp. grown in the electrochemical bioreactor may be a chemoautotroph capable of fixing CO2 with the electrochemical reducing power. Meanwhile, various articles have reported that *Alcaligenes* sp. grew autotrophically (Frete and Bowien, 1994; Doyle and Arp. 1987; Leadbeater and Bowien, 1984) or heterotrophically (Reutz *et al*., 1982). According to these articles, *Alcaligenes* spp. are capable of growing autotrophically with a gas mixture of H2, CO2, and O2, as well as heterotrophically under air on a broad variety of organic substrates. *Alcaligenes* spp. metabolically oxidize H2 to regenerate the reducing power during autotrophic growth under H2-CO2 atmosphere (Hogrefe *et al*., 1984). The essential requirement for the autotrophic growth of both *Achromobacter* spp. and *Alcaligenes* spp. under CO2 atmosphere is to regenerate reducing power in conjunction with metabolic H2 oxidation, which may be replaced by the electrochemical reducing power on the basis of the results obtained in this research. The electrochemical reducing power required for the cultivation of carbon-dioxide fixing bacteria can be produced completely by the solar panel, by which atmospheric carbon dioxide may be fixed by same system to the photosynthesis.
