Enhancement of Power Generation in Microbial Fuel Cells through Supplementation of Platycodon grandiflorum in Doraji Roots

The current study reports the evidence of enhancement in power generation from cellulosic biomass in microbial fuel cell (MFC) systems by supplement-ing dried Doraji (Platycodon grandiflorum) roots powder. Mediator-less two chamber H-type MFCs were prepared using rumen fluid as anode inocula to convert finely ground pine tree (Avicel) at 2% (w/v) to electricity. Dried Doraji roots were ground to pass 1 mm sieve and added to the anode of MFC at 0.1% w/v dosage for treatment. MFC power and current across an external resistor were measured daily for 10 d. At the end of incubation on d10, collected gases were measured for total gas volume and analyzed for gas composition on gas chromatography. Supplementation of Doraji roots powder to MFC anode chamber increased power generation and CO 2 production. Over the 10d experimental period, power density normalized to anode surface area were between 17.0 and 37.7 with average of 32.5 mW/m 2 in Doraji MFCs, and between 16.8 and 19.8 with average of 18.2 mW/m 2 in control group. CO 2 production increased and methane to CO 2 ratio decreased in Doraji root treatment comparing to control group. These observations imply that Doraji root components would inhibit methanogenesis and alter microbial fermentation of cellulose compounds favorable to produce bioenergy efficiently in MFC.


Introduction
Fossil fuels have served as the main energy resources for industrialization and economic growth for the past century [1], and represented 80% of the global primary energy use [2], but the use of these also contributed 94% to 96% of greenhouse emission in the USA [3] including CO 2 , methane, CO and nitrous oxide (N 2 O), which cause global warming and pollution [4]. Thus, greater efforts are currently being undertaken to develop technologies generating clean and sustainable energy sources that would replace and/or displace fossil fuels [5].
Cellulosic biomass is particularly attractive renewable resources because of its relatively low cost, plentiful supply [15] [16], and neutral carbon balance [17] and furthermore cellulose is a significant component in the annual production of 250 million tons municipal solid wastes and 40 billion cubic meters waste water [18]. To utilize cellulosic biomass in MFC, the anodic process requires cellulose degradation, but often the microorganisms that are electrochemically active did not show cellulolytic activity, thus, it requires products of cellulose fermentation as electron donors to generate electricity [19] [20]. Therefore, rumen fluid from cow [14] [21] or goat [22] had been studied for electricity generation from cellulose or cellulosic biomass because rumen microorganisms include both strict and facultative anaerobes, which effectively hydrolyze cellulose, and conserve energy via anaerobic respiration or fermentation [23]. However, one of major products from cellulose fermentation is acetic acid, and the acetate concentration and anaerobic condition influence the growth of methanogens which contribute significantly to limiting power generation in MFC. Methanogenesis diverts electron from the anode and methanogens act as substrate competitors to the exoelectrogens; acetoclastic methanogens compete for electron donors and hydrogenotrophic methanogens utilize the hydrogen produced in the reactor [24].
We hypothesized that supplementation of Doraji root in anolyte would inhibit methanogenesis and (or) proliferate cellulolytic microorganisms in anode chamber and subsequently ferment cellulosic biomass more efficiently and (or) enhance power generation in MFC. In the current study, MFCs were constructed Journal of Sustainable Bioenergy Systems with stained rumen fluid as anolyte and cellulose as electron donor, and effects of Doraji root supplementation on power generation and fermentation gas production were investigated.

Microorganisms and Culture Media
For MFC anode compartment inoculum (anolyte), rumen fluid was collected from a dry dairy cow and while flushing of CO 2 gas, filtered through 4 layers of cheesecloth to remove feed debris and transferred to an Erlenmeyer flask, then bubbled with CO 2 gas for 10 min and closed with cotton ball and stored in an incubator at 39˚C until inoculated to MFCs.

Microbial Fuel Cells
H-type MFCs consisted two 125 mL-volume glass bottles joined at branched tube.
Cation exchange membrane (CMI-7000S, Membranes International Inc., NJ) was placed and clamped between branched tube of anode and cathode compartments.
Two gram of cellulose (Avicel PH-101, Sigma-Aldrich, MO) was weighed into anode chamber and 80 mL of culture medium, 20 mL strained rumen fluid were transferred, then suspended using a magnetic bar on agitator. Graphite plates (12 cm 2 ) were used as electrodes for both anode and cathode. Electrode connected with copper wire fixed to butyl rubber stopper was placed in anode chamber. 100 mL of PBS was transferred to cathode chamber and electrode connected with copper wire was placed in the middle of cathode. Rubber stopper was capped on cathode but left open to air through tubing. Anode and cathode were connected externally through a copper wire with a resistor (300 ohm). MFCs were placed in a water bath at 39˚C for operation.
After 9 d of MFC pretrial operation, 100 mg of dried Doraji root (C&M Food, Seoul, Korea) ground to pass through 1 mm screen was added into anode chamber of treatment group. Two L-volume Mylar balloons were connected to each anode to collect biogas produced during experiment.

Measurements and Calculation
Using a digital multimeter, voltage across an external resistor (closed circuit voltage), open circuit voltage, and current were recorded daily for 10d. The power density normalized to electrode surface area was calculated using following equations.
where, I(A) is the current, V(V) is voltage, R(ohm) is the external resistance, and A(m 2 ) is the projected area of the anode.
The volume of biogases produced in anode collected in Mylar balloons were measured using a 250 mL glass syringe, and gas components were analyzed using an Agilent 6890 series gas chromatograph equipped with a thermal conductivity detector and a stainless steel packed column prepared with 60/80 Carboxen 1000 (12390-U Supelco, Sigma-Aldrich, MO).

Statistical Analyses
Effects of Doraji root addition to anode chamber of MFC on electricity generation, fermentation gas production and gas composition were analyzed using the one way ANOVA procedure of JPM 14.1.0 (SAS Institute Inc., NC). When the effect was significant (P < 0.05), means between treatments were separated using Student's t-test (P < 0.05). Means for operation time (d) were separated using Tukey HDS where the operation time effects exist (P < 0.05).

Power Generation
MFCs were stabilized and operational prior to treatment addition. Voltage across resistor, current density and power density were 96.4 ± 4.87 mV, 186.8 ± 9.43 mA/m 2 and 18.0 ± 0.64 mW/m 2 , respectively.
In control group, voltage across resistor (Table 1) were steady (P = 0.9766) with operation time and average was 96.8 ± 5.22 mV ranged from 93 to 101 mV, and end point potential (Table 2) also did not change (P = 0.9865) with operation time and average was 392 ± 33.6 mV ranged from 374 to 423 mV during 10d operation.
Voltage across resistor in MFCs received Doraji root powder increased (P < 0.05) at d 2 and maintained through d10 (Table 1), but greater (P < 0.05) end point potentials than d 0 were observed only at d 2, 5, 7 and 8 ( Table 2). In comparison to control group, MFCs received Doraji root powder had greater (P < 0.05) voltage across resistor (Table 1)    probabilities that treatments effect is not significant within the day. 3 P-value: probabilities that day effect is not significant within the treatment. Journal of Sustainable Bioenergy Systems probabilities that treatments effect is not significant within the day. 3 P-value: probabilities that day effect is not significant within the treatment.
of Doraji root powder supplementation that most likely causes favorable anode respiration by exoelectrogens by methanogenesis inhibitions [25] [26] [27] and (or) by cellulolysis extension with proliferation of fiber degrading bacteria [28] in MFCs constructed with rumen fluid as anolyte and cellulose as electron donor.

Biogases Production
Total gas productions in anode for 10d MFC operation were similar (P = 0.7072) and the volumes were 328 and 335 mL in control and Doraji root treatment, respectively ( Figure 1). Biomethane production were also similar (P = 0.0248) and amounts were 220 and 197 mL in control and Doraji root treatment, respectively. However CO 2 production was greater (P = 0.0305) in Doraji root treatment (138 mL) than in control group (108 mL). The increase in CO 2 and numerical difference in biomethane were clearly reflected in biomethane to CO 2 ratio, and the ratio was less (P = 0.0461) in Doraji root treatment (1.44) than in control group (2.03).
Biogas is produced from biomass fermentation, and it was from cellulose in the current study. Because of the identical carbon balance between methane and CO 2 and no difference in total gas production between treatment, the extend of  to exoelectrogens which transfer electrons to anode [24]. Aacetoclastic methanogens compete for electron donors (

Conclusion
Microbial Fuel Cell is one of the technologies that generate clean sustainable bioenergy from cellulosic biomass; however, methanogenesis remains as a major factor limiting MFC performance. In the current study, rumen microorganisms were employed as anolyte and cellulose served as electron donors, and Doraji (Platycodon grandiflorum) roots powder was tested at 0.1% of anolyte as methanogenesis inhibitor and cellulolytic microorganisms growth promoter. Cellulolysis did not change, however enhanced power generation and decreased methane to CO 2 ratio were observed with Doraji roots powder addition to anode in MFC which were hypothesized on the basis of saponine effects on rumen microbial fermentation. Results from the current study imply that Doraji root addition would inhibit methanogenesis and enhance MFC efficiency in cellulosic electricity generation. Optimal dose and preparation methods of Doraji roots may be elucidated by further studies to maximize the methanogenesis inhibition and power generation in MFCs from cellulosic biomass.