Continuously converting the fossilized carbon into atmospheric CO2 through expeditious extraction and consumption has left the carbon loop unbalancd leading to adverse climate changes and a dearth of raw material (fossils) (Hoornweg et al., 2013; Dowson and Styring, 2017). With substantial increase in the world population, need for sustainable feedstocks has also increased. In the lookout for alternative feedstocks, CO2 has gained ground as a virtuous feedstock for the production of chemicals, fuels, and energy (Venkata Mohan et al., 2016a). Utilizing CO2 as feedstock through biotechnological routes has the potential to sustainably solve the problem of global climate change primarily occuring due to increase in atmospheric CO2 levels (Butti and Mohan, 2017; Kant, 2017). Increase in the atmospheric CO2 concentration has a harmful influence on the environment like acidification of oceans which develops a higher homeostasis stress on the phytoplaktons to maintain their redox conditions, acidification of soil, and acid rains (Rodrigues et al., 2016). Photosynthesis provides a possible solution with its natural ability to biocapture CO2 using solar energy and produce valuable chemicals along with several other advantages to mitigate environmental pollution (Benemann et al., 1977; Demars et al., 2016; Keenan et al., 2016).

Amongst the photosynthetic organisms microalgae have achieved higher significance as the primary responders to the climate change with their efficient carbon assimilation mechanism, high growth rate, bioactive, and biofuels production and the ability to grow on non-arable lands. Microalgae can grow under different trophic modes of nutrition and hence can be cultivated using both inorganic and organic carbon in light and dark conditions under photoautotrophic, mixotrophic, and heterotrophic modes (Venkata Mohan et al., 2014; Rohit and Mohan, 2015). Understanding these resourceful abilities several researchers have progressed toward microalgal photo-biotechnology encompassing biosequestration of CO2, wastewater treatment and biobased products synthesis. Different microalgal strains have robust capabilities to overcome climatic changes by CO2 sequestration and production of diverse products owing to their competent metabolism. However, through an integrated approach or genetic modifications the spectrum of products synthesized can be expanded like biocrude through hydrothermal liquefaction, bioalcohols through anaerobic digestion, bioelectricity through microbial electrochemical technologies, biohydrogen/biomethane through acidogenic fermentation, biofertilizers through pyrolysis (Perez-Garcia et al., 2011; Ooms et al., 2016; Venkata Mohan et al., 2016b). Under autotrophic mode of nutrition, substrate (CO2) availability governs the photosynthetic efficiency and biomass productivity at optimum operational parameters. The incidence of a positive influence on the biomass growth and lipogenesis under higher CO2 for different microalgal strains like Chlorella, Scenedesmus, Nannochloropsis, Dunaliella, Botryococcus, and plants has been previously discussed in different studies (Riebesell et al., 1993; Hein and Sand-Jensen, 1997; Chiu et al., 2009; Yoo et al., 2010; Price and Howitt, 2014; Peng et al., 2016a; Watson-Lazowski et al., 2016). Improving the energy conversion efficiency in photosynthesis is a multivariable problem, where, it is greatly influenced by an array of operational and physiological parameters like phases of operation, nutrients/substrate availability, photosynthetically active radiation (PAR) exposure, temperature, pH, the source of essential compounds and gases (industrial exhausts; Stewart et al., 2015; Chiranjeevi and Mohan, 2016).

We hypothesize that microalgae can be optimized to selectively enhance the carbon dioxide uptake and the photobiocommodities productivity at an economic scale under photoautotrophic cultivation by regulating the inlet gas concentrations (10% CO2 + 10% H2, 20% CO2, 20% H2, 0.04% CO2) at an elevated pressure. The varying headspace gas concentrations would ensure controlled compartmentalization of essential metabolites and determine the possibility of selective photo-products synthesis. Also, in this study a monophasic cultivation strategy with a synergistic role of CO2 and H2 was incorporated taking lead from Gaffron who suggested the possibility of CO2 reduction by molecular hydrogen in microalgae (Gaffron, 1940). While most of the preceding studies in this domain have largely focused on varying the inlet concentrations of CO2 and operating in two phases with growth and stress phase separately. The present study has the potential to provide a holistic solution to the climate change and feedstock limitation through microalgal CO2 biosequestration and pave a sustainable path for the future generation.

### MATERIALS AND METHODS

### Microalgal Isolation and Cultivation

The freshwater microalgae Chlorella sp. was isolated from a local ecological water body (Hussain Sagar, Hyderabad, 17.4239◦N, 78.4738◦E—India). Water samples were collected from five different locations around the banks of the lake, each sample was filtered through a 0.2µm hydrophilic nylon membrane filter (Spin-pure, India), and the leftover residue was washed to remove non-biological components and it was suspended into BG11 culture media. Equal aliquots of the grown culture were sub-cultured in tapered opening glass screw-cap bottles (2 l, Borosil) tightly sealed to prevent contamination. The cultures were domesticated by providing a periodic input of sterile CO2 enriched air (0.4 l.min−1 ) at atmospheric pressure for 4 weeks enabling the adaptive evolution at the laboratory scale. Effective illumination was continuously provided by nonheating fluorescent white lights (95 µmol.m−2 .s−1 ) measured by photon density lux meter (Extech LT-300 Light meter) with a photoperiod of 12 h. The reactors were operated in an open top reciprocal shaker (120 rpm) maintained at 25 ± 2 ◦C and initial pH 8, this culture was labeled as pre-culture. A total of 10 ml pre-culture from suspension in log phase was added into autoclaved screw cap glass bioreactors (500 ml, Borosil) prefilled with modified BG11 growth medium which has the concentrations of nitrates (1.1 g.L−1 ), phosphates (0.03 g.L−1 ), and sulfates (0.060 g.L−1 ) with the other components same as the BG11 media. Pre-filled bioreactors were operated at the same operational conditions with a stepping increase in CO2 enriched air input (0.03–20% V/V) monitored by gas flow meter to acclimatize the pre-culture for high CO2 tolerance and uptake conditions for 4 weeks (only for CO2 and CO2 + H2 conditions). Homogeneity of species was confirmed microscopically (Nikon Eclipse-80i) and the images were captured on digital camera (YIM-smt, 5 MP) using NIS-elements (D3.0) software, primary observations suggest the occurrence of Chlorella sp.

### Experimental Setup and Operational Parameters

The specifically designed photobioreactor (height 25 cm, base diameter 11 cm, opening diameter 5 cm, glass thickness 3 mm, equipped with separate sampling and gas exchange ports i.d/o.d- −0.9/1.1 and 0.4/0.64 mm, respectively) with the ability to sustain pressures of upto 2 bar and having maximum light penetration. The working volume of the reactor was 400 ml with an effective headspace of 100 ml. Four identical reactor setups were evaluated with specific sterile gases; CO2 (20% v/v), H2 (20% v/v), CO2 + H2 (20% v/v + 20% v/v), and Air (as control with 0.03% CO2 v/v) input with initial pH 8 (**Table 1**). The gas inputs were provided from GC grade gas cylinders through pneumatic pressure and flow control values to maintain constant pressure and flowrate in the reactors. The different inlet CO2, H2, and N2 gases concentrations in the reactors were monitored using gas chromatography with a thermal conductivity detector (Nucon-5765, Centurion Scientific, India) operated under specific conditions: packed (1/8′′ × 2 m Heysep Q) column, injection volume 0.5 ml, argon as a carrier gas (47 ml.min−1 ), the injector and detector temperatures were maintained at 60◦C and the oven was operated at 40◦C isothermally. Culture and gas sampling were done through specifically provided ports using sterile needles on alternate days to determine the exponential growth, cellular components, carbon dioxide fixation, and occurrence of contamination was preventively monitored.

### Dry Weight and Growth Kinetics

Microalgal samples were collected at every 48 h interval to evaluate growth by measuring optical density at 750 nm using a UV-Vis Spectrophotometer (Thermo Electron). Dry cell weight (g.L−1 ) was determined after the biomass was centrifuged twice at 10,000 rpm at 4◦C for 5 min (XT/XF centrifuge, Thermo Scientific) the resulting pellet was washed twice with Milli-Q water and dried in hot air oven at 65◦C for 24 h. The resulting biomass dry cell weight was used to calculate growth rate µ (g.L−1 .d−1 ) as mentioned in Equation (1) (Cabanelas et al., 2016).

$$\mu = \frac{\ln \text{(DW}\_{\text{tf}} - \text{DW}\_{\text{to}}\text{)}}{\text{tf} - \text{to}} \tag{1}$$

Where, DWtf and DWto represent dry cell weight of biomass (g.L−1 ), at time tf (the final sample time) and to (the initial sample time) in days respectively. Mean of the biomass concentrations was considered for measurement at specific time intervals.

### Photosynthetic Efficiency

The microalgal photosynthetic efficiency was evaluated using pulse-amplitude modulated fluorometry employing AquaPen-C (AP-C 100, Photon System Instruments, Czech Republic) which was monitored and controlled through PC using FluorPen software (PSI, Czech Republic). Chlorophyll fluorescence of PSII Quantum yield (Fv/Fm) was evaluated for samples directly

withdrawn from the reactors after the biomass density was adjusted to 0.025 g.L−1 using MilliQ water and placed in a closed chamber for dark adaptation (10 min). The maximum quantum yield was calculated using Equation (2),

$$\frac{\mathbf{F\_v}}{\mathbf{F\_m}} = \frac{\mathbf{F\_m} - \mathbf{F\_o}}{\mathbf{F\_m}} \tag{2}$$

Where, the minimum fluorescence (F0) and maximum fluorescence (Fm) were determined after the samples were dark adapted and variable fluorescence (Fv) was determined according to calculation Fv = Fm – F0 which gives the fluorescence difference between the closed and open reaction centers in PSII under real time photosynthetic active radiation (PAR) (de Mooij et al., 2016).

### Carbon Dioxide Fixation Rate

The biomass at the end of the cycle was collected (10 ml), washed with MilliQ water twice and left overnight for drying in a glass crucible. The carbon content in the biomass was determined by the CHNS analyser (vario MICRO cube, Elementar). The relation between biomass productivity and carbon fixation rates (g.d−1 .L−1 ), when cultivated under different CO2 concentrations, was determined by the Equation (3), average biomass productivities were considered (Gonçalves et al., 2016).

$$\mathbf{R\_{CO\_2}} = \mathbf{C\_{c}} \mathbf{P\_{max}} (\mathbf{M\_{CO\_2}/M\_c}) \tag{3}$$

Where CC was the carbon content of the microalgal cells (% w/w), Pmax was the maximum biomass productivity (g.L−1 .d−1 ), MCO2 was the molar mass of CO2 (44 g.mol−1 ), and MC was the molar mass of carbon (12 g.mol−1 ).

### Cellular Components Analysis Lipids and Fatty Acids Composition

The total lipid content in the dry algal biomass was determined by the modified Bligh and Dyer method (Bligh and Dyer, 1959), where, 10 ml of 2:1 chloroform: methanol was added to 100 mg of dry biomass and sonicated at 40 KHz using probe Sonicator (Qsonica, Q55) for 2 min. Later, the solution is centrifuged at 8,000 rpm for 10 min and the supernatant was transferred into pre-weighed tubes and left overnight at 50◦C in hot air oven. The neutral lipid content was determined using the same method with n-Hexane as the solvent (Venkata Mohan and Prathima Devi, 2012). The percentage gain in weight determined by the gravimetric means was used to conventionally quantify the total and neutral lipid content in the biomass. The lipid productivity in the batch culture was calculated using Equation (4) (Han et al., 2013).

$$\text{Lipid productivity (mg.g}^{-1}.\text{d}^{-1}) = (\text{C}\_{\text{t}}\text{L}\_{\text{t}} - \text{C}\_{\text{o}}\text{L}\_{\text{o}}) .1000/\text{T} \tag{4}$$

Where, T was the culture time in days, Ct and Lt were the biomass concentration and lipid content at T and C0 and L0 were the initial biomass concentration and initial lipid content. Fatty acid composition was determined by acidic transesterification of the lipids using acid saturated methanol. The dried lipid was refluxed with acidified methanol for 2 h in hot water bath at 80◦C. The esterified mixture was washed with ethyl acetate and water until the solution became alkaline. Later, it was filtered through an anhydrous Na2SO3 impregnated filter paper to remove moisture. The solvent recovery is done using rotary evaporator (Hei-VAP, Heidolph) and the pooled fame mix obtained is dissolved in chloroform and injected into GC for compositional analysis. The quantification of fatty acid methyl esters is done by GC-FID (Nucon-5765) with a capillary wax column (Valcobond 30 mm) using hydrogen as fuel and nitrogen as carrier gas. The oven temperature was initially maintained at 140◦C later ramped to 240◦C with 4◦C/min increase, the detector and injector were maintained at 300 and 280◦C, respectively with a split ratio of 1:10. Obtained FAME composition was compared to the standard FAME mix (C8-C22; LB66766, SUPELCO; Rohit and Mohan, 2015). The average carbon chain length (CCL) and average degree of unsaturation (DU) observed in the fatty acid profile were calculated using the equations

$$\text{Avg CCL} = \sum\_{i=0}^{n} \left(\frac{\text{FA}\_{i}}{100}\right) \ast \text{C}\_{i} \tag{5}$$

$$\text{Avg DU} = \sum\_{i=0}^{n} \left(\frac{\text{FA}\_{i}}{100}\right) \ast \text{D}\_{i} \tag{6}$$

Where, CCL and DU represent the CCL and DU observed in the fatty acids synthesized, Ci and Di are the chain lengths and unsaturation of the particular fatty acid and FAi is the fatty acid abundance (Hoekman et al., 2012).

### Carbohydrates Estimation

The carbohydrate content was determined by a phenol-sulfuric acid method where the pelleted biomass after centrifugation was powdered and dried (DuBois et al., 1956). The dry powder (10 mg) was acid hydrolyzed with 5 ml 2.5N HCl in a boiling water bath for 30 min. The solution volume is made upto 10 ml with distil-water, then centrifuged at 5,000 rpm for 5 min. To 0.2 ml of supernatant 1 ml 5% phenol solution and 5 ml 96% sulfuric acid is added. The contents are vortexed and placed in water bath for 20 min at 30◦C later optical density was measured at 490 nm to determine the carbohydrate concentration.

### Proteins, Nitrates, and Chlorophyll Estimation

Proteins were extracted from the microalgal cells using 0.5N NaOH for 24 h followed by heating upto 40◦C followed by lysis buffer (Rausch, 1981; Fernández-Reiriz et al., 1989). Quantitative estimation of protein content in the biomass is determined by the Lowry's method (Lowry et al., 1951) and the nitrate removal is determined by the standard APHA methods (Chandra et al., 2015). Chlorophyll was estimated using spectrophotometry, the 10 ml of cell biomass was pelleted using a centrifuge at 5,000 rpm for 5 min. To the pellet 10 ml of acetone and ethanol (in 1:1 ratio) were added and sonicated at 40 kHz for 2 min using probes sonicator (Qsonica, Q55). The cell debris was separated by centrifugation at 8,000 rpm for 5 min, the resulting supernatant was used to quantify chlorophyll a and chlorophyll b by measuring the OD at 647 and 664 nm respectively using Equations (7–9) (Chiranjeevi and Mohan, 2016).

Chla = (−1.93∗OD647) + (11.93∗OD664) (7)

$$\text{Chl}\,\text{b} = \text{(20.36}^{\*}\text{OD}\_{647}) - \text{(5.5}^{\*}\text{OD}\_{664}) \tag{8}$$

$$\text{Total Chloophyll} = \text{Chl a} + \text{chl b} \tag{9}$$

### Bioprocess Monitoring Redox Conditions

Redox conditions (pH) was continuously monitored (Eutech, Thermo Scientific) to determine the effective CO2 uptake and the dissolved inorganic carbon fraction in the media under elevated pressure and gas inputs.

### RESULTS AND DISCUSSION

The different experimental conditions are given codes for ease of understanding and are uniformly followed throughout the manuscript. Where, experimental variations with Air, CO2, CO2 + H2, and H2 are coded as AI, CC, CH, and HY, respectively based on the gas provided in the headspace. Correlative analysis of the photosynthetic activity and the cellular metabolite synthesis are delineated to understand the functional role of the specific gas inlets.

### Photosynthetic Efficiency vs. CO2 Biofixation

Biomass growth, quantum yield, CO2 fixation rate and chlorophyll pigment were monitored for all the experimental variations to assess the photosynthetic efficiency and understand the influence of controlled gas composition. CH condition has resulted in relatively higher biomass productivity of 2.5 g.L−1 with a positive growth rate of 0.3 g.L−1 . d −1 on the 2nd day of cultivation. The maximum biomass growth was followed by CC, AI and HY with 2.3 g.L−1 (0.27 g.L−1 . d −1 ), 1.9 g.L−1 (0.21 g.L−1 . d −1 ), and 1.5 g.L−1 (0.17 g.L−1 . d −1 ), respectively (**Figure 1**).

Biosequestration of CO2 was evaluated in correlation with the quantum yield for all the experimental conditions. Rate of CO2 sequestration was observed to be higher in the CH condition with 0.39 g.L−1 . d −1 followed by CC (0.37 g.L−1d −1 ), AI (0.33 g.L−1d −1 ), and HY (0.30 g.L−1d −1 ) conditions respectively. The rate of CO2 sequestration is well in correlation with the biomass growth were higher CO2 uptake under the photoautotrophic conditions has led to higher biomass productivity. Efficiency of the photosynthetic machinery responsible for light energy harvestation is quantified by monitoring the quantum yield (Fv/Fm). Higher quantum yield was observed in CH- 0.77 followed by CC (0.75), AI (0.66), and HY (0.61) which supports the higher biomass growth and CO2 fixation rate (**Figure 2**). The marked increment in biomass productivity with CH condition might be attributed to the enhanced photosynthetic activity with the function of key photoautotrophic enzymes (carbonic anhydrase and Rubisco) associated with light reactions (Sun et al., 2016b). Enzymatic catalysis of CO2 into usable form is catalyzed by carbonic anhydrase which increases the dissolved

inorganic carbon, augmenting its accessibility for the cellular metabolism under CH condition followed by CC, AI, and HY (Moroney and Ynalvez, 2007). Calvin cycle operates by utilizing the reducing energy generated during the light reactions (in form of NADH and ATP) for the reduction of CO2 catalyzed by Rubisco to form carbohydrates (Blankenship, 2002). Maintaining elevated CO2 gas concentrations in the CH and CC conditions, the CO2/O2 ratio gets escalated (compared to the ambient condition) thereby negating the oxygenase activity which has a detrimental effect on the CO2 uptake (Kitaya et al., 2003; Lohman et al., 2015; Mortensen and Gislerod, 2015). The improved uptake of CO2 through photocatalytic carbon fixation releases ADP and NAD+ rapidly making them readily available for accepting inorganic phosphorous and electrons by the electron transport chain thereby increasing the photosynthetic efficiency (correlating well with the quantum yields). The addition of H2 gas along with elevated CO2 maintains the cellular redox conditions with abundant proton concentrations that reduce the reactive oxygen species thereby increasing the photosynthetic efficiency as observed in CH condition. The increment in biomass productivity with elevated CO2 gas has been reported in different studies (Bowes, 1991; Hu and Gao, 2006; Mohammadi, 2016; Sun et al., 2016a). The supplementation of hydrogen along with higher CO2 also has a governing influence on higher biomass productivity and photosynthetic efficiency where hydrogen could be acting as an energy source as reported by Gaffron (1940). Apart from being an energy source it also maintains cellular redox conditions which allows the effective availability of dissolved inorganic carbon for uptake and reduces the reactive oxygen species (Gaffron, 1940; Cuellar-Bermudez et al., 2015). On the contrary, much higher levels of CO2 or H2 causes acidification below the optimal level thus causing decrement in microalgal growth. However, in the HY conditions the non-availability of carbon as compared to other conditions has resulted in lower biomass yields.

Chlorophyll content (both a and b) was estimated for all the experimental variations, as the primary light harvesting pigment it correlates with the photosynthetic efficiency. The total chlorophyll quantified was observed to be higher in CH followed by CC, AI, and HY conditions (**Figure 3**). High chlorophyll a concentration was observed in CH conditions during the eighth day of operation with 5.1µ.mg−1 followed by CC, AI, and HY with 4.1, 2.9, and 0.66 µ.mg−1 respectively. Similar trends were observed in the case of chlorophyll b where CH (1.8µ.mg−1 ) was quantified to be higher followed by CC (1.5µ.mg−1 ), AI (1.3µ.mg−1 ), and HY (0.58µ.mg−1 ). Based on the mode of operation and nutrient availability the concentration of chlorophyll varies. Hence, the chlorophyll content showed a decremental trend toward the end of the cycle as the result of nitrate limitation and lower pH (Matich et al., 2016). The photosynthetic efficiency also correlates with the Chl a/b ratio as the chlorophyll a is the primary light harvesting pigment and chlorophyll b is the accessory pigment. Positive and higher chl a/b ratio symbolizes the effective photosynthetic activity and light conversion efficiency (Karpagam et al., 2015; Li et al., 2015). CH and CC showed ratio above 2, whereas, AI and HY showed ratio in between 1 and 2 correlating to the biomass productivity and quantum yields.

## Lipogenesis Under Nutrient and Redox Stress Conditions

To understand the role of varying gas conditions (CH, AI, CC, and HY) on lipid biosynthesis. Total lipids, neutral lipids, and lipid productivity are evaluated in correlation with nitrate utilization and pH variation. The elevated CO2 levels enhanced the overall lipogenesis, possibly thorough the upregulated activities of acetyl CoA carboxylase and the fatty acid synthase complexes (Yasmin Anum Mohd Yusof et al., 2011; Yu et al., 2011; Peng et al., 2016b). The total lipid content was observed to be higher in CH condition with 26% followed by CC, AI, and HY with 21, 19, and 13%, respectively. The trend of higher total lipids positively correlated with sufficient carbon supply and nitrate stress in the case of CH and CC compared to AI and HY. Single stage non-replete nitrogen conditions were employed in all the experimental conditions where, lower nitrogen (120 mg/l) was provided which enables simultaneous biomass growth and lipogenesis (Klok et al., 2013; Benavente-Valdés et al., 2016). The maximum lipid productivity of 29, 19, 12, and 9 mg.g−1 .d−1 were observed in CH, CC, AI, and HY conditions, respectively. The nitrate removal showed a steady decremental trend in the CH, CC, and AI conditions compared to HY condition where the nitrate utilization was relatively lower (Velmurugan et al., 2014). The nitrates concentrations dropped from 120 mg/l at the initial day of operation to 10 ± 1 mg/l at the end of operation in CH, CC, and AI conditions, whereas in the HY condition the concentration has only dropped to 70 mg/l which explains the lower biomass as well as lipid productivity. Carbon availability has played the major role in lipid synthesis under CH condition followed by CC and AI as the nitrate utilization trend is similar (**Figure 4**). Initial pH was setup at 8.0 to increase the system buffering at elevated CO2 and H2 conditions. The pH was observed to drop in all the conditions from 8 to 6.89, 6.99, 7.09, and 7.38 in HY, CH, CC, and AI, respectively (**Figure 4**). The nutrient stress in the form nitrogen limitation and pH variations governed the lipogenesis along with carbon availability.

The elevated CO2 gas concentrations have a governing influence on the lipogenic enzymes (phosphoenolpyruvate carboxylase, carbamoyl-phosphate synthase, and pyruvate carboxylase) along with anaplerotic carbon assimilation reactions which could have catalyzed the enhanced lipid synthesis (Peng et al., 2016b). The intracellular and extracellular redox conditions have a marked influence on the neutral lipids synthesis which are primarily storage lipids (TAGs and esters; Juneja et al., 2013). The neutral lipids were dominant under the CH condition followed by CC, AI, and HY condition with 14, 11, 9, and 6%, respectively. Under acidic conditions, to limit the proton gradient across the cell membrane, the microalgal cells discontinue the membrane polar lipid synthesis, and surge the storage lipid synthesis which are primarily neutral lipids (Juneja et al., 2013). The role of pH apart from governing the lipogenesis is also to regulate the carbonaceous species availability for cellular metabolism, where at high alkaline pH

the carbon is in the inaccessible form of carbonates (Azov, 1982).

### Photofuels and Bioactives

Transesterified fatty acid profile of the lipids produced in all the experimental conditions were quantified to determine the photofuels and bioactive compounds production under each experimental variation. The Fatty Acid Methyl Esters (FAME) composition showed synthesis of fatty acids with CCL ranging from C10 to C20 and having unsaturation (mono/poly). Polyunsaturated fatty acids (PUFA; C18:3 and C18:2) were observed with relative abundance of 17 and 15%, 14 and 13%, 10 and 14%, and 8 and 9% in CH, CC, AI, and HY conditions, respectively (**Figure 5**). PUFA have a multitude of applications as bioactive compounds that are of high nutrient value, edible oil supplements, pharmaceutical precursors which help in lowering cardiovascular diseases apart from being used as a intermediates for various industrial chemicals. Monounsaturated Fatty Acids (MUFA; C18:1, C16:1, and C15:1) were observed, amongst which C18:1 was quantitatively higher in the case of CH and AI (11%) followed by HY (10%) and lowest was observed in CC (8%). C18:1 fatty acids has a property of being used as a food and dietary supplement and pharmaceutical ingredient. C16:1 and C15:1 were observed to have relative abundance of 11 and 5%, 7 and 3%, 6 and 2%, and 5 and 2% in the case of CH, AI, CC, and HY, respectively which have fuel properties and is applied in detergent industries as emulsifiers and also as a cosmetics for topical application. Saturated Fatty Acids (SFA; C18:0, C16:0, and C20:0) are relatively higher along with low concentrations of fatty acids like C14:0, C13:0, C12:0, and C10:0. The quantitative variations in C18:0, C16:0, and C20:0 are 11, 16, and 15% in the case of CC, 13, 16, and 11% in the case of HY, 10, 15, and 17% in the case of CH and 11, 17, and 8% in the case of AI, respectively. SFAs find application as biofuels, soaps, medicines, military lubricants and photographic plates. The lipogenic potential of microalgae is observed to be regulated through CO2 and H2 variation enabling the selective production of photofuels and bioactive compounds which have a significant role in developing a sustainable future analogous to the petroleum based precursor chemicals.

### Fatty Acid Chain Length and Saturation

The average CCL was observed to be higher in the case of CH condition with 17.58 followed by CC with 17.33, HY with 17 and AI with 16.84. Along with the variations in chain length the additional CO2 and H2 influenced the DU observed in the fatty acids. Average DU was quantified, which portrayed higher unsaturation in the case of CH (0.99) followed by CC (0.88), AI (0.85), and HY (0.73). Though the variations in the average CCL is modest, a noteworthy influence of elevated CO2 and H2 was observed on the DU. The supplementation of CO2 influenced the shift of saturated fatty acids toward unsaturation which could be

FIGURE 4 | The total lipid and neutral lipid contents in the dry biomass at the end of the cycle along with specific lipid productivity (mg.g−1 .d−1 ) under nutrient and redox stress are depicted for the varied gas concentrations in the headspace.

compositions is presented for all experimental variations.

to maintain the membrane fluidity allowing efficient CO2 uptake. Most of the unsaturated fatty acids are membrane bound lipids and hence the increase in unsaturation could be observed in the CH, HY, and CC conditions sequentially as compared to AI. The predominant shifts of saturated fatty acid to unsaturated fatty acids was observed in C18:0 fatty acid to C18:1, C18:2, and C18:3 fatty acids (**Table 2**). Similar trends of unsaturation under elevated CO2 conditions were reported in different studies with microalgae, which suggested the upregulation of desaturase enzymes catalytic activity that results in more of unsaturation (Tsuzuki et al., 1990; Nagaich et al., 2014; Cuellar-Bermudez et al., 2015). However, the lowering of pH has shown an increase in the saturated fatty acids that is specifically observed in the change of C18 and C16 fatty acids which could protect the cells from the extracellular low pH (Santos et al., 2014). Total SFA:MUFA:PUFA was observed to be 47:18:32 in the case of CH, 49:21:17 in the case of HY, 51:16:27 in the case of CC and 47:14:24 in the case of AI depicting an increased chain length and unsaturation of fatty acids in the case of CH (Yasmin Anum Mohd Yusof et al., 2011; Yu et al., 2011).

### Essential Cellular Metabolites Synthesis

Under photoautotrophy the primary metabolites synthesized are carbohydrates and proteins which serve as the energy source for cellular metabolism in dark/stress conditions. The carbohydrate synthesis has shown a linear increase with time correlating to the photosynthetic activity, as the energy for CO2 hydrogenation catalyzed by Rubisco is drawn from the light reactions and supplemented hydrogen. Relatively higher carbohydrates synthesis was observed in CH conditions (274 mg.g−1 ; 8th day) followed by CC (243 mg.g−1 ; 9th day), AI (197 mg.g−1 ; 9th day), and HY (120 mg.g−1 ; 8th day) which has correlating trends with

TABLE 2 | FAME compositional distribution with desaturation, average carbon chain length, average degree of unsaturation for lipids extracted from samples grown under different headspace gas concentrations.

photosynthetic activities and CO2 fixation. Post the maximum productivity carbohydrate concentration showed a decremental trend in all the conditions with 231, 180, 174, and 101 mg.g−1 in CH, CC, AI, and HY conditions, respectively. The decrease in carbohydrate concentration maybe due to its consumption for lipids synthesis under stress conditions correlating with nutrient uptake and lowering of pH conditions (**Figure 6**). Proteins are synthesized during biomass growth most of which are essential enzymes, membrane bound electron shuttlers and redox mediators required for the cellular functioning. The higher protein levels were observed in CH followed by AI, CC, and HY conditions with 48, 46, 45, and 26 mg.g−1 , respectively. Owing to the reduced biomass growth during the nutrient stress conditions the protein concentrations have also shown a decrement in concentrations toward the end of the operation reaching 24, 37, 43, and 44 mg.g−1 in HY, CC, AI, and CH conditions, respectively.

### Elemental Distribution in Biomass

The dry algal biomass at the end of the experiment was analyzed to determine the carbon, hydrogen, nitrogen, and sulfur distribution for all the experimental conditions (**Figure 7**). The carbon and hydrogen distribution correlates with the carbohydrate synthesis and CO2 fixation as the reactors are operated in photoautotrophic mode of nutrition amounting to 85 ± 1% of the total elemental composition in all the experimental conditions. The distribution of nitrogen was modestly varying in CH (12.82%) compared to the other CC, AI, and HY (11 ± 0.5%) conditions which could be the result of enhanced protein synthesis in CH condition supporting the hypothesis that elevated CO2 inlets under photoautotrophic mode resemble the heterotrophic mode of nutrition. The sulfur content showed similar distribution (1.5 ± 0.2%) amongst all the experimental variations (Gonçalves et al., 2016). The increase in CO2 uptake had the influence on the carbon accumulation along with the protein synthesis which govern the C:N distribution of the biomass.

### CONCLUSIONS

Present study shows the possibilities of selectively producing microalgal based bioactives and photofuels from industrial effluent gasses. The influence of elevated CO2 and H2 gas concentrations on both biomass growth and lipogenesis was specifically evident from the fatty acids compositions and the quantum yield. This study unwraps the possibility of cultivating microalgae in integration with industrial effluent treatment by utilizing the flue gases with low investments and also help in generating additional revenue, making the process economically feasible. However, deeper insights for growth and metabolite regulation in photosynthesis can be determined through molecular level quantification and transcriptomics of the targeted proteins/enzymes. The recent advances in photosynthetic research provide a ray of hope to overcome climate change and progress toward sustainable development with the multifaceted applications of microalgae.

variations.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## ACKNOWLEDGMENTS

SKB acknowledges University Grants Commission (UGC) for providing research fellowship.

### REFERENCES

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microalga Coccomyxa subellipsoidea C-169. Biotechnol. Biofuels 9, 1–17. doi: 10.1186/s13068-016-0571-5

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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# Granular Carbon-Based Electrodes as Cathodes in Methane-Producing Bioelectrochemical Systems

Dandan Liu1 , Marta Roca-Puigros 1 , Florian Geppert 2 , Leire Caizán-Juanarena1 , Susakul P. Na Ayudthaya3 , Cees Buisman1 and Annemiek ter Heijne1 \*

1 Sub-Department of Environmental Technology, Wageningen University & Research, Wageningen, Netherlands, 2 Fraunhofer Institute for Environmental, Safety, and Energy Technology UMSICHT, Oberhausen, Germany, 3 Laboratory of Microbiology, Wageningen University & Research, Wageningen, Netherlands

#### Edited by:

Andrea Schievano, Università degli Studi di Milano, Italy

### Reviewed by:

Sunil A. Patil, Indian Institute of Science Education and Research Mohali, India Matteo Grattieri, University of Utah, United States Miriam A. Rosenbaum, Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie, Hans Knöll Institut, Germany

> \*Correspondence: Annemiek ter Heijne<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Bioengineering and Biotechnology

> Received: 29 March 2018 Accepted: 29 May 2018 Published: 12 June 2018

#### Citation:

Liu D, Roca-Puigros M, Geppert F, Caizán-Juanarena L, Na Ayudthaya SP, Buisman C and ter Heijne A (2018) Granular Carbon-Based Electrodes as Cathodes in Methane-Producing Bioelectrochemical Systems. Front. Bioeng. Biotechnol. 6:78. doi: 10.3389/fbioe.2018.00078 Methane-producing bioelectrochemical systems generate methane by using microorganisms to reduce carbon dioxide at the cathode with external electricity supply. This technology provides an innovative approach for renewable electricity conversion and storage. Two key factors that need further attention are production of methane at high rate, and stable performance under intermittent electricity supply. To study these key factors, we have used two electrode materials: granular activated carbon (GAC) and graphite granules (GG). Under galvanostatic control, the biocathodes achieved methane production rates of around 65 L CH4/m2catproj/d at 35 A/m2catproj, which is 3.8 times higher than reported so far. We also operated all biocathodes with intermittent current supply (time-ON/time-OFF: 4–2′ , 3–3′ , 2–4′ ). Current-to-methane efficiencies of all biocathodes were stable around 60% at 10 A/m2catproj and slightly decreased with increasing OFF time at 35 A/m2catproj, but original performance of all biocathodes was recovered soon after intermittent operation. Interestingly, the GAC biocathodes had a lower overpotential than the GG biocathodes, with methane generation occurring at −0.52 V vs. Ag/AgCl for GAC and at −0.92 V for GG at a current density of 10 A/m2catproj. 16S rRNA gene analysis showed that Methanobacterium was the dominant methanogen and that the GAC biocathodes experienced a higher abundance of proteobacteria than the GG biocathodes. Both cathode materials show promise for the practical application of methane-producing BESs.

Keywords: methane production, intermittent current supply, low cathode overpotential, bioelectrochemical system (BES), granular carbon-based electrode

## INTRODUCTION

The expansion of global energy demand results in an increasing utilization of fossil fuels, which leads to unwanted CO2 emissions (Rogelj et al., 2016). To mitigate CO2 emissions, the energy transition from fossil fuels to renewable energy is necessary. In the Energy Roadmap 2050 released by European Commission in 2011, the share of renewable energy in the final gross energy consumption will grow from 10% of today, to 30% in 2030, and at least 55% in 2050 (Commission, 2011). The substantial rise of renewable electricity demand requires new technologies for electricity storage, because the renewable electricity produced is fluctuating and intermittent due to the intermittent nature of wind and sun (Hu et al., 2018).

Power to Gas (PtG) technologies have been reported as a flexible option to convert and store excess renewable electricity from the power grid (electricity) into the gas grid (CH4) (Bailera et al., 2017). CH4 can be generated by reduction of CO2 through thermochemical or biological methanation. Methaneproducing bioelectrochemical systems (BESs) are one form of biological methanation (Geppert et al., 2016). In methaneproducing BESs, H2O is typically used as an electron donor, and oxidized at the anode (Van Eerten-Jansen et al., 2012). At the cathode, CO2 is reduced to CH4 by microorganisms. This assembly of cathode and microorganisms is called a biocathode.

Since the concept of methane-producing BESs has been shown in 2009 (Cheng et al., 2009), methane-producing BESs have mainly been studied at constant external electricity supply. The electricity generated by the renewable sources is, however, intermittent. So far, performance of biocathodes under intermittent electricity supply has not been studied. Intermittent operation has been performed with capacitive anode electrode materials in the form of activated carbon granules (GAC) for wastewater microbial fuel cells (MFCs) (Borsje et al., 2016; Santoro et al., 2017). These capacitive bioanodes can store electrons generated by electroactive microorganisms in the charging period (open circuit), and afterwards, these stored electrons could be harvested in the discharging cell (closed circuit) (Deeke et al., 2015). Use of granular electrodes with this capacitive property (storage of electrons) might benefit methane-producing BESs operated with intermittent electricity supply, so that the capacitance can act as an electron buffer when current peaks occur.

Besides the capacitance property of GAC, use of granular carbon-based electrodes is in general beneficial to the performance of biocathodes. The reason behind this may be that carbon-based materials have good biocompatibility, and the 3D granular structure can provide benefits for the attachment of microorganisms and increase mass transfer between the bulk solution and the electrode (Guo et al., 2015; Jourdin et al., 2015). In addition, GAC has been proven to stimulate methane production in anaerobic digestion, as it probably promotes direct interspecies electron transfer from Geobacter (Liu et al., 2012), Sporanaerobacter, and Enterococcus (Dang et al., 2016) species to methanogens. Addition of pre-acclimated GAC as inoculum has also been shown to enhance methane production and decrease startup time in the methane-producing BESs, although carbon brushes were used as cathode electrode (LaBarge et al., 2017).

In this paper, we report the use of GAC and graphite granules (GG) in a packed bed as the cathode electrode. Three intermittent current supply modes with time-ON/time-OFF (4– 2 ′ , 3–3′ , and 2–4′ ) were performed at two different current densities (10 and 35 A/m2 catproj). The effect of intermittent current supply with different time intervals was studied. We tested both granule types in duplicate reactors for 137 days and assessed performance in terms of methane production rate and current-to-methane efficiency. We also analyzed the microbial community composition.

## MATERIALS AND METHODS

### Experimental Setup

We operated four bioelectrochemical reactors (see Figure S1 in the Supporting Information). Each reactor contained an anodic and cathodic chamber, each with a volume of 33 cm3 (11 × 2 × 1.5 cm). A cation exchange membrane (FumaTech GmbH, Ingbert, Germany) was used with a projected surface area of 22 cm2 (11 × 2 cm). As cathode materials, we used GAC with a specific surface area of 764 m2 /g (Cabot Norit Nederland B.V., Zaandam, the Netherlands; 1–3 mm diameter) and GG with a specific surface area of 0.438 m2 /g (Carbone Lorraine Benelux BV, Wemmel, Belgium; 3–5 mm), leading to a substantially higher capacitance property in GAC compared with GG (Borsje et al., 2016).

Two cathodic chambers were packed with GAC granules (GAC1 with 8.5 g and GAC2 with 8.4 g). The other two cathodic chambers were packed with GG granules (GG1 with 26 g and GG2 with 29.2 g). All the granules were washed by distilled water before use. The current collector at the cathode was a plain graphite plate. The projected cathode surface area was 22 cm2 (11 × 2 cm), and was equal to the membrane surface area. The granule bed was tightly packed to ensure good contact between granules and current collector. The anodic chambers contained a 22-cm2 platinum-iridium-coated titanium plate as electrode (Magneto Special Anodes BV, Schiedam, the Netherlands). The anodic chambers were filled with glass beads with a 7-mm diameter (Hecht-Assistent, Sondheim v. d. Rhön, Germany) to further ensure tight packing of the carbon granules. The reference electrodes (3 M KCl Ag/AgCl, QM710X, QIS, Oosterhout, the Netherlands, +0.205 V vs. standard hydrogen electrode) were connected to the anolyte and catholyte solutions. Throughout this paper, all potentials are expressed against Ag/AgCl reference electrode.

Each cathodic chamber was connected to a liquid-gas separation bottle (60 mL) with a gas bag of 2 L (Cali-5-BondTM). After the separation bottle, the catholyte was channeled into the recirculation bottle (500 mL), where CO2 was sparged. The excess CO2 went through a water lock and was released into the environment. All four anode chambers shared the same anolyte that was pumped via a recirculation bottle (5 L). Anolyte and catholyte flow rates were 7 mL/min.

### Electrolytes and Microorganisms

The catholyte consisted of a 50 mM phosphate buffer (2.77 g/L NaH2PO4·2H2O and 4.58 g/L Na2HPO4) with 0.2 g/L NH4Cl, 0.13 g/L KCl, 10 mL/L Wolfe's vitamin solution and 10 mL/L Wolfe's modified mineral solution (Wolin et al., 1963). Catholyte pH was 7.1. The fresh catholyte was flushed with N2 gas for 30 min before each use. In order to keep the catholyte with sufficient CO2 and stable pH simultaneously, the catholyte in the recirculation bottle was sparged with CO2 for 2 h/day during weekdays. After day 71, the catholyte was sparged with CO2 continuously.

All cathode chambers were inoculated with 10 mL of an anaerobic mixed culture (volatile suspended solids = 12.9 ± 1.3 g/L), which contained 50% anaerobic granular sludge from the paper industry wastewater treatment facility in Eerbeek (the Netherlands) and 50% anaerobic sludge from the municipal wastewater treatment facility in Ede (the Netherlands).

The anolyte consisted of a 50 mM phosphate buffer at pH 7. The anolyte was continuously flushed with N2 gas in the recirculation bottle to keep O2 levels at a minimum.

### System Operation

Experimental conditions are shown in **Figure 1**. All reactors were galvanostatically controlled (fixed current) by a potentiostat (Ivium n-Stat, Eindhoven, the Netherlands), which collected the cathode potential data from all reactors at intervals of 1 min. In this way, methane production rates can be regulated more directly than with cathode potential control, as the current determines the electrochemical reaction rate (Jörissen and Speiser, 2015). After inoculation, all reactors were operated at a fixed current of 5 A/m2 catproj as startup period. The current of all reactors was increased from 5 to 10 A/m2 catproj on day 37 and from 10 to 35 A/m2 catproj on day 71. All cathodes were operated in batch. Half of the catholyte was replaced on days 31 and 70 to replenish buffer, nutrients and vitamins. The pH of each reactor was monitored daily by pH measurement of liquid samples (0.5 mL per sample) taken from anode and cathode chamber. All reactors were operated inside a temperature-controlled cabinet at 30◦C.

For intermittent operation, a cycle time of 6 min (′ ) was used at three different current time-ON/time-OFF ratios: 4–2′ , 3–3′ , and 2–4′ . Each ratio was tested for 20 h and was performed twice. After intermittent operation, all biocathodes were supplied with constant current for 20 h to investigate recovery after intermittent operation.

### Electrochemical Analysis

Polarization curves were recorded before inoculation and on day 30 and day 90 after inoculation. For the polarization curve before inoculation, the cathode potential was controlled from −0.5 to −1.0 V with steps of 0.1 V; for the polarization curve after inoculation, the cathode potential was controlled from −0.1 to −0.7 V with steps of 0.05 V. Each potential step lasted 600 s for the GAC biocathodes, and 300 s for the GG biocathodes, as the latter required a shorter equilibrium time.

### Chemical Analyses

The liquid and gas samples were taken from each reactor twice a week. Volatile fatty acids (VFAs), including formate, acetate and lactate, were determined in the liquid phase by high-performance liquid chromatography (HPLC) (Lindeboom et al., 2016), whereas the gas composition was measured by gas chromatography (GC) (Liu et al., 2016). The gas volume was quantified by emptying the gas bags with a syringe. The methane production rate was calculated and normalized to the projected surface area of the cathode (Equation 1) and the volume of the cathodic chamber (Equation 2), as follows:

$$\gamma\_{CH4-A} = \frac{\mathbf{V\_T \times C\_{CH4}}}{A\_{proj} \times t} \tag{1}$$

$$\gamma\_{CH4-V} = \frac{V\_T \times C\_{CH4}}{V\_{electrode} \times t} \tag{2}$$

Here, γCH4−A (L CH4/m2 catproj /d) and γCH4−V (L CH4/m3 cat /d) represent methane production rates; VT (L) is the total volume by summing up the volume of the gas bag and the headspace (0.015 L); CCH4 (%) is the methane fraction in the headspace; Aproj(m2 ) is the projected surface area of the graphite plate current collector and membrane; Velectrode(m3 ) is the cathodic chamber volume; t (d) is the experimental time between each headspace measurement (d).

### Current-to-Methane Efficiency

This indicates which percentage of the electrons ended up in the form of methane and is calculated as Equation 3.

$$
\eta\_{\rm CH4} = \frac{\mathbf{n}\_{\rm CH4} \times \mathbf{z}\_{\rm CH4} \times \mathbf{F}}{\int\_{t=0}^{t} \mathbf{I} \, \text{d}t} \tag{3}
$$

F is the Faraday constant (96485 C/mol e−); nCH4 (mol) is total moles of CH4 produced; zCH4 is moles of electrons per mole of CH4 (8); I (A) is the current.

### Microbial Community Analysis

After operation at a current density of 35 A/m2 catproj, all reactors were disassembled inside an anaerobic chamber, and 0.5 g (wet weight) of the granules was taken from each cathode. In addition, 300 mL of the catholyte was taken from each reactor and filtered it over a 0.22µm MF-MilliPore filter. Genomic DNA was extracted from each reactor samples with a Mo Bio PowerSoil DNA isolation kit for 0.5 g of the granular electrode and a Mo Bio PowerWater DNA isolation kit for the filter, according to the manufacturer's instructions. To investigate both bacteria and archaea, firstly amplification of 16S rRNA gene fragments was carried out by using a two-step PCR protocol, and then DNA was quantified using a Qubit R dsDNA BR Assay Kit and a DeNovix DS-11 FX spectrophotometer/fluorometer (DENovix Inc., Wilmington, DE, USA), finally the 16S rRNA gene Miseq sequencing data were analyzed using Galaxy/NG-Tax, an in-house pipeline (see detailed information in Supportive Information, under B). Bray-Curtis similarities were calculated between reactors (biocathodes and used catholytes) from the microbial community relative abundance data with Primer-E software, version 7 (LaBarge et al., 2017).

### RESULTS AND DISCUSSION

### High Methane Production Rates Directly Linked to Current Density

We determined methane production rates at two different current densities of 10 and 35 A/m2 catproj. At the current density of 35 A/m2 catproj, the methane production rates were around 65 L CH4 /m2 catporj/d for both cathode materials (**Figure 2A**). As methane production rates were directly related to current density, they were almost four times higher than at 10 A/m2 catproj. The current-to-methane efficiencies for the GAC and GG reactors (**Figure 2B** increased from 55% at 10 A/m2 catproj to 67% at 35 A/m2 catproj. No H2 and volatile fatty acids were detected in any of the reactors at these two current densities, which suggests that during the stable performance period, the methanogenic activity was high enough to utilize these components if they were produced. Possible other electron sinks are biomass growth (Geppert et al., 2016), or loss of methane via membrane, tubes, and connections within the reactor (Skovsgaard and Jacobsen, 2017), especially considering our relatively long sampling intervals (3–4 days). Also, reduction of oxygen generated at the anode could play a role (Van Eerten-Jansen et al., 2012). The methane production rates achieved with GAC and GG at constant current in this study were compared with similar carbon-based electrodes in other studies (Geppert et al., 2016; **Table 1**). One the one hand, methane production rate at 35 A/m2 were several times

higher than those in other studies. On the other hand, methane production rate at 10 was similar with other studies, but it is interesting to note that the cathode potentials of GAC biocathodes were quite different in this case, −0.55 V for GAC biocathodes compared to −1.1 V for biocathodes, i.e., Villano et al.

deviation of four samples for each reactor and current density.

### Methane Production Was Related to Total Charge Also in Intermittent Mode

After all the biocathodes achieved a stable methane production rates at a constant current supply of 10 A/m2 catproj, intermittent current (at the same current density) was supplied to all biocathodes with three different time intervals: 4–2′ , 3–3′ , and 2–4′ . Methane production rate of each biocathode is shown in **Figure 4A**, calculations based on the 20 h period for each time interval. Higher current time-ON/time-OFF interval supplied to the biocathodes resulted in higher methane production

TABLE 1 | Comparison of methane production rates for similar 3D carbon-based electrodes in methane-producing BESs when water was used as electron donor.

rates, with 9.5 L CH4/m2 catproj/d at 4–2′ , 5.5 L CH4/m2 catproj/d at 3–3′ and 4.0 L CH4/m2 catproj/d at 2–4′ , meaning that charge provided during ON-time was used to generate methane.

When the current density was increased from 10 to 35 A/m2 catproj, the methane production rate at continuous current supply increased from 15 L CH4/m2 catproj/d at 10 A/m2 (**Figure 3A**) to 90 L CH4/m2 catproj/d at 35 A/m2 (**Figure 3B**). Again, an increase in methane production rate was observed along with increasing time-ON/time-OFF ratios. Moreover, we compared our experimental data with the theoretical methane production calculated for the different current time-ON/time-OFF ratios (Figure S4 in Supporting Information, under D). The close fit between measured and calculated data shows that methane generation is closely linked to the charge provided to the biocathode, for both GG and GAC.

### Intermittent Current Operation Does Not Influence Biocathode Activity

Studies on bioanodes have shown that GAC can store charge in the electric double layer when used in Microbial Fuel Cells (Deeke et al., 2015; Lu et al., 2015), whereas GG with low capacitance does not show this charge storage behavior. This higher capacitance of GAC biocathodes was expected to result in smaller fluctuations in cathode potential, and as a possible electron buffer, compared to GG biocathodes during intermittent operation (Borsje et al., 2016). As shown in Figure S5, the cathode potentials of GAC biocathodes during intermittent current indeed kept stable around −0.5 V, whereas the cathode potentials of GG biocathodes changed in the range from −0.6 to −1.0 V. These results might indicate that intermittent current operation would affect GAC biocathodes less than GG biocathodes. However, at a current density of 10 A/m2 catproj, all biocathodes, operated under different current time-ON/time-OFF intervals, had a similar current-to-methane efficiency of 50–60% (**Figure 4A**). When the current density was increased from 10 to 35 A/m2 catproj, the current-to-methane efficiency was also constant with a slight decrease along with the longer time-OFF intervals (**Figure 4B**). After these intermittent operations, an additional constant current supply for 20 h was operated for all biocathodes to verify if the initial activity was restored after intermittent operation. As shown in **Figure 4**, the currentto-methane efficiencies of all biocathodes after intermittent operations were similar to those at constant current supply, indicating that biocathodes were not affected by the intermittent operation at these two current densities of 10 and 35 A/m2 catproj, for both materials. In addition, the methane production rate of all biocathodes in our study kept stable, even though all biocathodes had experienced around 60 min of open circuit period during each headspace and pH sampling time. Our results are, however, different from those results found in the previous study that the methane production rate decreased by 87% after an open circuit period of 45 min, and it took 4 months before performance was back at the original level (Bretschger et al., 2015). The discrepancy could be due to that the quantity and/or bioactivity of biofilm growth on our granular carbon-based electrodes is higher than those on the carbon cloth electrode used in that study, as higher current density was found on our biocathodes (5 A/m2 ) compared with their biocathodes (0.06 A/m2 ). To conclude, current-to-methane efficiency (%) remained quite stable under the different current supply modes at 10 A/m2 , and showed a slight decrease with increasing OFF-time at 35 A/m2 .

It is worth notifying that current-to-methane efficiencies obtained at continuous current density of 35 A/m2 catproj in **Figure 4B**, are even higher than those achieved at the same current density reported in **Figure 2B**. This discrepancy could be due to the different durations between headspace sampling: 20 h for **Figure 5** and 3–4 days for **Figure 3**. Shorter duration between headspace sampling could mitigate losses via H2 or O2 leakage from the joints of the experimental setup.

### Biocathodes With Granular Activated Carbon Produced Methane at Low Overpotentials

Although there is no difference between GAC and GG biocathodes during constant and intermittent operation in terms

of methane production rate (**Figures 2A**, **3**) and current-tomethane efficiency (**Figures 2B**, **4**). Interestingly, the cathode potentials of the GAC biocathodes were different from the GG biocathodes.

Directly after inoculation, all the reactors had similar cathode potentials of about −0.90 V (**Figure 5**). The cathode potential of GAC1 changed from −0.90 to −0.52 V between day 7 and 10, whereas the cathode potential of GAC2 changed from −0.80 to −0.52 V between day 30 and 37. The cathode potentials of the GG reactors remained stable around −0.92 V long after inoculation and became slightly more negative around day 37 and day 70 due to the increases in current density. These potential differences between GAC and GG biocathodes were also seen in the polarization curves at day 30 (Figure S2B in the Supporting Information, under C) and day 90 (Figure S2C in the Supporting Information, under C). These polarization curves show that the onset of current for GAC biocathodes occurred at a more positive potential from −0.5 V to even −0.4 V, whereas the current densities of GG biocathodes were negligible in the whole range of cathode potentials tested (−0.7 to −0.3 V). For the current obtained in GAC at −0.5 V during the polarization experiment, other possible secondary reactions, e.g., hydrogen, acetate or formate, could play a role. As with these intermediates, quick consumption by methanogens could lead to undetectable levels of these intermediates. Nevertheless, the onset potentials of the bare GAC electrodes (Figure S2A in the Supporting Information, under C) were around −0.7 V, the difference indicating the catalytic effect of the cathodic microorganisms growth on the GAC electrodes.

To exclude that the measurement of cathode potential was influenced by the fact that the reference electrode was placed outside the cathode compartment, we inserted a new Ag/AgCl reference electrode into one of GAC cathodic chamber as close as possible to the granular bed. The cathode potential was around −0.43 V, which was 100 mV less negative than the cathode

potential (−0.52 V) measured outside the cathode chamber, pointing out that the actual cathode potential was even less negative than that was measured. To our knowledge, these cathode potentials for GAC are the least negative ones (i.e., lowest overpotential) reported in the literature for methaneproducing BESs (Geppert et al., 2016). It is likely that methane production at a cathode potential of −0.52 V has not been reported before due to the fact that all methane-producing biocathodes in other studies were operated at a constant potential rather than at a constant current. Actually, most of the studies have used cathode potentials more negative than −0.7 V vs. Ag/AgCl to supply a sufficiently high overpotential for methane generation (Siegert et al., 2014; Villano et al., 2016; LaBarge et al., 2017). Switching from galvanostatic control to potentiostatic control with an active biocathode resulted in similar rates and efficiencies (results are shown as Figure S6 in the Supporting Information, under F). Galvanostatic control is thus useful to achieve methane production at low overpotential, but can be changed to potentiostatic control once an active biocathode is present, without loss in activity.

At this point, it is unclear why the cathode potential of GAC changed to −0.52 V, while cathode potential of GG remained at −0.90 V. The high specific surface area and average smaller size of GAC (764 m2 /g, 1–3 mm) relative to GG (0.438 m2 /g, 3–5 mm) may have played a role, but does not explain the mechanism of methane formation. It is worth notifying that similar phenomenon had been shown in a previous study where the presence of GAC in anaerobic digestion stimulated methane production rate, whereas graphite electrode did not affect the performance, for reasons not yet understood (Dang et al., 2016). In our study, the cathode potential of −0.52 V is 0.1 V more positive than the thermodynamic equilibrium potential for hydrogen evolution (−0.62 V) under the biological conditions (T = 30◦C, P = 1 bar, pH = 7) (Beese-Vasbender et al., 2015). Such less negative cathode potential and the undetectable hydrogen in GAC biocathodes suggests that the change in potential for methane production on GAC biocathodes observed here could be related to direct electron transfer. However, hydrogen as an intermediate for methane production at GAC biocathode cannot be excluded as the local hydrogen pressures and local pH values are not known. Additional research is needed to measure the actual values of local pH and hydrogen pressure on the biocathode by using microsensor, and therefore, providing insight into the relationship between mechanisms of electron transfer and different conductive materials.

The changes in the biocathode potentials of the GAC reactors occurred on different days (**Figure 5**). The reason for that may be that in GAC1, which had been operated and adjusted

FIGURE 5 | Average daily cathode potentials of all the reactors after inoculation. Both GAC biocathodes showed a steep increase in cathode potential, whereas the cathode potential for both GG biocathodes remained constant, and decreased with increased current density.

during 2 months before inoculation to perform electrochemical measurements, the catholyte and/or electrode may already have contained methanogens before inoculation. Indeed, a minor amount of CH4 was already detected in the headspace of GAC1 during the phase before inoculation (data not shown). The fluctuations of the cathode potentials, especially at current densities of 5 A/m2 catproj and 10 A/m2 catproj, were probably the result of fluctuations in catholyte pH due to intermittent CO2 supply (Jourdin et al., 2015). After changing to continuous CO2 supply and a current density of 35 A/m2 catproj on day 71, the pH of the catholyte and the cathode potentials remained more stable (Figure S3, supporting information, under C).

### Microbial Community Analysis Revealed Methanobacterium as Dominant Species

Microbial communities of biofilm and catholyte were characterized for all reactors to investigate whether different microbial communities developed on the two cathode materials. Table S1 in the Supporting Information shows the community similarity results for all granules. All cathodic communities (both in biofilm and catholyte) were dominated by hydrogenotrophic methanogens (Methanobacterium), which has been found in many other studies (Van Eerten-Jansen et al., 2013; Cai et al., 2016; LaBarge et al., 2017) regardless of electrode material and inoculum source (**Figure 6**). Another hydrogenotrophic methanogen, namely Mthanocorposculum, was detected 21% in the catholyte of GG1. The GAC electrode samples showed a greater relative abundance of Proteobacteria (Deltaproteobacteria and Betaproteobacteria) with 14% for GAC1 and 47% for GAC2, relative to 8.7% for GG1 and 3.4% for GG2, As exoelectrogens like Geobacter sp. belong to the proteobacteria, the most common phylum of bacteria found on the anode of microbial fuel cells (Hasany et al., 2016), this may be related to the lower overpotentials measured for GAC.

### CONCLUSION

In this paper, we have shown that both GAC and GG are suitable cathode materials for high methane production rates in methane-producing BESs. Intermittent current operation resulted in stable methane production for both materials, and original performance was restored directly after intermittent operation. GAC biocathodes showed lower overpotentials than GG; the mechanism behind this needs to be further investigated. Granular biocathodes thus hold promise for the practical application of methane-producing BESs for renewable electricity storage.

### AUTHOR CONTRIBUTIONS

AtH conceived the original idea and together with CB supervised the project. DL and MR-P carried out the experiment. DL wrote the manuscript with support from MR-P, SN. LC-J, and FG,

### REFERENCES

helped supervise the project. SN helped conducted the microbial community analysis.

### ACKNOWLEDGMENTS

The authors thank Nurul Azyyati Sabri for her help with DNA extraction, and Caroline Plugge for facilitating the microbial community analyses. The authors are also grateful for financial support from Dutch companies (Alliander, DMT Environmental Technology), the Chinese Scholarship Council (File No.<PHONE_NUMBER>43) and the Cluster of Excellence RESOLV (EXC 1069), which is funded by the Deutsche Forschungsgemeinschaft.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe. 2018.00078/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Liu, Roca-Puigros, Geppert, Caizán-Juanarena, Na Ayudthaya, Buisman and ter Heijne. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Sulfate-Reducing ElectroAutotrophs and Their Applications in Bioelectrochemical Systems

Valeria Agostino1 and Miriam A. Rosenbaum1,2,3 \*

1 Institute of Applied Microbiology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany, 2 Bio Pilot Plant, Leibniz Institute for Natural Product Research and Infection Biology – Hans-Knöll-Institute, Jena, Germany, 3 Faculty of Biological Sciences, Friedrich Schiller University, Jena, Germany

Electroautotrophs are microbes able to perform different biocathodic reactions by using CO2 as sole carbon source and electrochemical reducing power as a sole energy source. Electroautotrophy has been discovered in several groups of microorganisms, including iron-oxidizing bacteria, iron-reducing bacteria, nitrate-reducing bacteria, acetogens, methanogens and sulfate-reducing bacteria. The high diversity of electroautrophs results in a wide range of Bioelectrochemical Systems (BES) applications, ranging from bioproduction to bioremediation. In the last decade, particular research attention has been devoted toward the discovery, characterization and application of acetogenic and methanogenic electroautotrophs. Less attention has been given to autotrophic sulfate-reducing microorganisms, which are extremely interesting biocatalysts for multiple BES technologies, with concomitant CO2 fixation. They can accomplish water sulfate removal, hydrogen production and, in some case, even biochemicals production. This mini-review gives a journey into electroautotrophic ability of sulfate-reducing bacteria and highlights their possible importance for biosustainable applications. More specifically, general metabolic features of autotrophic sulfate reducers are introduced. Recently discovered strains able to perform extracellular electron uptake and possible molecular mechanisms behind this electron transfer capacity are explored. Finally, BES technologies based on sulfate-reducing electroautotrophs are illustrated.

Keywords: bioelectrochemical systems, electroautotrophs, sulfate-reducing microorganisms, biocathodes, bioremediation, bioproduction

## INTRODUCTION

During the last decade, much research focused toward the use of electroautotrophic microorganisms in Bioelectrochemical Systems (BES). While exoelectrogens have evolved to use extracellular insoluble minerals or electrodes as terminal electron acceptors, electroautotrophs are able to acquire energy by taking up electrons from extracellular solid compounds or electrodes, while using carbon dioxide (CO2) as inorganic carbon source (Tremblay et al., 2017).

Electroautotrophy was first discovered in the model exoelectrogen genus Geobacter (Gregory et al., 2004; Gregory and Lovley, 2005). As the majority of exoelectrogenic biocatalysts are dissimilatory iron-reducing bacteria, researchers hypothesized that iron-oxidizing bacteria could be able to accept electrons from a cathodic electrode. Indeed, Acidithiobacillus ferrooxidans, Mariprofundus ferrooxydans PV-1, and Rhodopseudomonas palustris have been designated

#### Edited by:

Deepak Pant, Flemish Institute for Technological Research, Belgium

#### Reviewed by:

Mirella Di Lorenzo, University of Bath, United Kingdom Feng Zhao, Institute of Urban Environment (CAS), China Mohita Sharma, University of Calgary, Canada

> \*Correspondence: Miriam A. Rosenbaum<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Energy Research

Received: 03 April 2018 Accepted: 04 June 2018 Published: 20 June 2018

#### Citation:

Agostino V and Rosenbaum MA (2018) Sulfate-Reducing ElectroAutotrophs and Their Applications in Bioelectrochemical Systems. Front. Energy Res. 6:55. doi: 10.3389/fenrg.2018.00055 as electroautotrophs (Carbajosa et al., 2010; Summers et al., 2013; Bose et al., 2014). Moreover, the chemolithoautotrophic archea Methanococcus maripaludis and Methanobacterium-like archaeon strain IM1, isolated with metallic iron as sole electron donor, are able to perform electromethanogenesis (Lohner et al., 2014; Beese-Vasbender et al., 2015a). Several acetogenic bacteria, including Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Sporumosa acidovorans, Sporumosa malonica, Clostridium ljungdahlii, Clostridium aceticum, and Moorella thermoacetica, can utilize the cathodic current for CO2 reduction to organic acids (Nevin et al., 2011; Aryal et al., 2017). Also, some autotrophic sulfate-reducing microorganisms (SRM) have shown the ability to consume electrons from the cathode to accomplish sulfate reduction and hydrogen (H2) production (Rodrigues and Rosenbaum, 2014; Beese-Vasbender et al., 2015b). However, overall fairly little research has been devoted toward this last group of electroautotrophic biocatalysts.

Pioneering discoveries regarding cathodic electron consumption of SRM are related to anaerobic microbial induced corrosion (MIC) studies (Widdel, 1992). SRM can stimulate not only a chemically-influenced corrosion of iron through the production of corrosive hydrogen sulfide (Widdel, 1992), but also an electrochemical-induced corrosion by the consumption of "cathodic hydrogen" formed on iron in contact with water (von Wolzogen Kühr and van der Vlugt, 1934; Pankhania, 1988), or by directly uptaking electrons from iron (Dinh et al., 2004; Gu et al., 2009; Gu and Xu, 2010; Xu and Gu, 2011). The readers are referred to several excellent reviews on to role of SRM in MIC (Enning and Garrelfs, 2014; Anandkumar et al., 2016; Li et al., in press).

It should be noticed that early BES studies with SRM have been focused on their anodic exploitation for electricity generation and sulfate removal using organic substrates (Habermann and Pommer, 1991; Liang et al., 2013; Zheng et al., 2014). However, it was soon clarified that the production of electricity with SRM-based anodes was mainly due to the abiotic oxidation of biologically produced sulfide to elemental sulfur (Zhao et al., 2008). Consequently, sulfur-oxidizing bacteria have started to be applied in anodic oxidation processes for current generation (Sun et al., 2009; Gong et al., 2013; Lee et al., 2014; Zhang et al., 2014).

Another very recent and promising BES application of SRM is the cathodic electrofermentation of short chain organic acids into more valuable compounds as alcohols and acetone (Sharma et al., 2013a,b, 2014, 2015).

**Figure 1A** summarizes the application of the different metabolic capabilities of SRM in various BES. Despite these different attractive SRM-based BES technologies, the specific focus of this mini-review is to summarize the current understanding and trends in biocathodic applications of electroautotrophic SRM, using CO2 as inorganic carbon source (highlighted with a red box in **Figure 1A**).

### SULFATE REDUCING MICROORGANISMS

SRM are a heterogeneous group of anaerobic microorganisms, widely distributed in anoxic environments with essential roles in the global cycling of carbon and sulfur (Jørgensen, 1982). Most cultured SRM belong to four bacterial (Deltaproteobacteria, Nitrospirae, Firmicutes, Thermodesulfobacteria) and two archaeal phyla (Euryarchaeota, Crenarchaeota) (Rabus et al., 2006; Muyzer and Stams, 2008). SRM have the ability to use sulfate, the most oxidized sulfur specie, as terminal electron acceptor for the oxidation of organic compounds or hydrogen in a process named dissimilatory sulfate reduction (Widdel and Hansen, 1991). This is an intracellular pathway that requires an eight-electron reaction for the reduction of sulfate to sulfide, with sulfite as intermediate. After crossing microbial membranes, sulfate is "activated" to form adenosine 5′ -phosphosulfate (APS) by the enzyme Adenosine Triphosphate (ATP) sulfurylase (Peck, 1959). APS is then reduced to sulfite in a two-electron reaction performed by the enzyme APS reductase (AprBA) (Lampreia et al., 1994). The final step of sulfite reduction to sulfide is catalyzed by the dissimilatory sulfite reductase complex (Dsr), with the involvement of an energy-conserving membrane complex (DsrMKJOP or DsrMK) (Fike et al., 2016).

### Carbon Assimilation and Electron Donors

SRM can grow on more than one hundred organic compounds, including monocarboxylic acids, dicarboxylic acids, sugars, alcohols, ketones, amino acids, aromatic compounds, and hydrocarbons (Barton and Fauque, 2009).

SRM can either perform incomplete or complete oxidation of organic compounds (Rabus et al., 2006). Incomplete oxidation of organic substrates results in the excretion of acetate as main product, due to a deficiency for the terminal oxidation of acetyl-CoA (Widdel, 1988). On the contrary, complete oxidizers degrade organic compounds to CO2, oxidizing acetate with two different pathways: a modified citric acid cycle (e.g., Desulfobacter spp., Brandis-Heep et al., 1983), or the acetyl-CoA pathway (e.g., Desulfobacterium spp., Schauder et al., 1986). Both of these groups can also utilize H2 as electron donor during sulfate reduction. Despite the prospects for chemolithoautotrophic growth on H2, most SRM require acetate in addition to CO2 for growth (Badziong et al., 1979). Nevertheless, true autotrophic growth with H2 as electron donor was discovered in some SRM (Pfennig et al., 1981; Jansen et al., 1985; Klemps et al., 1985; Brysch et al., 1987; Rozanova et al., 1988; Schauder et al., 1989). Of these facultative chemolithoautotrophic SRM all but Desulfosporosinus orientis are complete oxidizers (Brysch et al., 1987). Thereby, CO2-fixation proceeds through reverse reactions of the pathways used by SRM for acetyl-CoA oxidation during heterotrophic growth. D. hydrogenophilus assimilates CO2 via a reductive citric acid cycle (Schauder et al., 1987), while D. autotrophicum and D. orientis use the reductive acetyl-CoA pathway (Wood Ljungdahl–pathway) (Schauder et al., 1989), the only autotrophic microbial route able to simultaneously fix CO2 and yield ATP by converting acetyl-CoA to acetate (Wood et al., 1986; Fuchs, 2011). This pathway consists of two separate branches: one molecule of CO2 is reduced to carbon monoxide (CO) in the carbonyl branch and another CO2 molecule is reduced to a methyl group in the methyl branch. The acetyl-CoA is generated from the combination of the CO and methyl group with the coenzyme A (Wood et al., 1986). Recent reviews

FIGURE 1 | (A) Metabolic classification of the sulfate-reducing microorganisms (SRM) presented in this Mini-Review, whereby the review focus is on electroautotrophic SRM. (B,C) Schematic model of possible extracellular electron uptake (EEU) mechanisms in electroautotrophic SRM with (B) Direct EEU: the cathodic electrons can enter SRM cells through outer membrane (OM) multiheme cytochromes (Cyts) or via Cyt-covered nanowires (Deng et al., 2018). Cyt-rich group of SRM (Deltaproteobacteria and Nitrospirae) are characterized by periplasmatic tetraheme Type I cytochrome c3 (TpIc3) that could act as periplasmatic electron shuttle component, like cytochrome Cyc-1 in A. ferroxidans (Ishii et al., 2015) and the monoheme c-Cyt PccH in Geobacter sulfurreducens (Strycharz et al., 2011). TpIc3 serves then as electron donor for several inner membrane redox complexes that reduce the menaquinone pool or are involved in transmembrane electron transfer; and (C) Indirect EEU: SRM belonging to Cyt-poor group (Archaea and Clostridia) have no periplasmatic multiheme c-Cyts and they most probably perform a mediated electron transfer, using H2 and formate as soluble electron donors for their hydrogenases (Hase) and formate dehydrogenases (Fdh) associated to the inner membrane (IM). The abiotic electrochemical production of H2 and formate can be enhanced by extracellular Hase and Fdh, excreted by viable cells or released after microbial death (Deutzmann et al., 2015). MES, Microbial electrosynthesis; MIC, microbial induced corrosion; MQ/MQH2, menaquinone/menaquinol; TM cmplx, transmembrane complex.

give an exhaustive biochemical description of these CO2-fixation pathways (Berg, 2011; Fuchs, 2011; Schuchmann and Müller, 2014).

### Energy-Conservation and Electron Transport Pathways

In chemolithoautotrophic SRM, sulfate reduction must be coupled to energy conservation by oxidative phosphorylation. This implies an electron transport chain that allows the production of a transmembrane proton motive force for the chemiosmotic synthesis of ATP (Thauer et al., 2007; Grein et al., 2013). While APS and sulfite reduction are two strongly exoergonic reactions, the enzymes responsible, AprBA and DsrAB/DsrC, are cytoplasmic soluble reductases and, thus, cannot be directly involved in the formation of a transmembrane proton gradient. Instead, the quinone-interacting membranebound oxidoreductase complex (QmoABC) (Pires et al., 2003) and the DsrMKJOP complex (Pires et al., 2006) represent the membrane complex candidates that can act as electron donor for AprBA and DsrAB/DsrC, respectively (Ramos et al., 2012; Grein et al., 2013). Both complexes are strictly conserved across SRM (Pereira et al., 2011). Other energy-conserving membrane complexes, capable of ion translocation, are present in SRM, but they are less conserved (Pereira et al., 2011).

Due to the high variability of organic and inorganic electron donors used by SRM, there is no unifying theory for their electron transport chain. However, one classification method is based on the content of periplasmic c-type cytochromes (c-Cyt) (Rabus et al., 2015). The cytochromerich group has numerous multiheme c-Cyt and includes SRM belonging to Deltaproteobacteria (e.g., Desulfovibrio spp., Desulfobulbus spp., Desulfomicrobium spp.) and Nitrospira (e.g., Thermodesulfovibrio spp.), while the cytochrome-poor group has few or no c-Cyt and comprises Archaea (e.g., Archaeoglobus spp.) and Clostridia SRM (e.g., Desulfosporosinus spp., Desulfotomaculum spp.) (Pereira et al., 2011; Rabus et al., 2015). Cytochrome-rich SRM have soluble periplasmatic hydrogenases and formate dehydrogenases that lack an integral membrane subunit. These soluble enzymes use a periplasmatic multiheme c-Cyt, usually the tetraheme cytochrome c3 (TpIc3), as electron acceptor (Louro, 2007; da Silva et al., 2012; Romaõ et al., 2012) (**Figure 1B**). These SRM also contain a set of inner membrane redox complexes that reduce the menaquinone pool (Qrc, Nhc and Ohc) or are involved in transmembrane electron transfer (Tmc and Hmc) (Rabus et al., 2015). On the contrary, cytochrome-poor SRM have membrane-bound hydrogenases and formate dehydrogenases associated to the inner membrane through a b-type cytochrome that directly reduce the menaquinone pool (Pereira et al., 2011) (**Figure 1C**).

### Electroautotrophic SRM

The discovery of direct electron uptake capacity of some Fe(0)-corroding SRM (Dinh et al., 2004; Gu et al., 2009; Gu and Xu, 2010; Xu and Gu, 2011) suggested the researchers to start employing these microorganisms for biocathodic BES applications, turning, thus, this negative metabolic feature into positive and sustainable biotechnological solutions. However, so far only few pure culture SRM are elucidated as electroautotrophs.

In 2008, Desulfovibrio desulfuricans ATCC 27774 was shown to form an electroactive cathodic biofilm at an applied cathodic potential (Ecath) of −0.169 V vs. SHE. A stable negative current was obtained after 20 days, but lactate was supplied as carbon source, not CO2 (Cordas et al., 2008). Subsequently, other species of the genus Desulfovibrio were tested for cathodic current generation and H2 production, using bicarbonate or lactate as carbon source and Ecath that allow abiotic H2 evolution: D. paquesii and D. caledoniensis (Yu et al., 2011; Aulenta et al., 2012).

As first pure culture SRM to really show electroautotrophy, we identified Desulfosporosinus orientis and Desulfovibrio piger at Ecath = −0.31 V vs. SHE, which is much more positive than the H2 evolution redox potential at neutral conditions (E0 ′ H+/H2 = −0.41 V vs. SHE), with gaseous CO2 as sole inorganic growth substrate (Rodrigues and Rosenbaum, 2014). D. orientis is a spore-forming SRM in the class Clostridia and is able to perform anaerobic sulfate respiration but also acetogenesis. It can utilize a wide range of energy sources, such as H2/CO2, CO, formate, lactate, pyruvate, methanol, ethanol, and medium chain fatty acids (Klemps et al., 1985; Robertson et al., 2001), and different terminal electron acceptors, such as sulfate, thiosulfate, sulfite, sulfur dioxide (Cypionka and Pfennig, 1986).

D. piger is a non-spore-forming, H2-oxidizing Gram-negative SRM Deltaproteobacterium. It can oxidize organic compounds, such as ethanol, lactate, and pyruvate, incompletely to acetate. Like for the other Desulfovibrio species, autotrophic growth on CO2 was not reported before.

Desulfopila corrodens strain IS4 is the first Fe(0)-corroding SRM characterized in BES (Beese-Vasbender et al., 2015b). This Gram-negative Deltaproteobacterium was isolated from marine sediment using metallic iron as the sole electron donor. Using iron as energy source, this strain is able to perform very rapid sulfate reduction and hydrogen production compared to the conventional hydrogen-scavenging Desulfovibrio species (Dinh et al., 2004). In BES, direct electron uptake was achieved at Ecath = −0.4 V vs. SHE with CO2 in the headspace as carbon substrate (Beese-Vasbender et al., 2015b).

Very recently, electroautotrophic activity was reported also in the fully sequenced sulfate-reducing bacterium Desulfobacterium autotrophicum HRM2, using a Ecath = −0.5 V vs. SHE (Zaybak et al., 2018). This Deltaproteobacterium, isolated from marine mud, is a complete oxidizer SRM belonging to the c-Cyt rich group and harboring a bidirectional Wood-Ljungdahl pathway (Brysch et al., 1987; Strittmatter et al., 2009). D. autotrophicum HRM2 exhibited acetate bioelectrosynthesis ability, with an extremely high coulombic efficiency of 83 ± 6% (Zaybak et al., 2018).

### EXTRACELLULAR ELECTRON UPTAKE IN SRM

Little is known about the molecular mechanisms beyond extracellular electron uptake (EEU) in SRM and in electroautotrophic microorganisms in general. As for the extracellular electron transfer (EET) from microbes to the anode, direct and indirect pathways can be employed. So far, direct EEU mechanisms have been elucidated in the oxygen-reducers Shewanella oneidensis, which directly uses electrons from the cathode via the reversed anodic Mtr pathway (Ross et al., 2011), and Acidithiobacillus ferroxidans, which utilizes a cascade of outer membrane (OM) cytochrome reductases (Ishii et al., 2015). Indirect electron transfer pathway has been demonstrated for the anaerobic methanogen Methanococcus maripaludis (Choi and Sang, 2016). Here, the electron uptake seems dependent on extracellular formate dehydrogenases and hydrogenases that catalyze the cathodic production of hydrogen and formate, which act as soluble electron donors for microbial activity (Deutzmann et al., 2015).

### Indirect Mechanism

Generally, in BES for bioremediation and bioproduction a power input is given to reduce cathodic potential and drive thermodynamically unfavorable bioelectrochemical reductions. If the Ecath is more negative that the standard redox potential of the H+/H2 couple, molecular H2 is generated at the

cathode. The majority of acetogens, methanogens as well as SRM are able to use H2 as electron donor. Consequently, abiotic H2 can easily mediate the EET from cathode to hydrogenotrophic microorganisms. In addition, similar to the case of M. maripaludis, solubles enzymes, released by SRM after cell lysis, could increase the abiotic production rate of H2 and even formate by decreasing the overpotentials of these abiotic electroreduction processes (**Figure 1C**). Indeed, SRM are characterized by a high amount of soluble periplasmatic and cytoplasmatic hydrogenases and formate dehydrogenases (Rabus et al., 2006).

### Direct Mechanism

As discussed above, D. orientis, D. piger, and D. corrodens strain IS4 have shown ability to grow in cathodic environments with applied potentials too positive for the abiotic H2 evolution, suggesting a direct EEU requiring a physical interaction between the electrode and the microbial cells (**Figure 1B**).

D. piger and D. corrodens strain IS4 belong to the cytochromerich group of SRM, both having the periplasmatic TpIc3, but not D. orientis (Rabus et al., 2006). Nevertheless, the OM proteins that permit the entrance of extracellular electrons inside SRM cells and the subsequent reduction of periplasmatic redox components have yet to be elucidated. Electrochemical and infrared spectroelectrochemical analyses identified c-Cyt as redox active components associated with the OM of D. corrodens strain IS4 and are, thus, possibly involved in direct EEU (Beese-Vasbender et al., 2015b). These results are supported by the very recent study of the electron uptake mechanism of another iron-corroding SRM, Desulfovibrio ferrophilus IS5 (Deng et al., 2018). This strain was isolated from marine sediment with D. corrodens IS4 (Dinh et al., 2004). Biochemical, transcriptomic, and microscopic analyses of D. ferrophilus IS5 pointed out a high expression of different OM multiheme cytochromes in response to organic electron donor limitation. Moreover, transmission electron microscopy revealed segmented nanowire structures, strongly positive for cytochrome staining and very similar to the ones of S. oneidensis, suggesting that also D. ferrophilus IS5 can use nanowires for EEU (Deng et al., 2018).

### BES TECHNOLOGIES BASED ON ELECTROAUTOTROPHIC SRM

With growing interest in Microbial Electrolysis Cell (MEC) (Logan and Rabaey, 2012), SRM have started to be applied in biocathodic systems. This section outlines the recent applications of electroautotrophic SRM-based biocathodes, using CO2 as inorganic carbon source.

### Sulfate-Rich Waters Treatment

Sulfate-rich wastewaters require treatment before being discharged to the environment, as this anion may create acute laxative effects in humans and it may increase dissolution of metals in water resources (Gomez et al., 1995). Biological sulfate removal technologies are based on the exploitation of SRM. Sulfate-rich wastewaters are usually deficient in organic matter and, thus, external electron donors are required to achieve complete sulfate reduction in bioreactors (Liamleam and Annachhatre, 2007). While methanogens are generally more competitive to use organic electron donors, autotrophic SRM are generally more efficient in H2 utilization. However, the application of hydrogen reports several disadvantages, like cost and safety aspects of H2 storage. BES can overcome some of these limitations since the production/consumption of H2 occur in the same reactor, without H2 waste, and with operations at atmospheric temperature and pressure. Electricity-driven autotrophic sulfate-reduction has been reported by several authors (Su et al., 2012; Coma et al., 2013; Luo et al., 2014; Pozo et al., 2015, 2017a; Blázquez et al., 2017) and an overview is presented in **Table 1**. The first sulfate-reducing biocathode used a mixed culture originated from wastewater and at Ecath = −0.2 V vs. SHE a maximum sulfate reduction rate of 0.02 g L−1 day−1 was achieved (Su et al., 2012). Thereafter, many studies, mostly with Desulfovibrio, have investigated different Ecath, BES operation conditions cathodic electrode materials, inoculum source and start-up strategies (**Table 1**). To date, the highest bioelectrochemical sulfate reduction rate of 5.6 g L−1 day−1 was obtained with a mixed microbial community collected from previous sulfate-reducing BES reactors at an Ecath of −1.1 V vs. SHE (Pozo et al., 2017a).

Sulfide is the principal product of biological sulfate respiration and it may lead to significant issues such as corrosion, bad odors and human health toxicity. For real application of BES in sulfaterich water treatment, a second step of sulfide transformation to elemental sulfur should be integrated. Two recent studies have combined the bioelectroreduction of sulfate to sulfide with the recovery of elemental sulfur (S0 ) through the use of sulfuroxidizing bacteria in the anodic chamber of a separate BES, resulting in a S0 recovery of 74% (Pozo et al., 2017b), or in the same biocathodic system by using part of the anodic-produced oxygen that partially diffuse to the cathode through the ion exchange membrane (Blázquez et al., 2016).

### Hydrogen Production

H2 was the first value product generated through BES technology (Liu et al., 2005; Rozendal et al., 2006). Compared to conventional methods (dark fermentation, biophotolysis, water electrolysis and water photolysis), H2 production with MECs shows multiple advantages. Firstly, MECs can theoretically produce hydrogen with an energy input much lower than for industrial electrolyzers, 1 kWh m−3H2 (Rozendal et al., 2008) vs. 4.5–5 kWh m−3H2 (Wang et al., 2014). Secondly, no precious metals catalysts are needed since both anodic and cathodic reactions can be catalyzed by electroactive microorganisms (Jafary et al., in press). Thirdly, cathodic biocatalysts can use the CO2 originating from organic matter as inorganic carbon source for cathodic H2 production. MEC biocathodes are usually inoculated with the effluent of running BES or by directly transferring used bioanodes or biocathodes to new cathodic chambers (Hasany et al., 2016). Microbial community analysis of several H2-producing biocathodes revealed SRM as amongst the dominant bacteria (**Table 1**). SRM, indeed, have an extremely high hydrogenase activity and in sulfate limitation conditions can produce H2

TABLE 1 | Overview of mixed-community electroautotrophic SRM-based biocathodes for sulfate removal and H2 production.

Except for the work of Jafary et al. (2017), all the BES studies reported in the table operated in MEC mode.

CE, coulombic efficiency; Rext, external resistance.

fermentatively (Rabus et al., 2006). Very recently, Jafary and coworkers have purposely enriched a palm oil mill effluent sample for autotrophic SRM and then used this as inoculum source for biocathodic H2 production. The SRM enriched-biocathode was able to generate 1.85 m3 H2/ (m3 ·d) in acidic catholyte conditions (pH = 4) (Jafary et al., 2017).

It should be highlighted that the sulfate concentration in the catholyte of SRM-based MEC has to be limited not only to encourage SRM fermentative metabolism, but also to avoid the generation of a harmful off-gas mixture of H2S and H2. Researchers should start to focus their attention on the purification of the produced H2, especially in the case of mixedcommunity biocathodes.

### Microbial Electrosynthesis Enhancement

BES research on biocathodic production of alternative fuels and higher value chemicals from CO2 has caught much attention in the last years. Methane and acetate are, usually, the main products of this microbial electrosynthesis (MES), particularly in pure culture-based systems (Tremblay and Zhang, 2015). For practical implementation, the generation of molecules with higher value than acetate, such as longer carbon-chain organic compounds and alcohols, is desirable. Approaches with mixedculture MES that exploit intermediate metabolite transfer and microbial cooperation show some success to extend the product spectrum to n-butyrate, propionate, ethanol, isopropanol and caproate (Ganigué et al., 2015; Arends et al., 2017; Batlle-Vilanova et al., 2017; Jourdin et al., 2018). In addition, the low rate EEU of electroautotrophic acetogens and methanogens still constitute a big limit for application on a commercial scale. Especially in undefined mixed community biocathodes, high negative potentials are applied to allow H2-mediated bioproduction processes, resulting in high energetic efficiency losses.

Very recently, researchers have evaluated the enhancement of MES rates through syntrophic growth of SRM with acetogens or methanogens (Deutzmann and Spormann, 2017; Song et al., 2017; Xiang et al., 2017). By adding low concentration of sulfate in the catholyte of a MES, Xiang et al. enriched the biocathodic community in Desulfovibrionaceae (37%), resulting in a 2.7-fold increase in acetate production in comparison to a MES with lower abundance of SRM (Desulfovibrionaceae 7.3%). As proof of concept, the Spormann group evaluated two defined co-culture biocathodes: D. corrodens strain IS4 as high rate electron uptaking and H2 producing strain was combined with Acetobacterium woodi, as acetogenic biocatalyst, or with M. maripaludis, as methane producer (Deutzmann and Spormann, 2017). A. woodi is not able to directly consume electrons from the cathode to produce acetate (Nevin et al., 2011), but in this case the co-culture showed acetate production rate of 0.21–0.23 µmol cm−2 h −1 at Ecath = −0.4 V vs. SHE. The D. corrodens-M. maripaludis co-culture exhibited a methane production 20-times higher (0.6–1.2 µmol h−1 cm−2 ) (Deutzmann and Spormann, 2017) compared to a pure culture M. maripaludis cathode poised at −0.6 V (0.05 µmol h−1 cm−2 ) (Lohner et al., 2014). This study opens the door for the exploitation of defined co-cultures for microbial electrosynthesis of higher value chemicals from CO2. Thereby, naturally efficient electroautotrophic strains, can be coupled with acetogenic engineered strains. Importantly, this combination of an efficient cathodic EEU catalyst with an efficient bioproduction catalyst in the bulk liquid allows for a better use of the entire volume of the cathodic reactor. For the real scale-up of the MES process using SRM, researchers should consider also undesired MIC activities of these biocatalysts and, thus, avoid the use of metallic materials in the reactor design.

### CONCLUDING REMARKS AND OUTLOOK

With the aim to mitigate climate change, much research efforts have been initiated toward the development of new biotechnologies able to convert CO2-rich waste gases into valuable products. MES represents one of these technologies. Thereby, the exploitation of autotrophic SRM in CO2-based cathodic bioprocesses has just begun. With this mini-review, we highlight that SRM-based biocathodes represent a very promising technology for sustainable and environmentally friendly bioremediation and bioproduction applications. Further

### REFERENCES

understanding and characterization of these electroautotrophic biocatalysts will enable successful realization of SRM-based BES technologies. First of all, more effort should be addressed toward the screening of other Fe(0)-corroding strains, in order to likely discover SRM with superior EEU rate capacity. Moreover, the complete elucidation of the molecular mechanisms beyond the EEU transport chain will allow genetic and metabolic engineering of these biocatalysts for the incrementation of their natural electron uptake rate and to extend the product spectrum. From an engineering point of view, the most critical challenge to achieve the commercialization of BES technologies is the development of a cost-effective and scalable reactor design. The combination of the recovery of multiple value-added products could possibly help to cut down the implementation cost. The versatility of electroautotrophic SRM could positively contribute in accomplishing this goal. For example, the sulfide produced by SMR biocathodes can be exploited for the precipitation of heavy metals, combining, thus, the treatment of sulfate-rich wastewaters and metal-rich industrial effluents with the recovery of precious metals. Thus, with the highlighted new developments and emerging technologies the formerly considered destructive process of MIC might open new ways to biotechnological productions and environmental engineering strategies.

### AUTHOR CONTRIBUTIONS

VA performed the literature search for this review and prepared the first draft of the manuscript. MR discussed the content and structure and revised the review manuscript.

### ACKNOWLEDGMENTS

The authors acknowledge financial support through the Cluster of Excellence Tailor-Made Fuels from Biomass EXC 236, which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Agostino and Rosenbaum. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Highly Conductive Poly(3,4-ethylenedioxythiophene) Polystyrene Sulfonate Polymer Coated Cathode for the Microbial Electrosynthesis of Acetate From Carbon Dioxide

#### Nabin Aryal 1,2†, Pier-Luc Tremblay 1 , Mengying Xu1 , Anders E. Daugaard3 and Tian Zhang1,2 \*

#### Edited by:

Andrea Schievano, Università degli Studi di Milano, Italy

#### Reviewed by:

Pascal E. Saikaly, King Abdullah University of Science and Technology, Saudi Arabia Matteo Grattieri, University of Utah, United States

> \*Correspondence: Tian Zhang<EMAIL_ADDRESS>
#### †Present Address:

Nabin Aryal, Biological and Chemical Engineering, Anaerobic Digestion Technologies, Aarhus University, Aarhus N, Denmark

#### Specialty section:

This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Energy Research

> Received: 14 April 2018 Accepted: 03 July 2018 Published: 20 July 2018

#### Citation:

Aryal N, Tremblay P-L, Xu M, Daugaard AE and Zhang T (2018) Highly Conductive Poly(3,4-ethylenedioxythiophene) Polystyrene Sulfonate Polymer Coated Cathode for the Microbial Electrosynthesis of Acetate From Carbon Dioxide. Front. Energy Res. 6:72. doi: 10.3389/fenrg.2018.00072 1 School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan, China, 2 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark, 3 Department of Chemical and Biochemical Engineering, Danish Polymer Centre, Kongens Lyngby, Denmark

Microbial electrosynthesis (MES) is a bioelectrochemical technology developed for the conversion of carbon dioxide and electric energy into multicarbon chemicals of interest. As with other biotechnologies, achieving high production rate is a prerequisite for scaling up. In this study, we report the development of a novel cathode for MES, which was fabricated by coating carbon cloth with conductive poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) polymer. Sporomusa ovata-driven MES reactors equipped with PEDOT:PSS-carbon cloth cathodes produced 252.5 ± 23.6 mmol d−1 acetate per m2 of electrode over a period of 14 days, which was 9.3 fold higher than the production rate observed with uncoated carbon cloth cathodes. Concomitantly, current density was increased to −3.2 ± 0.8 A m−2 , which was 10.7-fold higher than the untreated cathode. The coulombic efficiency with the PEDOT: PSS-carbon cloth cathodes was 78.6 ± 5.6%. Confocal laser scanning microscopy and scanning electron microscopy showed denser bacterial population on the PEDOT:PSS-carbon cloth cathodes. This suggested that PEDOT:PSS is more suitable for colonization by S. ovata during the bioelectrochemical process. The results demonstrated that PEDOT: PSS is a promising cathode material for MES.

Keywords: microbial electrosynthesis, carbon dioxide, PEDOT:PSS, acetogens, acetate

## INTRODUCTION

Reductive bioelectrochemical reactions rely on the transfer of electrons from a cathode to a microbial catalyst for the reduction of a substrate with protons coming from an anodic reaction (Rabaey and Rozendal, 2010; Tremblay and Zhang, 2015). The substrate can be inorganic carbon molecules such as CO2 that will be reduced to multicarbon compounds or CH4 via microbial electrosynthesis (MES) (Cheng et al., 2009; Nevin et al., 2010; Lovley, 2012; Ganigué et al., 2015; Bajracharya et al., 2016, 2017; Tremblay et al., 2017). The MES process requires electric energy that could be generated by a combination of renewable sources such as wind and solar with biological

oxidation reactions at the anode (Villano et al., 2010, 2013; Tremblay and Zhang, 2015). In the actual energetic and environmental context, MES could become a flexible approach for the capture of the greenhouse gas CO2 and the storage of intermittent clean energy into compounds of interest such as biofuels and commodity chemicals (Zhang and Tremblay, 2017).

Since the beginning of the decade, multiple strategies have been deployed to increase the productivity of MES reactors. For instance, better microbial catalysts capable of synthesizing a wider range of products have been developed and electrolyte solution have been optimized (Ganigué et al., 2015; Tremblay et al., 2015; Ammam et al., 2016; May et al., 2016; Aryal et al., 2017b; Lehtinen et al., 2017; Krieg et al., 2018). Additionally, several research groups have focused their effort on the development of optimal cathode materials to establish highly efficient electron transfer to microbes (Aryal et al., 2017c). Most commonly-used materials for MES cathodes are carbon base such as graphite, carbon cloth and carbon felt as well as metals including stainless steel and nickel (Nie et al., 2013; Soussan et al., 2013; Tremblay and Zhang, 2015; Cui et al., 2017). More recently, a highly performant MES cathode made of multiwalled carbon nanotube coated onto reticulated vitreous carbon was developed (Jourdin et al., 2014, 2015, 2016). The potential of graphene, a robust, cheap and highly conductive material, was also explored for MES (Aryal et al., 2016, 2017a; Chen et al., 2016; Song et al., 2017).

The ideal cathode material for MES application should have some critical properties such as high conductivity, excellent chemical stability, high mechanical strength, good biocompatibility, high surface area and low cost (Aryal et al., 2017c). The poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) polymer and its derivatives, which have high conductivity at room temperature, are electrochemically stable, flexible and easy to process as well as relatively inexpensive could be interesting materials for the fabrication of performant MES electrodes (Groenendaal et al., 2000; Kirchmeyer and Reuter, 2005; Song et al., 2014). PEDOT:PSS consists of polycationic PEDOT chains incorporated into a polyanionic PSS matrix (Nardes et al., 2008). Combining PEDOT with PSS enables the formation of aqueous dispersion that can be cast into thin, optically transparent, conductive films. PEDOT:PSS films have been used for many applications including photovoltaics, circuits, field-effect transistors and light-emitting diodes (Chen et al., 2003; Kok et al., 2004; Ko et al., 2007; Yoo and Dodabalapur, 2007). PEDOT:PSS alone or in combination with other materials has also been employed in the fabrication of anodes to improve the performance of microbial fuel cell (MFC), a technology developed for the production of electric energy from the microbial oxidation of organic carbon (Wang et al., 2013; Chou et al., 2014; Antolini, 2015; Jiang et al., 2015; Webb et al., 2015; Pang et al., 2018).

This work reports the fabrication of a cathode made of carbon cloth coated with PEDOT:PSS. The PEDOT:PSS-carbon cloth cathode was tested in MES reactors with the well-characterized electroautotrophic bacteria Sporomusa ovata as microbial catalyst (Nevin et al., 2010). The composite cathode covered with the conductive polymer was shown to be suitable for bacterial colonization resulting in microbial acetate production from CO2 and current density nearly one order of magnitude higher than the unmodified electrode.

## EXPERIMENTAL PROCEDURE

## Bacterial Strain and Growth Condition

S. ovata DSM 2662 was ordered from the Deutsche Sammlung Mikroorganismen und Zellkulturen (DSMZ) and grown in 311 medium with 40 mM betaine as substrate under a N2:CO2 (80:20) atmosphere (Möller et al., 1984). Bacteria were successively transferred four times in autotrophic condition with H2 as the electron source and CO2 as the carbon source omitting yeast extract, betaine, casitone, sodium sulfide, and resazurin from 311 medium. Cultures from the fourth transfer were used to inoculate the cathode chamber of MES reactors. Cysteine was omitted from 311 medium during MES experiments (Aryal et al., 2017b).

### PEDOT:PSS-Carbon Cloth Electrode Fabrication

Carbon cloth (Jiangsu Tongkang Special Activated Carbon Fiber & Fabric Co., Ltd, China) was first pretreated by immersion into 3M HNO3 during 12 h before being extensively washed with ultrapure water (Aryal et al., 2016). The conductivity of carbon cloth is ca. 110 S cm−1 (Stolten et al., 2016). The acidtreated electrode was then dried under nitrogen gas. The dip and dry method described by Hou et al. (2012) was used to coat carbon cloth with PEDOT:PSS. Briefly, the carbon cloth material was dipped into an aqueous solution of commercially available PEDOT:PSS (3.0–4.0% in H2O, high-conductivity grade above 200 S cm−1 , Sigma-Aldrich, USA) for 2 h to ensure that the porous carbon substrate was completely soaked. Then, the electrode was taken out of the solution and left at room temperature in air for 15 min to remove excess PEDOT:PSS. The process was repeated a second time before drying the electrode in an oven at 105◦C for 5 h. In the last step, the PEDOT:PSS carbon cloth electrode was cleaned continuously for 15 min with running ultrapure water (Hou et al., 2012).

### Operation of MES Reactor

Dual-chambered three-electrode system bioreactors were operated at room temperature with S. ovata grown in the cathode chamber as described previously (Nevin et al., 2010; Tremblay et al., 2015). Unmodified or PEDOT:PSS-coated carbon cloth cathodes (20.25 cm2 ) and the graphite stick anode (36 cm2 ) were both immerged into 250 ml of 311 medium and were separated by a Nafion 115 ion-exchange membrane (Ion Power, Inc., New Castle, DE, USA). A platinum wire and a marine wire were used as current collector for the cathode and the anode, respectively. Platinum wires were not in contact with the electrolyte solution to avoid unwanted H2 evolution reaction. An Ag/AgCl electrode model ET072 (eDAQ, Denmark) was used as reference electrode and the cathode potential was set at −690 mV versus Standard Hydrogen Electrode (SHE) during MES with a CH Instrument potentiostat (CH Instruments, Inc, USA). The cathode potential was chosen because it works well for S. ovata-driven MES and it enables performance comparison

with other electrodes tested under the same MES operational conditions (Tremblay et al., 2015; Ammam et al., 2016; Aryal et al., 2017a,b; Lehtinen et al., 2017). After the inoculation of MES reactors with S. ovata, bacterial population were established with a N2:CO2:H2 (83:10:7) gas mix. After three fresh medium swaps, the gas mix was switched to N2:CO2 (80:20) and acetate production as well as current data started being collected (Aryal et al., 2017a). All MES experiments were done in triplicate.

### Cyclic Voltammetry

The cyclic voltammetry (CV) experiments were performed with a Gamry potentiostat (Gamry Instruments, Warminster, PA). Electrochemical data for both MES and CV experiments were analyzed with the EC-Lab v.10.2 software (BioLogic, France) as described previously (Sharma et al., 2013; Lepage et al., 2014) CV of tested electrodes was performed at a scan rate of 1 mV s−1 within a potential range of 0 to −1000mV vs. Ag/AgCl.

## High Performance Liquid Chromatography and Gas Chromatography

High Performance Liquid Chromatography (HPLC) was used for the quantification of acetate as previously described (Tremblay et al., 2015). Gas chromatography (GC) was used for the quantification of H2 in the MES reactor headspace. Briefly, gas samples were collected in N2-flushed serum bottles and H2 was measured with a Trace 1300 gas chromatograph (ThermoFisher Scientific, Denmark). Argon was the carrier gas with a HP-PLOT Molesieve column (Agilent). The GC oven temperature was 130◦C. H2 was detected with a thermal conductivity detector (TCD).

### Microscopy

Confocal Laser Scanning Microscopy (CLSM) images and Scanning Electron Microscopy (SEM) images were taken after 14 days of MES operation to study bacterial population at the surface of the different cathodes. For CLSM, S. ovata cells present on cathodes were stained with the LIVE/DEAD R BacLightTM Bacterial Viability Kit (ThermoFisher Scientific). Images were taken with a Zeiss LSM 5 Pascal microscope and analyzed with the ZEN imaging software (Zeiss, Germany) (Aryal et al., 2016). For SEM images, cathodes from MES reactors were fixed with 0.1M buffer solution at pH 7.0 containing 2.5% glutaraldehyde

FIGURE 3 | Acetate production, electron consumption and current draw with (A) unmodified carbon cloth cathode and (B) PEDOT:PSS-carbon cloth cathode. Electron consumption curves correspond to the acetate concentration in mM if all the electrons transferred were converted to acetate. Acetate production curves in mM correspond to the real progression of acetate concentration in the MES reactor detected by HPLC. Results shown for acetate production and electron consumption are in triplicate. Current draw curves are from a representative example of three replicate MES reactors.

aCathode potential set at −690 mV vs. SHE.

bS. ovata is the wild type strain DSM-2662.

cEach value is the mean and standard deviation of three replicates.

dNot detected.

eNot applicable.

for 5 h at room temperature. Fixed samples were washed with the same buffer without glutaraldehyde and then immersed successively in acetonitrile and ethanol before being dried under nitrogen as described previously (Zhang et al., 2013). SEM images were taken with a Quanta 200 FEG scanning electron microscope (FEI) at an accelerating voltage of 10 V under high vacuum condition (Aryal et al., 2016).

### Analytical Methods

Specific surface area of carbon cloth and PEDOT:PSS-carbon cloth cathodes was measured with the Brunauer–Emmett–Teller (BET) method as previously described (Poreddy et al., 2015). X-ray photoelectron spectroscopy (XPS) was performed with an ESCALAB 250Xi XPS system (ThermoFisher Scientific) with an aluminum K-alpha (1486.6 eV) source. X-ray spot area measurement was set at 500µM.

### RESULTS AND DISCUSSION

### The PEDOT:PSS-Carbon Cloth Electrode

The novel PEDOT:PSS-carbon cloth electrode tested here for MES activities was fabricated via the dip dry method (Hou et al., 2012). SEM image showed that the coating of the carbon cloth cathode electrode with the PEDOT:PSS polymer was successful (**Figure 1**). After treatment, a PEDOT:PSS layer significantly increasing surface roughness could be observed on the carbon fibers comprised in the carbon cloth electrode (**Figure 1B** inset). This layer was not present on the untreated electrode (**Figure 1A** inset). XPS also confirmed the presence of PEDOT:PSS at the surface of carbon cloth. Characteristic S(2p) peaks were observed corresponding to the sulfur signal from PEDOT at a binding energy of 164.1 and 163.1 eV and to the sulfur signal from PSS at 167.2 eV (**Figure 2A**) (Crispin et al., 2006). Electrochemical performance of the PEDOT:PSS-modified carbon cloth electrode was investigated by CV with sterile 311 medium as the electrolyte (**Figure 2B**). Interestingly, the reductive current response with the sterile medium was improved from −0.988 to −5.506 mA after coating with PEDOT:PSS. Furthermore, PEDOT:PSS increased the specific surface area of the electrode from 0.5 to 1.1 m2 g −1 . This is consistent with results published before by Guzman et al. also showing a doubled specific surface area when carbon cloth material is coated with PEDOT (Guzman et al., 2017).

### MES With PEDOT:PSS-Carbon Cloth Cathode

MES systems equipped with a PEDOT:PSS-carbon cloth cathode poised at −690 mV vs SHE had an acetate production rate of 252.3 ± 17.7 mmol m−2 d −1 (n = 3) over a period of 14 days with a current density of −3.2 ± 0.8 A m−2 and a coulombic efficiency

of 78.6 ± 5.6% (**Figure 3**,**Table 1**). Acetate production rate was 9.2-fold faster and current density was 10.7-fold higher with the modified electrode compared to the untreated electrode. No H2 was detected in the headspace of MES reactors equipped with the PEDOT:PSS-carbon cloth cathode. In combination with the good coulombic efficiency, this suggested that most electrons coming from the cathode were harvested by the microbial catalyst.

To understand further the impact of PEDOT:PSS on MES productivity, SEM and CLSM images were taken after 14 days of reactor operation. The bacterial population found on the modified cathode was significantly denser than on the untreated electrode with the presence of numerous characteristic rodshaped S. ovata cells (**Figure 4**). The higher number of bacteria attached to the PEDOT:PSS-carbon cloth cathode electrode suggested that this material is more suitable for colonization by electroautotrophic bacteria than carbon cloth. As with other MES systems, this increase in the bacterial population density may correspond to a higher number of electric contacts between microbial catalysts and the modified cathode, which is probably one of the main reasons for the observed higher rates of current consumption and acetate production (Chen et al., 2016; Aryal et al., 2017a).

Until now, the three-dimensional graphene carbon felt electrode (3D-G-CF) was the best reported cathode material for MES with wild type S. ovata as microbial catalyst and a cathode potential set at −690 mV versus SHE. The PEDOT:PSS-carbon cloth cathode performance was similar to the 3D-G-CF, which had an acetate production rate of 231.4 ± 7.4 mmol d−1 per m2 of electrode (Aryal et al., 2016). The PEDOT:PSS-carbon cloth electrode is more performant than other carbon-based cathodes tested at the same potential with S. ovata wild type including carbon cloth, carbon paper, carbon felt and reduced graphene oxide (RGO) paper (Aryal et al., 2017a). Within this group, RGO paper cathode achieved the highest acetate production rate of 168.5 ± 22.4 mmol d−1 per m2 of electrode, which was ca. 1.37 times lower than with PEDOT:PSS-carbon cloth electrode. These comparisons showed the potential of PEDOT:PSS as an electrode material for MES.

### Electrochemical Performance of the PEDOT:PSS-Carbon Cloth Biocathode

The PEDOT:PSS-modified biocathode was further characterized via CV (**Figure 5**). The cathodic current of the PEDOT:PSScarbon cloth biocathode was significantly improved compared to the unmodified biocathode, showing that the PEDOT:PSS modification increased bioelectrocatalytic activity. No reversible redox peaks were detected on the current–potential curves before and after colonization by S. ovata during MES (**Figures 2**, **5**). This suggested that no electroactive species were present in the medium or excreted by S. ovata, acting as electron shuttles between the bacterial cells and the cathodes.

### CONCLUSION

The results presented here demonstrate that PEDOT:PSS is a suitable conductive polymer for the development of performant MES biocathode. The results of acetate production as well as current consumption were improved significantly in the presence of PEDOT:PSS. S. ovata population at the surface of PEDOT:PSS

### REFERENCES

was more substantial when compared with the uncoated carbon cloth electrode, which demonstrated that PEDOT:PSS is biocompatible and suitable for the development of robust bioelectrocatalyst. Beside its good conductivity, PEDOT:PSS has other interesting properties such as low cost, good mechanical flexibility and high environmental stability that could be harnessed in the future for the fabrication of more performant and robust MES reactor (Groenendaal et al., 2000; Wang, 2009).

### AUTHOR CONTRIBUTIONS

NA, TZ, and AD designed the study. MX and NA fabricated the cathodes and did the microscopy characterization. NA performed the bioelectrochemical experiments and did the analytical chemistry. MX did the XPS analysis. NA, TZ, and P-LT analyzed the data and wrote the manuscript.

### ACKNOWLEDGMENTS

TZ acknowledges the financial support of the Chinese Thousand Talents Plan Program and the Novo Nordisk Foundation.

improved electrode design and operating conditions. Enviro. Sci. Technol. 50, 1982–1989. doi: 10.1021/acs.est.5b04431

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Aryal, Tremblay, Xu, Daugaard and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effects of Applied Potential and Reactants to Hydrogen-Producing Biocathode in a Microbial Electrolysis Cell

Swee Su Lim1,2, Byung Hong Kim2,3, Da Li 4 , Yujie Feng4 , Wan Ramli Wan Daud2 , Keith Scott 1 and Eileen Hao Yu1 \*

*1 School of Engineering, Newcastle University, Newcastle Upon Tyne, United Kingdom, 2 Fuel Cell Institute, Universiti Kebangsaan Malaysia, Bangi, Malaysia, 3 Bioelectrochemistry Laboratory, Water Environment and Remediation Research Centre, Korea Institute of Science and Technology, Bongdong-eup, South Korea, 4 State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin, China*

#### Edited by:

*Deepak Pant, Flemish Institute for Technological Research, Belgium*

#### Reviewed by:

*Jung Rae Kim, Pusan National University, South Korea Mohanakrishna Gunda, Qatar University, Qatar*

\*Correspondence: *Eileen Hao Yu<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Green and Sustainable Chemistry, a section of the journal Frontiers in Chemistry*

> Received: *21 February 2018* Accepted: *10 July 2018* Published: *15 August 2018*

#### Citation:

*Lim SS, Kim BH, Li D, Feng Y, Daud WRW, Scott K and Yu EH (2018) Effects of Applied Potential and Reactants to Hydrogen-Producing Biocathode in a Microbial Electrolysis Cell. Front. Chem. 6:318. doi: 10.3389/fchem.2018.00318* Understanding the mechanism of electron transfer between the cathode and microorganisms in cathode biofilms in microbial electrolysis cells (MECs) for hydrogen production is important. In this study, biocathodes of MECs were successfully re-enriched and subjected to different operating parameters: applied potential, sulfate use and inorganic carbon consumption. It was hypothesized that biocathode catalytic activity would be affected by the applied potentials that initiate electron transfer. While inorganic carbon, in the form of bicarbonate, could be a main carbon source for biocathode growth, sulfate could be a terminal electron acceptor and thus reduced to elemental sulfurs. It was found that potentials more negative than −0.8 V (vs. standard hydrogen electrode) were required for hydrogen production by the biocathode. In additional, a maximum hydrogen production was observed at sulfate and bicarbonate concentrations of 288 and 610 mg/L respectively. Organic carbons were found in the cathode effluents, suggesting that microbial interactions probably happen between acetogens and sulfate reducing bacteria (SRB). The hydrogen-producing biocathode was sulfate-dependent and hydrogen production could be inhibited by excessive sulfate because more energy was directed to reduce sulfate (*E* ◦ SO2− 4 /H2S = −0.35 V) than proton (*E* ◦ H +/H2 = −0.41 V). This resulted in a restriction to the hydrogen production when sulfate concentration was high. Domestic wastewaters contain low amounts of organic compounds and sulfate would be a better medium to enrich and maintain a hydrogen-producing biocathode dominated by SRB. Besides the risks of limited mass transport and precipitation caused by low potential, methane contamination in the hydrogen-rich environment was inevitable in the biocathode after long term operation due to methanogenic activities.

Keywords: hydrogen-producing biocathode, microbial electrolysis cell, electron bifurcation, sulfate reduction, bicarbonate conversion

### INTRODUCTION