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Title: Selective enrichment establishes a stable performing community for microbial

electrosynthesis of acetate from CO2

Authors: Sunil A. Patil, Jan B.A. Arends, Inka Vanwonterghem, Jarne van Meerbergen, Kun

Guo, Gene W. Tyson, Korneel Rabaey

In: Environmental Science and Technology, 49 (14) 8833-8843

Link: http://pubs.acs.org/doi/abs/10.1021/es506149d

To refer to or to cite this work, please use the citation to the published version:

Patil, S.A., Arends, J.B.A., Vanwonterghem, I., Van Meerbergen, J., Guo, K., Tyson, G.W.,

Rabaey, K. 2015. Selective enrichment establishes a stable performing community for

microbial electrosynthesis of acetate from CO2. Environmental Science & Technology,

49(14), 8833-8843. DOI: 10.1021/es506149d

Selective enrichment establishes a stable performing community for

microbial electrosynthesis of acetate from CO2

Sunil A. Patil1#*

, Jan B.A. Arends1#

, Inka Vanwonterghem2, Jarne van Meerbergen

1, Kun

Guo1, Gene W. Tyson

2 and Korneel Rabaey

1*

1Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience

Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium

2Australian Centre for Ecogenomics (ACE) & Advanced Water Management Centre

(AWMC), The University of Queensland, Brisbane QLD 4072, Australia

AUTHOR CONTRIBUTIONS

#S.A.P. and J.B.A.A. contributed equally to this paper.

ASSOCIATED CONTENT

Supporting Information available. This information is available free of charge via the

Internet at http://pubs.acs.org.

TOC Art

ABSTRACT

The advent of renewable energy conversion systems exacerbates the existing issue of

intermittent excess power. Microbial electrosynthesis can use this power to capture CO2 and

produce multicarbon compounds as a form of energy storage. As catalysts, microbial

populations can be used, provided side reactions such as methanogenesis are avoided. Here a

simple but effective approach is presented based on enrichment of a robust microbial

community via several culture transfers with H2:CO2 conditions. This culture produced

acetate at a concentration of 1.29±0.15 gL-1

(maximum up to 1.5 gL-1

; 25 mM) from CO2 at a

fixed current of -5 Am-2

in fed-batch bioelectrochemical reactors at high N2:CO2 flow rates.

Continuous supply of reducing equivalents enabled acetate production at a rate of 19±2 gm-2

d-

1 (projected cathode area) in several independent experiments. This is a considerably high rate

compared with other unmodified carbon-based cathodes. 58±5% of the electrons was

recovered in acetate, whereas 30±10% of the electrons was recovered in H2 as a secondary

product. The bioproduction was most likely H2 based, however, electrochemical, confocal

microscopy and community analyses of the cathodes suggested the possible involvement of

the cathodic biofilm. Together, the enrichment approach and galvanostatic operation enabled

instant start-up of the electrosynthesis process and reproducible acetate production profiles.

Keywords: Electricity-driven bioproduction; methanogenesis inhibition; pre-enrichment;

CO2; acetate; fixed current

INTRODUCTION

The electricity-driven production of high-value chemicals and fuels from CO21-3

and/or

organic substrates4,5

using microorganisms is a developing technology concept for

bioproduction and storage of excess energy as chemicals.6 It is a far less land intensive

approach compared to a crop-based bioproduction scheme. This concept is referred to as

microbial electrosynthesis (MES) and typically relies on the capability of microorganisms to

grow on the cathode surfaces or in the bulk of bioelectrochemical systems (BESs) by

consuming reducing equivalents released from the electrode as their energy source.7,8

Due to

inadequate knowledge on microbial inoculum sources today, obtaining novel microorganisms

for cathodic bioproduction either through screening of pure strains or through enrichment-

selection strategies from mixed inoculum sources is of crucial importance for the

development of MES.9 The need for good inoculum sources as well as thoroughly engineered

reactor systems becomes clear as production rates of current systems are low (Table 1).

For acetate bioproduction processes starting from CO2, homoacetogens are the obvious

choice of microorganisms because of their ability to fix CO2 via the efficient Wood-

Ljungdahl pathway.6,10

Only a few pure strains have been reported to use electricity for

converting CO2 into valuable chemicals such as acetate.1,11

Mixed microbial inoculum sources

have also successfully been applied for MES of mainly acetate.12-15

The use of mixed

microbial inocula offers some advantages over pure cultures such as the possibility to operate

reactors with limited or no sterile conditions, no risk of strain degradation, resistance to

operational perturbations and adaptive capacity of the biocatalyst to its diverse composition.

However, when using mixed inoculum sources the production of acetate via MES has to

compete with the production of methane in the original inoculum. 13,16,17

The practice of using

methanogenic inhibitors such as 2-bromoethanesulfonate can result in improvement of acetate

production rates.13,16,17

Short-term suppression of methanogens by such inhibitors and the

need for continued addition of such inhibitors to the reactors limits their use for long-term and

large-scale applications.6 Moreover, delays in start-up of bioproduction (up to 40 days) have

been reported with the use of a mixed inoculum sources directly from their natural habitat.12-14

In most of these cases, microbial enrichments were directly achieved in BES cathodes.

Recently, a successful pre-enrichment strategy comprising the growth of inoculum first in

serum cultures and then in cathode chamber of BESs has been reported for enhancing the

start-up times of autotrophic biocathodes.15

However, glucose was used as carbon source

during these enrichment phases, which consequently affected the overall bioproduction into a

wide variety of products.15

It is noteworthy to stress here that this particular study reported a

generalized method for pre-enrichment using alternative electron donors to develop anaerobic

biocathodes for electrosynthesis in BESs.

The goal of this study was to investigate if (i) it would be possible to enrich a mixed

microbial community from an anaerobic environment on H2:CO2 for only acetate production

and (ii) how this enriched microbial community would behave in a galvanostastically

operated cathode. The enrichment approach comprised of the addition of 2-

bromoethanesulfonate only once in the initial phase followed by several culture transfers

across serum flasks in quick succession to eliminate methanogenic activity. A resulting

enriched community used in BES cathodes lead to an instant start-up of the bioproduction

process. Different from previous studies that applied potentiostatic control for a CO2 to

acetate process, here, the BESs were operated in galvanostatic mode to ensure a constant flux

of electrons towards the microorganisms.5,18

Table 1. Comparison of reported data on microbial electrosynthesis of acetate.

#calculated per projected cathode surface; *estimated based on linear production rate and reported flow rate; n.d.: not defined; ^based on 24 h production test;

$estimated

based on the data reported in the paper for autotrophic bioproduction (stoichiometric calculations based on consumed electrons); &based on the last phase of the batch

experiments (38 days/140 days); RVC: Reticulated vitreous carbon; ~data provided by Jourdin et al., 2014

£High N2:CO2 flow rate (>5 L d

-1);

+Low N2:CO2 flow rate (<1.5 L d

-1)

Note: It is practical to consider projected surface area of the cathode for calculating rates since this is the main driver determining the reactor geometry and size. Therefore the

rates are compared considering the projected cathode surface area (in addition to volume-based rates) irrespective of the used electrode material or reactor design.

J applied/produced

(A/m2)#

Ecathode (V vs SHE)Volumetric production rate

(mM/day (g/L/day))

Surface based rate

(g/m2cathode/day)

#

Max. product titer

(g/L)

Coulombic efficiency

in acetate (%)

Graphite stick -0.208 0.17 (0.045) 1.3 0.063* 85 1

Ni-coated graphite

stick -0.63 1.13 ( 0.068) 3.38 0.094* 82 ± 14 45

Sporomusa sphaeriodes -0.017 0.01 0.06 0.003* 84 11

Morella thermoacetica -0.009 0.01 0.10 0.005* 84 11

Acetobacterium woodii Stainless steel felt -1.5 -0.690 (0.046) 12.8 0.127 81 18

Brewery wastewater sludge 4.0 (0.240) n.d. 1.71 67 (incl. H2) 13

17.25 (1.0) n.d. 10.5 69 17

-0.800 52 (3.1) n.d. 8.7 n.d.

Graphite rod ~ - 1.2 -0.600 3.6 (0.22 ) 10.8 0.35 77 ± 9.8 (incl. H2)

Domestic WWTP sludge Carbon felt -2.96 -0.903 2.35 (0.14) 10 4.7 90 16

Domestic WWTP sludge^ Carbon felt ~ 19 -0.953 6.58 (0.39) 19 0.095 15 12

Enriched culture (Sediment from a bog)$ Carbon fiber rod -0.03 ± 0.006 -0.4 0.05 (0.0037) 0.063 ± 0.008 0.02 ± 0.0025 35.2 ± 4.4 15

Carbon plate -1.76 ± 0.01 0.38 ± 0.09 (0.02 ± 0.005) 5.9 ± 1.4 4.2~ 49.5

NanoWeb RVC -37 ± 3 0.42 ± 0.06 ( 0.03 ± 0.004 ) 195 ± 30 1.65~ 78.5

58 ± 5

30 ± 10 in H2

44 ± 5

5.5 ± 0.7 in H2

This

study£

-0.590

-0.85

Electrochemical variables of cathode Acetate production

-5.0 -1.26 ± 0.08 1.0 ± 0.09 (0.06 ± 0.006) 19±1.7 1.29 ± 0.15

Microbial inoculum/Reactor operation Cathode material

15.5±0.3 1.5 ± 0.2 This

study+

n.d.Graphite granules

Adapted brewery WW sludge

28

-1.28 ± 0.03 0.83 ± 0.02 (0.05 ± 0.001)

14

Pu

re c

ult

ure

s

Reference

Graphite stick

-0.4

fed

-ba

tch

or

ba

tch

Mix

ed c

ult

ure

s

Enriched culture (Labscale anode and

algae UASB sludge)

Carbon felt

con

tin

uo

us

Pond sediments and anaerobic WWTP

sludge&

Sporomusa ovata

MATERIALS AND METHODS

Enrichment experiments. As a source of microbial inoculum, several mixtures of anodic

effluent of a microbial fuel cell19

and the effluent of a lab-scale Upflow anaerobic sludge

blanket reactor (UASB) digesting microalgae20

were used. The selection of these two

inoculum sources was based on the theoretical possibility of the presence of the needed

functional traits within the microbial community i.e. interaction with an electrode (anode

biomass) and the formation of products from CO2 as a carbon source (UASB biomass). The

enrichment experiments were conducted in serum bottles (120 mL) filled with 40 mL growth

medium based on Leclerc et al.21

and with a H2:CO2 (70:30) headspace at 50 kPa

overpressure (data for three enrichment cultures E1-E3; Figure S2). The composition of the

modified growth medium is provided in Table S1. All incubations were done at 28°C on a

rotary shaker maintained at 100 rpm. 2-bromoethanesulfonate (0.5 mM) was added only once

in the medium during the start-up of enrichment phase to inhibit methanogenesis.22

The initial

transfers (during the enrichment phase) were based on the experimental observations of

acetate production (Figure S2). Subsequent quick culture transfers (1000x dilution every 2

days for 3 consecutive cycles) were then performed at the end of the enrichment phase (i.e

starting at day 110, Figure S2). In order to keep track of acetogenesis and methanogenesis, the

production of VFAs and headspace gas composition and pressure were monitored by

analysing samples every 2 days.

Microbial electrosynthesis experiments. All MES experiments were carried out under

galvanostatic control (Potentiostat/Galvanostat Model VMP3, Biologic Science Instruments,

France) using custom-made glass reactors (250 mL) with five necks (Figure S1; based

on23,24

). A modified homoacetogenic medium (125 mL) lacking yeast extract, tryptone,

resazurin and Na2S with pH 7.6±0.2 was used as production medium (referred to as catholyte)

in the cathode chamber (Table S1). CO2 was initially supplied in the form of sodium

bicarbonate (30 mM) as the sole carbon source in the medium. The anolyte was 0.5 M

Na2SO4 with pH 2.5 (adjusted with 1 M H2SO4). In order to avoid limitations from anodic

reactions, dimensionally stable anodes and abiotic conditions were used.18

To ensure similar

start-up conditions, the enriched acetogenic culture (E3) was revived from a -80 °C stock

culture (40% glycerol) under H2/CO2 atmosphere in serum flasks to check activity. Cathode

chambers were inoculated to a final density of ~107 intact cells per mL. The cell count was

determined by viability staining method using flow cytometry (Accuri C6 flow cytometer; BD

Biosciences).25

The procedure was adjusted to 4 μM propidium iodide (PI) and incubation for

13 min at 37°C. The cathode chamber was continuously purged with N2:CO2 (90:10) using

gas flow meters (OMA-1, Dwyer, UK) in order to maintain anaerobic conditions in reactors.

This also served as a constant source of CO2 and buffering agent in the medium. The effluent

gas from the cathode chamber was sent through the anode chamber to strip O2 produced at the

anode. Different N2:CO2 flow rates (high; >5 L d-1

and low; <1.5 L d-1

) were used in order to

investigate its effect on the electron recovery in acetate and residual H2 (Table S2). The gas

flow rates were monitored and determined by using the water displacement method. The

applied gas flow rates did not result in a change in catholyte volume due to evaporation.

Electrosynthesis was facilitated by providing a constant cathodic electron supply by means

of setting a fixed current of -5 A m-2

using chronopotentiometry i.e. galvanostatic conditions.

(With the reactors used in this study, 5 A m-2

translates, assuming 100% conversion to

acetate, to ~33 g m-2

cathode d-1

. The aim behind using -5 A m-2

was not to be limited on supply

of reducing equivalents which was clearly the case as maximum production rates in our

experiments were in the range of 19 g m-2

d-1

(see results & discussion)). The use of

galvanostatic operation leads to the production of H2 at the cathode. This is of high

importance to ensure quick consumption of large currents in the framework of excess

electrical energy storage. Despite the low solubility of H2 at ambient conditions (0.002 g L-1

water,26

although in situ production of H2 may lead to supersaturation), and its retention or

distribution in reactors,27

this approach offers some interesting possibilities. Firstly, it can

enable the participation of planktonic cells in electrosynthesis processes. Secondly, the excess

H2 can be coupled to conventional biorefineries for supporting further conversions.

Consequently, it can contribute in bringing down the cost of H2 production in conventional

chemical refineries. Finally, as H2 is a gaseous product, it separates naturally and therefore

does not interfere with the downstream processing of soluble products.

These experiments were conducted in a fed-batch or batch mode at 28±2°C. In batch mode,

all removed liquid during sampling was replaced with an equal amount of anaerobic sterile

medium. In fed-batch experiments, medium exchanges after each batch cycle were

accomplished by replacing 90% of the spent medium with an equal volume of fresh medium.

The remaining 10% of the spent medium acted as an inoculum source for the subsequent

cycle. Furthermore, both anolyte and membrane were replaced during medium

replenishments. Cyclic voltammograms (CVs) were recorded at a scan rate of 1 mV s-1

at

different time points in order to get mechanistic insights into the electrochemical reactions

occurring at the cathodes. All CVs were recorded under constant N2:CO2 purging conditions.

The CV scans were started from ‘0’ V vs Ag/AgCl and were recorded for two cycles in order

to obtain reproducible and stable voltammograms. The 2nd

cycle is reported in the paper. The

most important operational parameters and main objectives of each experiment with these

reactors (R1 to R6) are presented in Table S2.

Unless stated otherwise, all potentials provided in this manuscript refer to the Ag/AgCl

reference electrode [3M KCl, (0.240 V vs. standard hydrogen electrode (SHE)); RE-1B,

Biologic Science Instruments, France]. The observations and results of MES experiments

presented in this work are based on six independent reactors (R1 to R4, operated at high gas

flow rates of >5 L d-1

and R5 to R6, operated at low gas flow rate of <1.5 L d-1

). For details

on bioelectrochemical reactor set-up, chemical analyses and microscopy please refer to

section S1.

Analyses of the enriched acetogenic culture and electrosynthetic microbial communities

The enriched acetogenic culture grown in serum bottles with CO2:H2 as well as samples (both

catholyte and biocathode) from the MES reactors (R1 to R2) taken at different time points as

elaborated further below were processed for microbial community analysis. The samples were

taken at day 19 of the batch cycle 1 from both reactors, and at day 63 and day 70 of the batch

cycle 3 from R1 and R2, respectively (Figure 1A & 1B). In addition, a cathode sample was

taken at the end of the experiment of R2 (day 110; Figure S6). For further details on

molecular biology protocols please refer section S1.

RESULTS AND DISCUSSION

Enrichment of the mixed microbial culture lacking methanogenic activity. The

enrichment of an acetogenic culture was assessed by volatile fatty acids (VFAs) and methane

production in actively growing cultures provided with CO2 and H2. Methane production was

only detected after accumulation of acetate (data for three replicate enrichment cultures E1-

E3; Figure S2), leading to the hypothesis that acetoclastic methanogenesis was the dominant

process in the start-up inoculum used. Therefore, multiple transfers in quick succession (after

2 days incubation, 1000x dilution) were made in fresh medium, which eventually resulted in a

washout of methanogenic activity (Figures S2, S3).

A representative enriched acetogenic culture (E3), which was revived from -80°C storage

and used for subsequent tests, produced independent of the enrichment phase acetate and

other VFAs, but no detectable methane at static conditions (Table S3). Methane was not

detected even after incubation of these cultures for more than a month, thus clearly indicating

the inhibition of methanogenic activity. These observations were initially confirmed by PCR

analysis (Figure S3 for methods and results) and further corroborated by analysing enriched

communities through 16S rRNA gene amplicon sequencing (Figure 5). In the presented case

the enrichment phase lasted 110 days. However, we would like to stress here that such a long

duration is not needed for the initial enrichment phase. The quick culture transfer steps

(Figure S2) can be done at the moment acetate production occurs in the serum flasks thus

shortening the enrichment phase considerably.

Microbial electrosynthesis of acetate from CO2. Abiotic (un-inoculated, connected) and

biotic (inoculated but disconnected) control experiments were conducted for >2 weeks under

constant N2:CO2 flow but did not result in production or accumulation of acetate or other

VFAs (data not shown). In order to check the efficacy of the enriched acetogenic culture for

bioproduction of acetate from CO2 and electrical current, initially, two BESs, R1 and R2,

were operated for three fed-batch cycles. Acetate production started immediately in both

reactors (Figure 1). This can be attributed to the immediate activity of acetogenic

microorganisms in CO2 reduction and also to the fact a constant current was supplied to the

cathode. In both reactors, an increase in acetate concentration over time and eventual

stabilization in the range of 1 to 1.5 g L-1

(16 to 25 mM) in all fed-batch cycles was observed.

The maximum concentrations of acetate produced in R1 and R2 were 1.5 g L-1

and 1.2 g L-1

respectively. Other VFAs such as formate, propionate and butyrate were also produced, but in

low concentrations (˂0.06 g L-1

) ( Figure S4). No ethanol was detected at this stage of the

MES process. These results indicate the ability of the enriched acetogenic culture for the MES

of acetate as a major product. The production of acetate in both reactors was accompanied by

a gradual decrease in the pH of the catholyte (from 7.5 to <6.0), attributed to the accumulation

of VFAs in these reactors. The pH drop was not associated with the diffusion of protons from

the anode chamber as confirmed with abiotic control tests conducted with electrically-

disconnected reactors at similar conditions (data not shown). No methane was detected in

these reactors for two fed-batch cycles. However, during the prolonged duration of

experiments after attaining maximum production in the third batch cycle, methane production

was observed (though below quantification limit). It is important to note that methane was not

detected at the end of the second batch cycle even after closing the gas flow for two days.

Appearance of limited methane will likely influence the overall production efficiency on the

long run. When operating the MES reactors as a batch production process with reactor or

electrode cleaning in between, the influence of methanogenesis can be further limited.

Inoculation of a new or cleaned reactor with this particular enrichment delivers reproducible

results (see Fig S9).

In case of R1, 64±13% and 19±3% of the electrons were recovered in acetate and H2,

respectively (Figure 1C). Only 3.2±1.4% electrons were recovered in other VFAs. In addition,

10±3% electrons appeared in other products determined as soluble COD. With R2, 52±9%,

37±10% and 1.4±0.2% electrons were recovered in acetate, H2 and other VFAs, respectively

(Figure 1C). In this case also a similar amount of electrons (9±4%) appeared in other soluble

products. Biomass formation most likely accounted for the rest of the electrons. The electron

recovery calculations for H2 are based on its residual concentration in the headspace of the

reactors.

Figure 1. Acetate production and pH profiles of reactors (A) R1 and (B) R2 operated with

similar experimental conditions (except N2:CO2 flow rates). Arrows indicate medium

replenishments. (C) Electron recovery (coulombic efficiency) in different products achieved

with reactors R1 and R2. Electron recovery is based on the final measured products i.e.

soluble components and H2 in the effluent gas. The data are averages and standard deviations

based on three fed-batch cycles shown in A & B.

A

C

B

R1 R20

10

20

30

40

50

60

70

80

Ele

ctr

on

rec

ov

ery

(%

)

Acetate

H2

Other VFAs

Other products

0 10 20 30 40 50 60 70

0

300

600

900

1200

15005

6

7

8

Ac

eta

te (

mg

L-1)

Time (days)

pH

0

300

600

900

1200

15005

6

7

8

Ac

eta

te (

mg

L-1)

pH

Microscopic and electrochemical analyses of the biocathodes

Reproducibility of the enriched acetogenic culture for MES process. Two additional BESs

(R3 and R4) were operated under similar conditions to assess the reproducibility of the

enriched acetogenic culture for MES of acetate. Both reactors exhibited reproducible

behaviour compared to R1 and R2 in terms of start-up time, acetate production and pH drop

(Figure 2). Up to 1.3 g L-1

(21.5 mM) and 1.2 g L-1

(20 mM) of acetate production was

achieved with R3 and R4 respectively. The gradual increase in acetate concentration was

accompanied by the increase in OD610 in these reactors (from 0.03 to 0.27 in R3 and from

0.03 to 0.31 in R4). Other VFAs, mainly butyrate up to 0.06 g L-1

, were again present in low

amounts. Electron recoveries in acetate with R3 and R4 were up to 61% and 56%

respectively. Similar to R1 and R2, a large portion of the electrons was recovered in H2 (26%

in R3 and 41% in R4). A substantial amount of the electrons was also recovered in other

products as soluble COD (13% in R3 and 12% in R4). In agreement with earlier observations,

no methane was detected in the off-gas of these reactors. When acetate production reached its

maximum, these reactors were subjected to further analyses (microscopy and CV) as will be

discussed in the following sections.

Figure 2. Acetate production and pH profiles of reactors R3 and R4. The values in boxes

represent the electron recovery in acetate (Ac-) and H2 for respective reactors. Electron

56 % in Ac-

41 % in H2

61 % in Ac-

26 % in H2

0 5 10 15 20 250

300

600

900

1200

15005

6

7

8

Ac

eta

te (

mg

L-1)

Time (days)

pH

0 5 10 15 20 250

300

600

900

1200

15005

6

7

8

Ac

eta

te (

mg

L-1)

Time (days)

pH

R3 R4

recovery is based on the final measured products i.e. soluble components and H2 in the

effluent gas.

Analyses of electrochemical processes involved in bioproduction. The abiotic control CV

recorded in growth medium (without inoculum) confirmed that no redox active components

were present in the catholyte (Figure 3; trace a). The onset potential of H2 evolution at the

abiotic carbon felt cathode was around -0.9 V. No redox peaks were observed in the CVs

recorded immediately after inoculation of these reactors with the enriched culture (data not

shown). A clear shift in the onset potential of H2 evolution potential towards a higher

potential of approximately -0.8 V was observed at the time when maximum acetate

concentration was present in the catholyte (Figure 3; trace b). The pH of the catholyte was

close to 5.6 at the time the CV was recorded (R3 in Figure 2). The electrochemical potential

of the biological redox active components is pH dependent.24

Therefore, in order to clarify the

role of pH in the shift in H2 evolution potential and also the role of soluble mediators in the

electron exchange process, another CV was recorded in a fresh catholyte (pH 7.6) with the

same biocathode (Figure 3; trace c). As evident from this CV, the potential of H2 evolution

remained at around -0.8 V thereby suggesting that the shift was not associated with the pH

decrease nor with soluble mediators. It indicates that the pH did not affect the thermodynamic

performance considerably for the minor difference between CV traces b and c. These results

are well in agreement with the recent report by LaBelle et al.28

that highlights the possible

role of an enriched electrosynthetic community in lowering the H2 evolution overpotential by

0.250 V on biocathodes.

The CV data also reveals that the current draw at -1.2 V in the case of a biocathode (-2 to -

2.5 A m-2

) is 8-10 times higher than the abiotic cathode (-0.25 A m-2

). The increased current

production in the biotic scans strongly supports the involvement of microbial catalysis in

current draw from the electrode. This can be due to an enhanced removal of H2 from the

cathode surface at a faster rate by the microorganisms. In addition, Figure 3B reveals that for

the electrosynthetic biocathode in fresh medium, one redox reaction is found between -0.05

and -0.35 V, one irreversible oxidation reaction at -0.4 V and one oxidation reaction at -0.7 V.

It seems unlikely that these redox-active moieties played any important role in the electron

uptake process as the actual potential recorded during the MES experiments was -1.35±0.1 V

(Table S2). However, substantiating this hypothesis would require further investigation. The

sustained appearance of a redox active moiety upon replacing the reactor medium by fresh

medium (Figure 3B; trace c) indicates that no soluble mediator was involved in this particular

electron exchange process.29

Similar electrochemical behaviour was also observed in the

biocathode of reactor R4 as evident from the CVs recorded at comparable conditions and time

points (Figure S5).

Figure 3. A) Cyclic voltammograms recorded on the biocathode of reactor R3: before

inoculation; abiotic cathode (black trace; a), biocathode at the peak acetate concentration

(red trace; b) and biocathode immediately after medium change or in a fresh medium (blue

trace; c). Scan rate: 1 mV s-1

. B) Zoom in of small section of the Figure A.

Microscopic analyses of the biocathodes. Biofilm growth was clearly present on the cathode

surface of R3 (Figure 4A). Individual cells can be distinguished in the cathodic biofilm

(Figure 4B). The 3D image of the biocathode shows ~75 % coverage of the electrode by

microbial biomass (Figure 4C). Biofilm growth on the cathode and lowered overpotential of

cathode indicate microorganisms played an important role in the consumption of reducing

equivalents from the cathode.

Figure 4. A) Representative confocal microscopy images of the biocathode from R3

processed at the end of the experimental run (Figure 3). B) zoom in of the red frame of image

A, C) 3D view of 16 stacks of the selected section from image A. Green fluorescence indicates

intact microbes and red fluorescence indicates microbes with damaged membranes.

Composition of the enriched acetogenic culture and electrosynthetic microbial

communities. The enriched acetogenic culture grown on H2:CO2 was dominated by

populations belonging to the order Clostridiales at a total relative abundance of up to 77%

(Figure 5). Members of the order Clostridiales are metabolically versatile and known

(homo)acetogens.30,31

Methanogens were present at a low relative abundance (<0.1%) in

enriched cultures, consistent with the lack of methane production in serum cultures. These

results demonstrate the efficacy of performing multiple culture transfers in quick succession

for promoting acetogenic activity over methanogenic activity.32

The transfer of a similar

enriched culture to medium with fructose resulted in 2.5% and 14% relative abundance of

Methanosaeta and Methanobacterium, respectively, indicating that not all methanogens were

completely removed from the enrichment; rather, they were successfully repressed. The

presence of Methanobacterium, known for its hydrogenotrophic metabolism,32

indicates that

there might still be competition for the substrates of acetogenic metabolism in the serum

bottles.

The microbial communities in the effluents of the first and third batch cycles of reactors

(R1 and R2; see methods for sampling days) were dominated by populations belonging to the

bacterial order Rhodocyclales (up to 40% relative abundance). Up to 20% of the total

population belonged to Anaerovorax and Sphaerochateaceae in the samples of the first batch

cycle. The most abundant population after cycle 3 in both R1 and R2 belonged to the genus

Azovibrio which is capable of N2-fixation.33

Remarkably, the genus Arcobacter was present in

the effluent of R2 up to 12% relative abundance. This genus has been implicated to harbour

species capable of electron transfer to an electrode.47

It is likely that Arcobacter, in this case,

does not perform a unique, essential function as it was only present in the effluent of one

reactor and not in the biofilm. Known homoacetogens34

such as Sporomusa and

Acetobacterium were present at relatively low abundance (<10%), suggesting unknown or

biofilm based acetogens may have been involved in the MES process. Marshall et al.13,17

and

LaBelle et al.28

have also observed a low relative abundance of Acetobacterium in the

supernatant compared to a relative enrichment of Acetobacterium in the biofilm on the

electrodes. A decrease in species richness from the supernatant towards the biofilm was also

observed in this work (i.e., 31% less OTUs were recovered from the biofilm compared to the

effluent of reactor R2; Figure 5).

Methanogen abundance in the effluents of R1 and R2 was low at the end of the first batch

cycle (<0.02%), and increased to 17% after cycle 3. The relative abundance of

Methanobacterium was higher in the cathode biofilm and reached up to 47% towards the end

of the experiment. However, the total biomass of methanogens was relatively low compared

to the total bacterial biomass (Max. <13% cell dry weight; calculation S1).

As the cathode samples were analysed at the end of the experiments (>3.5 months; Figure

S6), the prolonged duration of the experiments may have resulted in the revival of

methanogens and other microbial groups that were present in low abundance in the initial

enriched culture. Interestingly, Methanobacterium has been enriched on anaerobic mixed

culture biocathodes before,35

also in conjunction with Acetobacerium.13,28

In this work an

increased relative abundance of Acetobacterium from 2.7% in the suspension to 16.7% in the

biofilm was observed. Although Methanobacterium is able to produce activated acetate

(Acetyl CoA) from CO2,36

large amounts of extracellular acetate production via this pathway

is rather unlikely because this seems merely an assimilatory process rather than an acetate

excreting process such as the Wood-Ljungdahl pathway. A more plausible explanation for the

high abundance of Methanobacterium on cathodes (here without quantifiable CH4 production)

is the enhanced production of H2 as proposed for a new Methanobacterium isolate IM1.37

In

the resulting model from this theory, current is converted into molecular H2 by means of the

hydrogenases on the cell wall of Methanobacterium or by other enzymes released in the

biofilm by the methanogenic biomass44,48

which is subsequently scavenged by acetogens

before it can be transformed into CH4. The IM1 isolate has recently been shown to produce

H2 instead of methane when confronted with an excess supply of reducing equivalents on a

cathode.38

The hypothesis put forward here is plausible since (i) there is a constant, relatively

high, supply of reducing equivalents due to galvanostatic conditions, (ii) the substrate

affinities (Ks) for H2 point to a possible competitive advantage of acetogens over

hydrogenotrophic methanogens e.g. Ks of Acetobacterium bakii (4 µM)39

is similar or an

order of magnitude lower compared to hydrogenotrophic methanogens (4-120 µM),40-43

and

(iii) also non-viable cell(component)s of methanogenic microorganisms can be used to

facilitate H2 and even formate production on cathodes.38,44,48

Since the reactors were operated

with live biomass in this study, the individual influence of cellular components and intact

cells on the cathodic biocatalysis remains to be investigated.48

To the best of our knowledge,

substrate affinities or half saturation constants are not widely available to make a definite

statement on this issue. The action of the hydrogenases and other enzymes on the electrode or

the cell wall of the methanogen can, in this case, be considered as merely (bio)catalytic

instead of being linked to the metabolism of the methanogen.44,48

This hypothesis can explain

the low production of methane (below detection limit) in the effluent due to competitive

scavenging of any produced H2. The fact that Methanobacterium was enriched on the cathode

indicates that it is still able to use part of the provided energy to sustain some growth. The

proposed (bio)catalysis pathway opens a new avenue for linking non-electroactive acetogens

onto a cathode, with possible implications for research on microbial induced corrosion

processes, but needs further thorough experimental verification.

Figure 5. Microbial community composition of the enrichment cultures grown on CO2:H2 and

fructose, R1 (effluents cycle 1 & 3), R2 (effluents cycle 1 & 3) and R2 (cathode electrode).

The heatmap shows the relative abundance of operational taxonomic units (OTUs) at >1%

relative abundance in at least one of the samples. Taxonomy at the phylum level is indicated

in the left column whereas the right column shows taxonomy at the lowest classification level.

High gas flow rates favours electron recovery in H2 over acetate. During the

aforementioned four reactor runs, different N2:CO2 flow regimes were applied. These gas

flow rates influenced the electron recovery in the main products, i.e. acetate and H2 (Figure

S7). At the low gas flow rate of 5 L d-1

, more electrons (64±13%) were recovered in acetate

and comparatively less electrons were recovered in H2 (19±3%). At increased gas flow rates

(6.0 and 6.5 L d-1

), the percentage recovery of electrons in H2 increased up to 37%. It

increased further to more than 40% at 7 L d-1

flow rate. Flushing H2 at faster rates from

reactors clearly affected the electron recovery in acetate as it restricted the retention of H2 in

the cathode. Very low gas flow rate (1.25±0.25 L d-1

for R5 and R6, see below) affected

electron recovery in H2 (<10%) but no effect was observed on the electron recovery in acetate

(44±5%). These observations highlight the importance of fine-tuning the gas flow rates

depending upon the product(s) of interest. However, gas flow rates had no substantial effect

on the concentration of acetate all these reactors (R1 to R6; 1.37±0.19 g L-1

).

pH, CO2, current and growth factors did not limit acetate concentrations. With the

enriched acetogenic culture, the acetate production in all these reactors was stabilized at

maximum 1.5 g L-1

. A decrease in the pH (˂6.0) of the catholyte (Figure 1 and 2) was initially

suspected to be the reason for the limited acetate production. Therefore, two additional BESs

were started (R5 and R6) to understand the influence of pH on the maximum acetate

concentration. In reactors R5 and R6, the catholyte pH was kept in the range of 7.6 to 8.4,

which resulted in acetate concentrations (1.4 to 1.7 g L-1

; Figure S8) in a similar range as

observed in R1-R4. These observations suggest that the observed low pH was not the major

factor limiting the acetate concentrations in these reactors. However, lower pH values (~5.0)

than observed in this work have been recently reported to limit acetate production.28

Furthermore, the bicarbonate concentration in these reactors remained in the range of 2.5 to

6.5 g L-1

(Figure S8), thereby ruling out possible carbon limitations. Preliminary observations

with additional supply of current and growth factors (vitamins and trace metals) also indicated

their non-limiting role in acetate production (data not shown). Continuing the operation of

batch cycle for longer period didn’t improve acetate concentrations (Figure S6). Instead, it

lead to the transition of production from acetate to ethanol during the third batch cycle of R2.

Further analyses of the products and mechanisms in this case were beyond the scope of this

study.

Therefore, acetate concentrations achieved at the end of each batch cycle in this study are

most likely due to the reactor design, the electrode to volume ratio, and retention or

availability of H2 that acts as an energy source for acetogenic microorganisms. On the basis of

our observations, strategies such as i) increasing the cathode surface area relative to the

production medium, ii) exploiting efficient H2 producing cathode materials, iii) testing

different reactor designs, and iv) more inocula from different sources need to be investigated

to establish the key factor limiting the concentration of the desired product.

Acetate production rates. On average, 19±2 g m-2

d-1

(maximum up to 28 g m-2

d-1

) acetate

was produced with carbon felt cathodes in reactors R1 to R4 (Figure S9). As presented in

Table 1, this is the highest, reproducible acetate production rate achieved so far with

unmodified carbon-based electrodes in MES process, irrespective of the microbial inoculum

source. This can be attributed to the porous and fibrous nature of the unmodified carbon felt

cathode that allows efficient mass transfer, possess high real surface area, provides habitat for

the growth of bacteria besides its conductive and biocompatible properties. Rates are

compared based on projected cathode surface area as this will likely be the main parameter for

scale up i.e. a parallel plate reactor with as high as possible surface to volume ratio. Although

Jiang et al.12

achieved similar surface based rates (19 g m-2

d-1

), they did not show the

robustness of their enrichment, moreover the start-up time to achieve these rates was in the

order of 40 days. In addition, similar to the work of Marshall and co-workers13,17

methane was

still present in the headspace or had to be suppressed by using 2-bromoethansulfonate. A 10-

fold higher acetate production rate (195±30 g m-2

d-1

) was achieved with multiwalled-carbon-

nanotubes modified reticulated vitreous carbon (nanoweb-RVC) cathodes.14

In this case, the

high rate was attributed to the nanostructure and the high surface area to volume ratio of the

modified RVC cathodes compared to their unmodified counterparts. Such nanostructured

electrodes14,45

and the use of an enriched culture as presented here, could result in improved

start-up times consequently leading to high production rates and stable, reliable process

operation over longer periods. In case of the use of electrodes with non-defined surface area,

the performance of MES processes can be compared considering the volumetric production

rates as presented in Table 1. Volumetric production rates of acetate via H2:CO2 conversions

by a genetically modified A. woodii49

have reached to 28 g L-1

d-1

whereas via chemical

synthesis starting from methanol can reach values, up to 7200-67680 g L-1

d-1

(Lab scale

representation of commercial process; 190 °C; 22-28 barg).50

These values indicate the

possibilities for development of MES and the need for positioning of MES in an appropriate

economical niche.

On the use of enrichment approach and mixed culture inoculum sources for MES

processes. Even though mixed cultures offer some operational advantages, similar to mixed

culture fermentations their use in MES processes is associated with the production of a

mixture of products. Such issues can be addressed by using an initial enrichment strategy (as

presented here), selective removal of the target product(s)46

and/or long-term culture

adaptation towards the production of specific products.17

Our strategy of pre-enrichment of

the acetogenic microbial community under selective pressure (H2:CO2 conditions) prior to

reactor operation shows promising results as far as start-up phase and the production of

acetate from CO2 and electrical current is concerned. The presence of methanogens in the

catholyte solution and in the biofilm on the cathode was evident after prolonged reactor

operation although this did not notably affect product outcomes. Though the methanogenic

activity was irrelevant for more than two months of reactor operation, these results also

suggest that the enrichment strategy presented here to eliminate methanogens on the long

term is not successful. These findings point to a need for better strategies to prevent

methanogenesis or methanogens on the longer term. Nevertheless, based on acetate

production rates and profiles, a batch production process where production is stopped upon a

maximum concentration of product, the reactor is cleaned and a new batch is started, as is

done in industrial fermentation processes, can be envisioned. Using this approach with an

active start-up inoculum and limited methanogenic activity will ensure a dedicated process

without interference of side reactions.

Bioproduction in a cathode can be facilitated by operating reactors either in a potentiostatic

or a galvanostaic mode. In a potentiostatic mode, the potential at the working electrode is set

at more positive value (usually >–600 mV vs Ag/AgCl) depending upon the electrode

materials to avoid abiotic H2 production and to facilitate direct electron uptake process. Lower

potentials can also be set using the potentiostatic mode. The energy input in using more

positive potentials is usually lower. In a galvanostatic mode of reactor operation, as presented

in this study, the current is fixed to enable a constant flow of electrons to the microorganisms.

However, this can lead to more negative cathode potentials leading to a higher energy input.

The ultimate measure to determine the optimal strategy is the amount of energy input per

gram of product produced.

In summary, in addition to providing a promising pre-enrichment strategy for a robust

microbial community for autotrophic acetate production, these results expand the existing

knowledge on electricity-driven bioproduction processes. The enriched acetogenic culture

exhibited consistency in the production of acetate via MES in several independent

experiments. The combined use of i) a galvanostatic approach for continuous supply of

reducing equivalents and ii) an enriched culture enabled rapid start-up of electrosynthetic

biocathodes and high acetate production rates (up to 28 g m-2

d-1

). The galvanostatic approach

also leads to the production of H2, the excess of which can be used for other catalytic or

production processes. These results also suggest that a useful bioproduction system could be

developed irrespective of the presence or the absence of direct electron uptake mechanism.

Overall, these two approaches pave the way for a fast conversion of excess electrical energy

into storable energy carriers.

ACKNOWLEDGEMENTS

SAP acknowledges the financial support by the European Commission within FP-7 via Marie

Curie Intra-European fellowship (Grant agreement n° PIEF-GA-2012-326869). JBAA was

supported by the European Community Seventh Framework Program FP7/2007-2013 under

grant agreement no.226532 (enrichment phase). KR and JA (MES phase) are supported by the

European Research Council via Starter Grant ELECTROTALK. Authors are grateful to Dr.

Marta Coma for analysing alcohol samples. We thank anonymous reviewers for their critical

and constructive comments on the paper.

REFERENCES

(1) Nevin, K. P.; Woodard, T. L.; Franks, A. E.; Summers, Z. M.; Lovley, D. R. Microbial

electrosynthesis: feeding microbes electricity to convert carbon dioxide and water to

multicarbon extracellular organic compounds. mBio 2010, 00103–10.

(2) van Eerten-Jansen, M. C. A. A.; ter Heijne, A.; Buisman, C. J. N.; Hamelers, H. V. M.

Microbial electrolysis cells for production of methane from CO2: long-term performance and

perspectives. Int. J. Energ. Res. 2012, 36 (6), 809–819.

(3) Soussan, L.; Riess, J.; Erable, B.; Delia, M.-L.; Bergel, A., Electrochemical reduction of

CO2 catalysed by Geobacter sulfurreducens grown on polarized stainless steel cathodes.

Electrochem. Commun. 2013, 28, 27–30.

(4) van Eerten-Jansen, M. C. A. A.; ter Heijne, A.; Grootscholten, T. I. M.; Steinbusch, K. J.

J.; Sleutels, T. H. J. A.; Hamelers, H. V. M.; Buisman, C. J. N. Bioelectrochemical production

of caproate and caprylate from acetate by mixed cultures. ACS Sus. Chem. Eng. 2013, 1 (5),

513–518.

(5) Dennis, P. G.; Harnisch, F.; Yeoh, Y. K.; Tyson, G. W.; Rabaey, K. Dynamics of

cathode-associated microbial communities and metabolite profiles in a glycerol-fed

bioelectrochemical system. Appl. Environ. Microbiol. 2013, 79 (13), 4008–4014.

(6) Desloover, J.; Arends, J. B. A.; Hennebel, T.; Rabaey, K. Operational and technical

considerations for microbial electrosynthesis. Biochem. Soc. T. 2012, 40, 1233–1238.

(7) Rabaey, K.; Rozendal, R. A. Microbial electrosynthesis — revisiting the electrical route

for microbial production. Nat. Rev. Microbiol. 2010, 8 (10), 706–716.

(8) Lovley, D. R.; Nevin, K. P. A shift in the current: New applications and concepts for

microbe-electrode electron exchange. Curr. Opin. Biotechnol. 2011, 22 (3), 441–448.

(9) Rabaey, K.; Girguis, P.; Nielsen, L. K. Metabolic and practical considerations on

microbial electrosynthesis. Curr. Opin. Biotechnol. 2011, 22 (3), 371–377.

(10) Ragsdale, S. W.; Pierce, E. Acetogenesis and the Wood–Ljungdahl pathway of CO2

fixation. BBA - Proteins Proteom. 2008, 1784 (12), 1873–1898.

(11) Nevin, K. P.; Hensley, S. A.; Franks, A. E.; Summers, Z. M.; Ou, J.; Woodard, T. L.;

Snoeyenbos-West, O. L.; Lovley, D. R. Electrosynthesis of organic compounds from carbon

dioxide is catalyzed by a diversity of acetogenic microorganisms. Appl. Environ. Microbiol.

2011, 77 (9), 2882–2886.

(12) Jiang, Y.; Su, M.; Zhang, Y.; Zhan, G.; Tao, Y.; Li, D. Bioelectrochemical systems for

simultaneously production of methane and acetate from carbon dioxide at relatively high rate.

Int. J. Hydrogen Energ. 2013, 38 (8), 3497–3502.

(13) Marshall, C. W.; Ross, D. E.; Fichot, E. B.; Norman, R. S.; May, H. D. Electrosynthesis

of commodity chemicals by an autotrophic microbial community. Appl. Environ. Microbiol.

2012, 78 (23), 8412–8420.

(14) Jourdin, L.; Freguia, S.; Donose, B. C.; Chen, J.; Wallace, G. G.; Keller, J.; Flexer, V.

A novel carbon nanotube modified scaffold as an efficient biocathode material for improved

microbial electrosynthesis. J. Mat. Chem. A 2014, 2 (32), 13093–13102.

(15) Zaybak, Z.; Pisciotta, J. M.; Tokash, J. C.; Logan, B. E. Enhanced start-up of anaerobic

facultatively autotrophic biocathodes in bioelectrochemical systems. J. Biotechnol. 2013, 168

(4), 478–485.

(16) Su, M.; Jiang, Y.; Li, D. Production of acetate from carbon dioxide in

bioelectrochemical systems based on autotrophic mixed culture. J. Microbiol. Biotechnol.

2013 23 (8), 1140–1146.

(17) Marshall, C. W.; Ross, D. E.; Fichot, E. B.; Norman, R. S.; May, H. D. Long-term

operation of microbial electrosynthesis systems improves acetate production by autotrophic

microbiomes. Environ. Sci. Technol. 2013, 47 (11), 6023–6029.

(18) Arends, J. B. A. Optimizing the plant microbial fuel cell: diversifying applications and

product outputs. Ph.D. Dissertation, Ghent University, Gent, 2013.

(19) Clauwaert, P.; van der Ha, D.; Boon, N.; Verbeken, K.; Verhaege, M.; Rabaey, K.;

Verstraete, W. Open air biocathode enables effective electricity generation with microbial fuel

cells. Environ. Sci. Technol. 2007, 41 (21), 7564–7569.

(20) Zamalloa, C.; Boon, N.; Verstraete, W. Anaerobic digestibility of Scenedesmus

obliquus and Phaeodactylum tricornutum under mesophilic and thermophilic conditions.

Appl. Energ. 2012, 92, 733–738.

(21) Leclerc, M.; Bernalier, A.; Donadille, G.; Lelait, M. H2/CO2 metabolism in acetogenic

bacteria isolated from the human colon. Anaerobe 1997, 3 (5), 307–315.

(22) Srikanth, S.; Venkata Mohan, S.; Lalit Babu, V.; Sarma, P. N. Metabolic shift and

electron discharge pattern of anaerobic consortia as a function of pretreatment method applied

during fermentative hydrogen production. Int. J. Hydrogen Energ. 2010, 35 (19), 10693–

10700.

(23) Carmona-Martinez, A. A.; Harnisch, F.; Fitzgerald, L. A.; Biffinger, J. C.; Ringeisen,

B. R.; Schröder, U. Cyclic voltammetric analysis of the electron transfer of Shewanella

oneidensis MR-1 and nanofilament and cytochrome knock-out mutants. Bioelectrochemistry

2011, 81 (2), 74–80.

(24) Patil, S. A.; Harnisch, F.; Koch, C.; Hübschmann, T.; Fetzer, I.; Carmona-Martínez, A.

A.; Müller, S.; Schröder, U. Electroactive mixed culture derived biofilms in microbial

bioelectrochemical systems: The role of pH on biofilm formation, performance and

composition. Bioresour. Technol. 2011, 102 (20), 9683–9690.

(25) Van Nevel, S.; Koetzsch, S.; Weilenmann, H.-U.; Boon, N.; Hammes, F. Routine

bacterial analysis with automated flow cytometry. J. Microbiol. Meth. 2013, 94 (2), 73–76.

(26) Kolev, N. Solubility of O2, N2, H2 and CO2 in water. In Multiphase Flow Dynamics 4;

Springer: Berlin Heidelberg 2012; pp 209–239.

(27) Hammonds, R. Bioelectrochemical reduction of carbon dioxide to acetate using a

microbial consortium derived from the cow rumen. Master dissertation, Clemson University,

2013.

(28) LaBelle, E. V.; Marshall, C. W.; Gilbert, J. A.; May, H. D. Influence of acidic ph on

hydrogen and acetate production by an electrosynthetic microbiome. PLoS One 2014, 9 (10),

e109935.

(29) Rabaey, K.; Boon, N.; Siciliano, S. D.; Verhaege, M.; Verstraete, W. Biofuel cells

select for microbial consortia that self-mediate electron transfer. Appl. Environ. Microbiol.

2004, 70 (9), 5373–5382.

(30) Lee, C.; Kim, J.; Shin, S. G.; Hwang, S. Monitoring bacterial and archaeal community

shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewater.

FEMS Microbiol. Ecol. 2008, 65 (3), 544–554.

(31) Sundberg, C.; Al-Soud, W. A.; Larsson, M.; Alm, E.; Yekta, S. S.; Svensson, B. H.;

Sørensen, S. J.; Karlsson, A. 454 pyrosequencing analyses of bacterial and archaeal richness

in 21 full-scale biogas digesters. FEMS Microbiol. Ecol. 2013, 85 (3), 612–626.

(32) Bonin, A.; Boone, D. The order Methanobacteriales. In The Prokaryotes, Dworkin, M.;

Falkow, S.; Rosenberg, E.; Schleifer, K.-H.; Stackebrandt, E., Eds. Springer: New York 2006;

pp 231–243.

(33) Reinhold-Hurek, B.; Hurek, T. The Genera Azoarcus, Azovibrio, Azospira and

Azonexus. In The Prokaryotes, Dworkin, M.; Falkow, S.; Rosenberg, E.; Schleifer, K.-H.;

Stackebrandt, E., Eds. Springer: New York 2006; pp 873–891.

(34) Drake, H. L.; Gößner, A. S.; Daniel, S. L. Old acetogens, new light. Ann. NY. Acad. Sci.

2008, 1125 (1), 100–128.

(35) Cheng, S.; Xing, D.; Call, D. F.; Logan, B. E. Direct biological conversion of electrical

current into methane by electromethanogenesis. Environ. Sci. Technol. 2009, 43,(10), 3953–

3958.

(36) Stupperich, E.; Fuchs, G. Autotrophic acetyl coenzyme A synthesis in vitro from two

CO2 in Methanobacterium. FEBS Lett. 1983, 156 (2), 345–348.

(37) Dinh, H. T.; Kuever, J.; Muszmann, M.; Hassel, A. W.; Stratmann, M.; Widdel, F. Iron

corrosion by novel anaerobic microorganisms. Nature 2004, 427 (6977), 829–832.

(38) Beese-Vasbender, P. F.; Grote, J.-P.; Garrelfs, J.; Stratmann, M.; Mayrhofer, K. J. J.

Selective microbial electrosynthesis of methane by a pure culture of a marine lithoautotrophic

archaeon. Bioelectrochemistry 2015, 102, 50–55.

(39) Kotsyurbenko, O. R.; Glagolev, M. V.; Nozhevnikova, A. N.; Conrad, R. Competition

between homoacetogenic bacteria and methanogenic archaea for hydrogen at low

temperature. FEMS Microbiol. Ecol. 2001, 38, (2-3) 153–159.

(40) Qu, X.; Vavilin, V. A.; Mazéas, L.; Lemunier, M.; Duquennoi, C.; He, P. J.; Bouchez,

T. Anaerobic biodegradation of cellulosic material: Batch experiments and modelling based

on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis. Waste

Manage. 2009, 29 (6), 1828–1837.

(41) Jud, G.; Schneider, K.; Bachofen, R. The role of hydrogen mass transfer for the growth

kinetics of Methanobacterium thermoautotrophicum in batch and chemostat cultures. J. Ind.

Microbiol. Biotechnol. 1997, 19 (4), 246–251.

(42) Grotenhuis, J. T.; Smit, M.; Plugge, C. M.; Xu, Y. S.; van Lammeren, A. A.; Stams, A.

J.; Zehnder, A. J. Bacteriological composition and structure of granular sludge adapted to

different substrates. Appl. Environ. Microbiol. 1991, 57 (7), 1942–1949.

(43) Kristjansson, J. K.; Schönheit, P. Why do sulfate-reducing bacteria outcompete

methanogenic bacteria for substrates? Oecologia 1983, 60 (2), 264–266.

(44) Yates, M. D.; Siegert, M.; Logan, B. E. Hydrogen evolution catalyzed by viable and

non-viable cells on biocathodes. Int. J. Hydrogen Energ. 2014, 39 (30), 16841–16851.

(45) Nie, H.; Zhang, T.; Cui, M.; Lu, H.; Lovley, D. R.; Russell, T. P., Improved cathode for

high efficient microbial-catalyzed reduction in microbial electrosynthesis cells. Phys. Chem.

Chem. Phys. 2013, 15 (34), 14290–14294.

(46) Andersen, S. J.; Hennebel, T.; Gildemyn, S.; Coma, M.; Desloover, J.; Berton, J.;

Tsukamoto, J.; Stevens, C. V.; Rabaey, K. Electrolytic membrane extraction enables fine

chemical production from biorefinery sidestreams. Environ. Sci. Technol. 2014, 48 (12),

7135–7142.

(47) Fedorovich, V.; Knighton, M.C.; Pagaling, E.; Ward, F.B.; Free, A.; Goryanin, I. Novel

electrochemically active bacterium phylogenetically related to Arcobacter butzleri, isolated

from a microbial fuel cell. Appl. Environ. Microbiol. 2009, 75 (23), 7326–7334.

(48) Deutzmann, J.S.; Sahin, M.; Spormann, A.M. Extracellular enzymes facilitate electron

uptake in biocorrosion and bioelectrosynthesis. mBio 2015, 6 (2), e00496–15.

(49) Straub, M.; Demler, M.; Weuster-Botz, D.; Dürre, P. Selective enhancement of

autotrophic acetate production with genetically modified Acetobacterium woodii. J.

Biotechnol. 2014, 178, 67–72.

(50) Sunley, G.J.; Watson, D.J. High productivity methanol carbonylation catalysis using

iridium: The cativa™ process for the manufacture of acetic acid. Catal. Today, 2000, 58 (4),

293–307.

Supporting information

Selective enrichment establishes a stable performing community for

microbial electrosynthesis of acetate from CO2

Sunil A. Patil1#*

, Jan B.A. Arends1#

, Inka Vanwonterghem2, Jarne van Meerbergen

1, Kun

Guo1, Gene Tyson

2 and Korneel Rabaey

1*

1Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience

Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium

2Australian Centre for Ecogenomics (ACE) & Advanced Water Management Centre

(AWMC), The University of Queensland, Brisbane QLD 4072, Australia

#These authors contributed equally to this paper.

*Corresponding Authors:

korneel.rabaey@ugent.be; sunil.patil@ugent.be

Phone: + 32 (0) 9/264 59 76; Fax: + 32 (0) 9/264 62 48

Number of pages: 18

Number of figures: 9

Number of tables: 3

Table of contents

S-1

Table S-1

Materials and methods

Composition of modified homoacetogenic medium

S3

S6

Table S-2 Experimental details and objectives for six microbial

electrosynthesis (MES) reactors (R1 to R6) used in this study

S8

Table S-3 VFAs produced by the enriched mixed microbial culture S9

Figure S-1 Schematic overview of the bioelectrochemical system used in this

work

S10

Figure S-2 (I) Product profiles for 3 replicate cultures during the initial

enrichment phase

S11

Figure S-3

(II) Optical density (OD620) data for three cultures (E1 to E3)

PCR detection of archaea during the quick-transfer phase of the

enrichments

S12

S12

Figure S-4 Production of VFAs in reactors R1 and R2 S13

Figure S-5 Cyclic voltammograms obtained with reactor R4 S14

Figure S-6 Microbial electrosynthesis with reactor R2 with a prolonged batch

cycle

S14

Figure S-7 Influence of gas flow rates on electron recoveries in acetate and H2 S15

Figure S-8 Acetate production and bicarbonate concentration profiles for

reactors R5 and R6

S15

Figure S-9 Acetate production rates achieved with all reactors S16

Calculation S-1 Estimation of biomass in the reactor S16

References S17

S-1 MATERIALS AND METHODS

All microbiological and bioelectrochemical experiments were conducted using anaerobic

culturing techniques.1 The medium replenishments were done under constant N2:CO2 gas

flow in the cathode chamber. The tubing and connections were assembled in such a way that

allowed easy replenishments and no oxygen intrusion due to a constant overpressure in the

cathodic chamber.

Bioelectrochemical reactor set-up. The bioelectrochemical experiments were conducted

using custom-made glass reactors (250 mL) with five necks (Figure S1) and a three-electrode

arrangement consisting of a working electrode, an Ag/AgCl reference electrode and a counter

electrode. The working electrode or cathode was a carbon felt (projected surface area, 4 cm2;

Alfa Aesar, Germany) and the counter electrode or anode was a dimensionally stable

titanium-coated TaO2/IrO2 (35/65 %) mesh or rod (5x2x0.1 cm or 15x1 cm; m-270, Magneto

Special Anodes, The Netherlands). Titanium wire (Ø1 mm, Advent Research Materials, UK)

was used to establish external connections between the working electrode and the

potentiostat. Before use, the carbon felt was pre-treated with 1 M HCl and then with 1M

NaOH (each step for 24 h), and finally washed with deionized water to remove any possible

organic and metal contamination. The Brunauer–Emmett–Teller surface area of the treated

carbon felt was 2.03 m² g-1

. The counter electrode was separated from the working electrode

compartment by a cation exchange membrane (0.8 cm2; Ultrex CMI-7000, Membranes

International Inc., USA) fixed with a punctured rubber stopper and an aluminium crimp seal

in a glass serum culture tube (Balch tube2). The other end of the tube was cut-off in order to

accommodate the counter electrode. This tube was assembled in the central neck of the

reactor. The membrane was pretreated by soaking it in 4% NaCl for at least 24 h. The

working volume of the catholyte was maintained at 125 mL. Separate sampling ports were

used for liquid and gas samples.

Chemical analyses and calculations. During the enrichment phase of the autotrophic

community, VFAs were extracted by using ether extraction protocol according to Holdeman

et al.3 Extracted samples were analysed on a Gas Chromatogrpah (GC-2014, Shimadzu) with

a flame ionization detector using an Alltech EC-1000 Econo-cap column (25m x 0.53 mm).

N2 was used as the carrier gas. Headspace CH4 and CO2 were analysed during the enrichment

phase on a GC (GC 14-B, Shimadzu) with a TCD using a Hayesep Q 80-100 column (2.74m

x 2 mm) connected to an integrator (C-R8A, Shimadzu).

During MES experiments, liquid samples were taken three times a week for monitoring

VFAs, pH and bicarbonate. Optical density of these samples was measured with a UV-VIS

spectrophotometer (Isis 9000, Dr Lange, Germany) at 610 nm. The filtered samples (0.22

µM) were analysed for VFAs (formate to butyrate) using a Dionex DX 500 ion

chromatography system (Dionex-IC, Wommelgem, Belgium) equipped with an IonPac ICE-

AS1 column and an ED50 conductivity detector. The mobile phase was 0.4 mM HCl with a

flow rate of 0.8 mL min-1

. Representative liquid samples from MES reactors were also

processed for detection of other organic acids (valerate & caproate) and alcohols. For organic

acids, the samples were processed by ether extraction protocol and then analysed with GC as

explained before. For alcohols, filtered samples were analysed using a high performance

liquid chromatography (Prostar Varian, Walnut Creek, USA) equipped with Rezex ROA-

Organic Acid-H+ column. The mobile phase was 5 mM H2SO4 with a flow rate of 0.6 mL

min-1

. The detection limit for ethanol was 200 mg L-1

.

The flow rate of N2:CO2 was monitored by water displacement measurements during each

experiment. Effluent gases from reactors were analysed on a Compact GC (Global Analyser

Solutions, Breda, The Netherlands), equipped with a Molsieve 5A pre-column and Porabond

column (CH4, O2, H2 and N2) and a Rt-Q-bond pre-column and column (CO2, N2O and H2S).

Concentrations of gases were determined by means of a thermal conductivity detector. The

carrier gases were N2 for H2 channel and He for all other gases. The detection limit for these

gases was 100 ppmv (1 % = 10000 ppmv). For estimating bicarbonate concentrations as CO2,

1 mL of spent catholyte was added to 1 mL 1M H2SO4 in a vacutainer. The contents of

vacutainers were mixed vigorously for a couple of minutes and then the headspace CO2 was

determined by Compact-GC. These CO2 values were correlated with a standard curve of

CO2/bicarbonate determined with the same method in order to calculate the bicarbonate

concentration. Suspended biomass was estimated using chemical oxygen demand (COD)

analysis as the difference between total and soluble (i.e., 0.22 µm filtered) COD using

standard kits according to the manufacturer’s instructions (Nanocolor® COD, Macherey-

Nagel, Germany). pH was measured using a handheld probe (SP10T, Consort, Belgium).

Electron recoveries were calculated by dividing electrons recovered in products by total

electrons supplied as current. “Total electrons supplied” was calculated by dividing

theoretical coulombs supplied (calculated using current and time relationship) by Faraday’s

constant. For the calculations, samples from periods where acetate concentrations reached at

the maximum or a stable plateau were considered. The electron recovery calculations for H2

are based on its residual concentration in the headspace of the reactors. The production rates

of acetate were calculated by dividing its concentration by projected cathode surface area and

the time to reach this concentration.

Confocal microscopy. At the end of the MES experiments the cathode samples from two

reactors (R3 and R4) were processed for biofilm growth analysis by confocal microscopy.

The cathode samples were treated with 20 μl of live/dead staining solution (a mixture of PI

(200 μM) and SYBR green (SG; 200X diluted from stock, Invitrogen) for 30 min at 37°C.

Subsequently, these stained biocathode samples were visualized and z-stacks were captured

using a confocal laser scanning microscope (CLSM, Nikon C1, The Netherlands). The CLSM

images were processed using open source software “Fiji”.

Analyses of the enriched acetogenic culture and electrosynthetic microbial communities.

Genomic DNA was extracted from 2 mL samples using a fastprep method according to the

protocol described by Vilchez-Vargas et al.4 The concentration of extracted DNA was

measured using a nanodrop ND1000 spectrophotometer (Thermo scientific, USA) and Quant-

iT dsDNA BR Assay kits with a Qubit fluorometer (Life Technologies, VIC Australia). The

V6 to V8 region of the 16S rRNA gene was amplified using the 926F (5’-

AAACTYAAAKGAATTGRCGG-3’) & 1392R (5’-ACGGGCGGTGWGTRC-3’) primers.5

Library preparation was performed according to the Illumina workflow (#15044223 Rev.B),

with the substitution of Q5 Hot Stat High-fidelity 2x Mastermix (NEB) in standard PCR

conditions.5 Indexing of the purified DNA with unique 8bp barcodes was performed using the

Illumina Nextera XT 96 sample Index kit (Illumina, CA, USA). Indexed amplicons were

sequenced at the Australian Centre for Ecogenomics on the MiSeq Sequencing System

(Illumina, CA, USA) using the MiSeq Reagent Kit v3 (600 cycle, #MS-102-3003) for paired-

end sequencing according to the manufacturer’s protocol. The forward and reverse reads were

independently examined to confirm the taxonomic composition of the communities, however,

only the results from the forward reads were used in the subsequent analysis. Adaptor

sequences were removed and reads were hard trimmed to 250bp using Trimmomatic,6

discarding reads below 190 bp. Sequence cluster representatives were selected using the

QIIME v.1.8.0 pick_open_reference_otus.py script7 with the Greengenes database

8 to assign

taxonomy. Sequences were clustered at 97 % identity and operational taxonomic units

(OTUs) matching less than 0.01 % of total reads were discarded. An OTU table was

generated and a heatmap showing relative abundances was created in RStudio v2.15.0 with

the RColorBrewer package.9 The sequences used to create the heatmap were deposited under

accession number SRR1577794 in the National Center for Biotechnology Information (NCBI)

short read archive.

Table S-1. Composition of modified homoacetogenic medium adapted from Leclerc et al.10

K2HPO4 0.2 g L-1

NH4Cl 0.25 g L-1

KCl 0.5 g L-1

CaCl2.2H2O 0.15 g L-1

MgCl2.6H2O 0.6 g L-1

NaCl 1.2 g L-1

NaHCO3 30 mL from 84 g L-1

stock

Trace metal solution* 1 mL

Vitamin solution** 2.5 mL

Tungstate-selenium solution*** 0.1 mL

*Composition of trace metal solution (g L-1

)11

Nitrilotriacetic acid (dissolve with KOH; pH 6.5) 1.5

Mg2Cl2.6H2O 3.0

MnCl2.2H2O 0.5

NaCl 1

FeCl2 0.1

CoCl2 0.1

CaCl2.2H2O 0.1

ZnCl2 0.1

CuCl2 0.01

AlCl3.6H2O 0.01

H3BO3 0.01

Na2MoO4.2H2O 0.01

**Composition of vitamin solution (mg L-1

)12

Sodium ascorbate 10

Biotin 4

Folic acid 4

Pyridoxine hydrochoride 20

Thiamine hydrocloride 10

Riboflavin 10

Nicotinic acid 10

DL-calcium pantothenate 10

Vitamin B12 0.2

p-aminobenzoic acid 10

Lipoic(thioctic) acid 10

Myo-inositol 10

Choline chloride 10

Niacinamide 10

Pyridoxal hydrochloride 10

***Composition of tungstate-selenium solution:

0.1mM Na2WO4 + 0.1mM Na2SeO3 in 20mM NaOH

Table S-2. Experimental details and objectives for six microbial electrosynthesis (MES)

reactors (R1 to R6) used in this study.

Reactors R1 R2 R3 R4 R5 R6

Ex

per

imen

tal

det

ail

s

Gas flow rate# (L d

-1) 5 6.5 6 7 1.25±0.25

Reported

experimental

duration (days)

63 72 24 24 36 38

No. of batch cycles 3 3 1 1 1 1

Catholyte pH (start-

up)

7.57 7.50 7.69 7.69 7.78 7.79

Experimental aims Production profiles,

Production rates,

Microbial

community analysis

Testing

reproducibility,

Electrochemical &

microscopic analyses

of biocathodes

Production capacity,

Investigate factors

limiting product

concentrations and rates

Cathode potential* -1.2±0.2 -1.2±0.2 -1.35±0.1 -1.3±0.1 -1.26±0.1 -1.3±0.08

#R1 to R4 were operated at high gas flow rates (>5 L d-1

). It is important to stress that

different gas flow rates didn’t affect the acetate concentrations in these reactors. However, as

discussed in results section in the main paper it affected the electron recoveries in acetate and

H2. For reactors R5 and R6, very low gas flow rates (>1.5 L d-1

) were used for checking its

influence on electron recovery.

* V (vs. Ag/AgCl, 3 M KCl) during MES experiments. The slight differences in measured

potential values can be attributed to real-time pH of the medium, more specifically close to

the electrode surfaces. Furthermore, the data for R1 and R2 is based on three batch cycles,

whereas for other reactors it is based on a single batch cycle. The long-term operation of these

reactors likely resulted in a decrease in the overpotential at biocathodes as also indicated by

the CV scans.

Note: The pH of the anolyte remained in the range of 1.75±0.75 during these experiments.

Table S-3. VFAs produced by the representative enriched mixed microbial culture (E3,

grown as static culture at 30 °C) in serum bottles with CO2:H2 growth conditions. The

analysis was conducted at different time intervals during incubation. No methane production

was observed with the enriched culture. Data is presented for the representative culture.

VFAs (mg/L) Day 3 Day 6 Day 16 Day 31

Formate 65 89 594 587

Acetate 602 1488 1923 1850

Propionate 7 106 135 160

Butyrate 46 343 730 743

Methane - - - -

Figure S-1. Schematic overview of the bioelectrochemical system used in this work (design

based on13, 14

). (RE/WE/CE: reference/working/counter electrode; CEM: cation exchange

membrane).

Figure S-2. I) Product profiles for three replicate cultures (denoted as E1, E2 and E3) during

the initial enrichment phase. A) Acetate concentration, B) Methane concentration in the

headspace, C) close-up view of B. Vertical dotted lines indicate a culture transfer step to fresh

medium. The earlier transfers (during enrichment phase until day 110) were based on the

experimental observations of acetate production. The quick culture transfers (every 2-3 days)

were done at the end of the enrichment phase (i.e starting at day 110) when it became clear

that methanogenic activity was still present in the culture flasks.

C

B

A

0 20 40 60 80 100 120

0.0

0.4

0.8

1.2

1.6

2.0

CH

4 (

mL

@ S

TP

)

Time (days)

0 20 40 60 80 100 120

0

2

4

6

8

10

12

14

CH

4 (

mL

@ S

TP

)

0 20 40 60 80 100 120

0

200

400

600

800

1000

1200

Aceta

te (

mg

/L)

E1 E2 E3A

0 20 40 60 80 100 1200.0

0.1

0.2

0.3

0.4

0.5

OD

62

0

Time (days)

E1

E2

E3

Figure S-2. II) Optical density (OD620) data for three replicate cultures (E1 to E3) during the

initial enrichment phase (data shown from transfer cycle 3). OD of 200 µl sample from serum

flasks was determined in a microtiter plate at 620 nm using a Tecan Sunrise absorbance

Reader with XFluor4 v.4.51 software (Tecan, Austria). Actively growing cultures were

distinguished by observing an increase in absorbance.

Figure S-3. PCR detection of archaea during the quick-transfer phase of the enrichments in

the serum bottles (S2). Odd numbers are samples taken before transfer, even numbers after

transfer. Archaeal PCR was conducted using primerset GC-ARCH 915/ Uni-B-rev according

to Yu et al.15

Figure S-4. Production of volatile fatty acids (VFAs) in reactors R1 and R2 operated at

similar experimental conditions (except N2:CO2 flow rates). Arrows indicate medium

replenishments.

0 10 20 30 40 50 60 70

0

300

600

900

1200

1500

VF

As

(m

g/L

)

Time (days)

Acetate

Formate

Butyrate

Propionate

R2

0 10 20 30 40 50 60 70

0

400

800

1200

1600

VF

As

(m

g/L

)

Time (days)

Acetate

Formate

Butyrate

Propionate

R1

Figure S-5. Cyclic voltammograms obtained with reactor R4: before the inoculation; abiotic

cathode (black trace; a), biocathode at the peak acetate production (red trace; b) and

biocathode immediate after medium change/in a fresh medium (blue trace; c). Scan rate 1

mV/s. Inset shows the zoom in of small section of the main figure.

0 20 40 60 80 100 120

0

200

400

600

800

1000

1200

Ace

tate

(m

g/L

)

Time (days)

50 to 60 mg/L

EtOH

Figure S-6. Microbial electrosynthesis with reactor R2 with a prolonged batch cycle, showing

the transition of production from acetate to ethanol during the third batch cycle (box with

dotted lines). Arrows indicate medium replenishments.

-0.8 -0.6 -0.4 -0.2 0.0-0.2

0.0

0.2

0.4

c

b

j (A

/m2)

E (V)

a

-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0

-4

-3

-2

-1

0

1

E (V vs. Ag/AgCl, 3M KCl)

j (A

/m2)

a Abiotic cathode

b Biocathode at peak acetate production

c Biocathode in fresh medium

a

b

c

5.0 5.5 6.0 6.5 7.00

20

40

60

80

Acetate

H2

N2:CO

2 flow rate (L d

-1)

Ele

ctr

on

re

co

ve

ry (

%)

Figure S-7. Influence of gas flow rates on the electron recoveries in acetate and H2 as

determined over R1-R4. The data for R1 & R2 (flow rates: 5.0 and 6.5 L d-1

) are averages and

standard deviations based on three fed-batch cycles shown in Figure 1.

Figure S-8. Acetate production and bicarbonate concentration profiles for reactors R5 and

R6. The pH in both reactors was maintained in the range of 8.0±0.4 by dosing 1N NaOH

and/or 1 N HCl as needed.

R6

0 10 20 30 400

300

600

900

1200

1500

1800

Time (days)

Ac

eta

te (

mg

L-1)

0

2

4

6

8

Acetate

HCO3

-

HC

O3

- (g

L-1)

0 10 20 30 400

300

600

900

1200

1500

1800

Acetate

HCO3

-

Time (days)

Ace

tate

(m

g L

-1)

0

2

4

6

8

HC

O3

- (g

L-1)

R5

R5

to R

6 (A

vg)

R1

to R

4 (A

vg)

R1

R2

R3

R4

R5

R6

0 5 10 15 20 25 30

Acetate production rate (g m-2 d

-1)

Figure S-9. Acetate production rates achieved with reactors R1 to R6. The rates are

calculated relative to the projected cathode surface area and based on the difference in

concentration and time between each sampling event. The data for R1 and R2 is based on the

production rates of three batch cycles calculated separately whereas the data for other reactors

(R3 to R6) is based on a single batch production cycle. Experiments with R1 to R4 were

conducted at high N2:CO2 flow rates (>5 L d-1

) and with R5 to R6 at low gas flow rates (<1.5

L d-1

). Multiply values with 0.0004 m2/0.125 L to obtain production rates in terms of g L

-1 d

-1.

The rates are calculated until maximum acetate concentration is reached.

Calculation S-1: Estimation of biomass in the reactor

Based on confocal analysis of the biofilm (thickness; ~20 µm, coverage 75%), relative

abundance of methanogens in the biofilm (~45%), a total outer electrode surface area of 10

cm2 (rectangle of 2.5*1.6*0.25 cm) and the example provided by Arends and Verstraete

16 an

approximation of the total methanogenic biomass in the reactor can be made. This

approximation is about 1.35 x 108 methanogenic cells in the biofilm.

Over the total reactor operation about 20 mgCOD L-1

could be quantified as suspended

COD in the reactor effluent which can be interpreted as biomass. Using the numbers and

uncertainties provided by Gaudy et al.17

and verified by Heddle and Tavener,18

the percentage

of methanogens of the total biomass max. 13%. However, this calculation is based on the

assumption that all unidentified COD is active biomass therefore, the number might still be

lower.

REFERENCES

(1) Plugge, C. M. Anoxic Media Design, Preparation, and Considerations. In Methods in

Enzymology; Jared, R. L., Ed.; Academic Press: 2005; Vol. 397, pp 3–16.

(2) Balch, W. E.; Wolfe, R. S. New approach to the cultivation of methanogenic bacteria:

2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium

ruminantium in a pressureized atmosphere. Appl. Environ. Microbiol. 1976, 32 (6), 781–791.

(3) Holdeman, L. V.; Cato, E. P.; Moore, W. E. C. Anaerobe Laboratory Manual 4th ed.;

Blacksburg, VA: Virginia Polytechnic Institute and State University: 1977.

(4) Vilchez-Vargas, R.; Geffers, R.; Suárez-Diez, M.; Conte, I.; Waliczek, A.; Kaser, V. S.;

Kralova, M.; Junca, H.; Pieper, D. H. Analysis of the microbial gene landscape and

transcriptome for aromatic pollutants and alkane degradation using a novel internally

calibrated microarray system. Environ. Microbiol. 2013, 15, (4), 1016–1039.

(5) Vanwonterghem, I.; Jensen, P. D.; Dennis, P. G.; Hugenholtz, P.; Rabaey, K.; Tyson,

G. W. Deterministic processes guide long-term synchronised population dynamics in replicate

anaerobic digesters. ISME J. 2014, 1–14.

(6) Bolger, A. M.; Lohse, M.; Usadel, B., Trimmomatic: A flexible trimmer for Illumina

Sequence Data. Bioinformatics 2014. doi:10.1093/bioinformatics/btu170

(7) Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.; Costello,

E. K.; Fierer, N.; Peña, A. G.; Goodrich, J. K.; Gordon, J. I.; Huttley, G. A.; Kelley, S. T.;

Knights, D.; Koenig, J. E.; Ley, R. E.; Lozupone, C. A.; McDonald, D.; Muegge, B. D.;

Pirrung, M.; Reeder, J.; Sevinsky, J. R.; Turnbaugh, P. J.; Walters, W. A.; Widmann, J.;

Yatsunenko, T.; Zaneveld, J.; Knight, R. QIIME allows analysis of high-throughput

community sequencing data. Nat Methods 2010, 7, 335–336.

(8) DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E. L.; Keller, K.;

Huber, T.; Dalevi, D.; Hu, P.; Andersen, G. L. Greengenes, a chimera-checked 16S rRNA

gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 2006, 72,

5069–5072.

(9) Neuwirth, E. RColorBrewer: ColorBrewer palettes, 2011.

(10) Leclerc, M.; Bernalier, A.; Donadille, G.; Lelait, M., H2/CO2 metabolism in

acetogenic bacteria isolated from the human colon. Anaerobe 1997, 3 (5), 307–315.

(11) Balch, W. E.; Fox, G., E.; Magrum, L. J.; Woese, C. R.; Wolfe, R. S. Methanogens:

reevaluation of a unique biological group Microbiol. Rev. 1979, 43 (2), 260–296.

(12) Greening, R. C.; Leedle, J. A. Z. Enrichment and isolation of Acetitomaculum

ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen. Arch. Microbiol.

1989, 151 (5), 399–406.

(13) Patil, S. A.; Harnisch, F.; Koch, C.; Hübschmann, T.; Fetzer, I.; Carmona-Martínez,

A. A.; Müller, S.; Schröder, U. Electroactive mixed culture derived biofilms in microbial

bioelectrochemical systems: The role of pH on biofilm formation, performance and

composition. Bioresour. Technol. 2011, 102 (20), 9683–9690.

(14) Carmona-Martinez, A. A.; Harnisch, F.; Fitzgerald, L. A.; Biffinger, J. C.; Ringeisen,

B. R.; Schröder, U., Cyclic voltammetric analysis of the electron transfer of Shewanella

oneidensis MR-1 and nanofilament and cytochrome knock-out mutants. Bioelectrochemistry

2011, 81 (2), 74–80.

(15) Yu, Z.; García-González, R.; Schanbacher, F. L.; Morrison, M. Evaluations of

different hypervariable regions of archaeal 16s rrna genes in profiling of methanogens by

archaea-specific pcr and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol.

2008, 74 (3), 889–893.

(16) Arends, J. B. A.; Verstraete, W. 100 years of microbial electricity production: three

concepts for the future. Microb. Biotechnol. 2012, 5 (3), 333–346.

(17) Gaudy, A. F.; Bhatla, M. N.; Gaudy, E. T., Use of chemical oxygen demand values of

bacterial cells in waste-water purification. Appl. Microbiol. 1964, 12 (3), 254–260.

(18) Heddle, J. F.; Tavener, A., Evaluation of the total carbonaceous biochemical oxygen

demand method, using a compensated recording respirometer. Appl. Environ. Microbiol.

1981, 41 (3), 580–584.