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Selective enrichment establishes a stable performing community for microbial electrosynthesis of acetate from CO2 Sunil A. Patil, Jan Arends, Inka Vanwonterghem, Jarne van Meerbergen, Kun Guo, Gene Tyson, and Korneel Rabaey Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506149d • Publication Date (Web): 16 Jun 2015 Downloaded from http://pubs.acs.org on June 19, 2015
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Selective enrichment establishes a stable performing community for
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microbial electrosynthesis of acetate from CO2
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Sunil A. Patil1#*, Jan B.A. Arends1#, Inka Vanwonterghem2, Jarne van Meerbergen1, Kun
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Guo1, Gene W. Tyson2 and Korneel Rabaey1*
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1
Laboratory of Microbial ecology and Technology (LabMET), Faculty of Bioscience
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Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium 2
Australian Centre for Ecogenomics (ACE) & Advanced Water Management Centre (AWMC), The University of Queensland, Brisbane QLD 4072, Australia
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#
These authors contributed equally to this paper.
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*Corresponding Authors:
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[email protected];
[email protected] 19
Phone: + 32 (0) 9/264 59 76; Fax: + 32 (0) 9/264 62 48
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ABSTRACT
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The advent of renewable energy conversion systems exacerbates the existing issue of
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intermittent excess power. Microbial electrosynthesis can use this power to capture CO2 and
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produce multicarbon compounds as a form of energy storage. As catalysts, microbial
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populations can be used, provided side reactions such as methanogenesis are avoided. Here a
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simple but effective approach is presented based on enrichment of a robust microbial
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community via several culture transfers with H2:CO2 conditions. This culture produced
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acetate at a concentration of 1.29±0.15 gL-1 (maximum up to 1.5 gL-1; 25 mM) from CO2 at a
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fixed current of -5 Am-2 in fed-batch bioelectrochemical reactors at high N2:CO2 flow rates.
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Continuous supply of reducing equivalents enabled acetate production at a rate of 19±2 gm-2d-
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1
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compared with other unmodified carbon-based cathodes. 58±5% of the electrons was
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recovered in acetate, whereas 30±10% of the electrons was recovered in H2 as a secondary
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product. The bioproduction was most likely H2 based, however, electrochemical, confocal
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microscopy and community analyses of the cathodes suggested the possible involvement of
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the cathodic biofilm. Together, the enrichment approach and galvanostatic operation enabled
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instant start-up of the electrosynthesis process and reproducible acetate production profiles.
(projected cathode area) in several independent experiments. This is a considerably high rate
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Keywords: Electricity-driven bioproduction; methanogenesis inhibition; pre-enrichment;
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CO2; acetate; fixed current
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INTRODUCTION
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The electricity-driven production of high-value chemicals and fuels from CO21-3 and/or
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organic substrates4,5 using microorganisms is a developing technology concept for
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bioproduction and storage of excess energy as chemicals.6 It is a far less land intensive
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approach compared to a crop-based bioproduction scheme. This concept is referred to as
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microbial electrosynthesis (MES) and typically relies on the capability of microorganisms to
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grow on the cathode surfaces or in the bulk of bioelectrochemical systems (BESs) by
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consuming reducing equivalents released from the electrode as their energy source.7,8 Due to
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inadequate knowledge on microbial inoculum sources today, obtaining novel microorganisms
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for cathodic bioproduction either through screening of pure strains or through enrichment-
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selection strategies from mixed inoculum sources is of crucial importance for the
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development of MES.9 The need for good inoculum sources as well as thoroughly engineered
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reactor systems becomes clear as production rates of current systems are low (Table 1).
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For acetate bioproduction processes starting from CO2, homoacetogens are the obvious
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choice of microorganisms because of their ability to fix CO2 via the efficient Wood-
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Ljungdahl pathway.6,10 Only a few pure strains have been reported to use electricity for
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converting CO2 into valuable chemicals such as acetate.1,11 Mixed microbial inoculum sources
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have also successfully been applied for MES of mainly acetate.12-15 The use of mixed
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microbial inocula offers some advantages over pure cultures such as the possibility to operate
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reactors with limited or no sterile conditions, no risk of strain degradation, resistance to
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operational perturbations and adaptive capacity of the biocatalyst to its diverse composition.
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However, when using mixed inoculum sources the production of acetate via MES has to
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compete with the production of methane in the original inoculum. 13,16,17 The practice of using
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methanogenic inhibitors such as 2-bromoethanesulfonate can result in improvement of acetate
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production rates.13,16,17 Short-term suppression of methanogens by such inhibitors and the 4 ACS Paragon Plus Environment
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need for continued addition of such inhibitors to the reactors limits their use for long-term and
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large-scale applications.6 Moreover, delays in start-up of bioproduction (up to 40 days) have
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been reported with the use of a mixed inoculum sources directly from their natural habitat.12-14
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In most of these cases, microbial enrichments were directly achieved in BES cathodes.
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Recently, a successful pre-enrichment strategy comprising the growth of inoculum first in
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serum cultures and then in cathode chamber of BESs has been reported for enhancing the
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start-up times of autotrophic biocathodes.15 However, glucose was used as carbon source
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during these enrichment phases, which consequently affected the overall bioproduction into a
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wide variety of products.15 It is noteworthy to stress here that this particular study reported a
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generalized method for pre-enrichment using alternative electron donors to develop anaerobic
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biocathodes for electrosynthesis in BESs.
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The goal of this study was to investigate if (i) it would be possible to enrich a mixed
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microbial community from an anaerobic environment on H2:CO2 for only acetate production
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and (ii) how this enriched microbial community would behave in a galvanostastically
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operated
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bromoethanesulfonate only once in the initial phase followed by several culture transfers
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across serum flasks in quick succession to eliminate methanogenic activity. A resulting
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enriched community used in BES cathodes lead to an instant start-up of the bioproduction
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process. Different from previous studies that applied potentiostatic control for a CO2 to
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acetate process, here, the BESs were operated in galvanostatic mode to ensure a constant flux
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of electrons towards the microorganisms.5,18
cathode.
The
enrichment
approach
comprised
of
the
addition
of
2-
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MATERIALS AND METHODS
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Enrichment experiments. As a source of microbial inoculum, several mixtures of anodic
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effluent of a microbial fuel cell19 and the effluent of a lab-scale Upflow anaerobic sludge 5 ACS Paragon Plus Environment
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blanket reactor (UASB) digesting microalgae20 were used. The selection of these two
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inoculum sources was based on the theoretical possibility of the presence of the needed
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functional traits within the microbial community i.e. interaction with an electrode (anode
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biomass) and the formation of products from CO2 as a carbon source (UASB biomass). The
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enrichment experiments were conducted in serum bottles (120 mL) filled with 40 mL growth
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medium based on Leclerc et al.21 and with a H2:CO2 (70:30) headspace at 50 kPa
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overpressure (data for three enrichment cultures E1-E3; Figure S2). The composition of the
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modified growth medium is provided in Table S1. All incubations were done at 28°C on a
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rotary shaker maintained at 100 rpm. 2-bromoethanesulfonate (0.5 mM) was added only once
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in the medium during the start-up of enrichment phase to inhibit methanogenesis.22 The initial
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transfers (during the enrichment phase) were based on the experimental observations of
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acetate production (Figure S2). Subsequent quick culture transfers (1000x dilution every 2
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days for 3 consecutive cycles) were then performed at the end of the enrichment phase (i.e
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starting at day 110, Figure S2). In order to keep track of acetogenesis and methanogenesis, the
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production of VFAs and headspace gas composition and pressure were monitored by
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analysing samples every 2 days.
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Microbial electrosynthesis experiments. All MES experiments were carried out under
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galvanostatic control (Potentiostat/Galvanostat Model VMP3, Biologic Science Instruments,
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France) using custom-made glass reactors (250 mL) with five necks (Figure S1; based
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on23,24). A modified homoacetogenic medium (125 mL) lacking yeast extract, tryptone,
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resazurin and Na2S with pH 7.6±0.2 was used as production medium (referred to as catholyte)
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in the cathode chamber (Table S1). CO2 was initially supplied in the form of sodium
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bicarbonate (30 mM) as the sole carbon source in the medium. The anolyte was 0.5 M
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Na2SO4 with pH 2.5 (adjusted with 1 M H2SO4). In order to avoid limitations from anodic
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reactions, dimensionally stable anodes and abiotic conditions were used.18 To ensure similar
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start-up conditions, the enriched acetogenic culture (E3) was revived from a -80 °C stock
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culture (40% glycerol) under H2/CO2 atmosphere in serum flasks to check activity. Cathode
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chambers were inoculated to a final density of ~107 intact cells per mL. The cell count was
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determined by viability staining method using flow cytometry (Accuri C6 flow cytometer; BD
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Biosciences).25 The procedure was adjusted to 4 µM propidium iodide (PI) and incubation for
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13 min at 37°C. The cathode chamber was continuously purged with N2:CO2 (90:10) using
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gas flow meters (OMA-1, Dwyer, UK) in order to maintain anaerobic conditions in reactors.
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This also served as a constant source of CO2 and buffering agent in the medium. The effluent
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gas from the cathode chamber was sent through the anode chamber to strip O2 produced at the
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anode. Different N2:CO2 flow rates (high; >5 L d-1 and low; 5 L d-1 and R5 to R6, operated at low gas flow rate of 2 weeks under
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constant N2:CO2 flow but did not result in production or accumulation of acetate or other
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VFAs (data not shown). In order to check the efficacy of the enriched acetogenic culture for
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bioproduction of acetate from CO2 and electrical current, initially, two BESs, R1 and R2,
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were operated for three fed-batch cycles. Acetate production started immediately in both
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reactors (Figure 1). This can be attributed to the immediate activity of acetogenic
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microorganisms in CO2 reduction and also to the fact that the constant supply of current at the
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cathode. In both reactors, an increase in acetate concentration over time and eventual
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stabilization in the range of 1 to 1.5 g L-1 (16 to 25 mM) in all fed-batch cycles was observed.
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The maximum concentrations of acetate produced in R1 and R2 were 1.5 g L-1 and 1.2 g L-1
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respectively. Other VFAs such as formate, propionate and butyrate were also produced, but in
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low concentrations (˂0.06 g L-1) ( Figure S4). No ethanol was detected at this stage of the
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MES process. These results indicate the ability of the enriched acetogenic culture for the MES
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of acetate as a major product. The production of acetate in both reactors was accompanied by
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a gradual decrease in the pH of the catholyte (from 7.5 to
