RSC Advances PAPER View Article Online View Journal | View Issue Composition and role of the attached and planktonic microbial communities in mesophilic Cite this: RSC Adv.,2018,8,3069 and thermophilic xylose-fed microbial fuel cells† a b a a Paolo Dess`ı, * Estefania Porca, Johanna Haavisto, Aino-Maija Lakaniemi, Gavin Collins b and Piet N. L. Lens ac A mesophilic (37 C) and a thermophilic (55 C) two-chamber microbial fuel cell (MFC) were studied and compared for their power production from xylose and the microbial communities involved. The anode- attached, membrane-attached, and planktonic microbial communities, and their respective active subpopulations, were determined by next generation sequencing (Illumina MiSeq), based on the presence and expression of the 16S rRNA gene. Geobacteraceae accounted for 65% of the anode- attached active microbial community in the mesophilic MFC, and were associated to electricity generation likely through direct electron transfer, resulting in the highest power production of À3 À3 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 1.1 W m . A lower maximum power was generated in the thermophilic MFC (0.2 W m ), likely due to limited acetate oxidation and the competition for electrons by hydrogen oxidizing bacteria and hydrogenotrophic methanogenic archaea. Aerobic microorganisms, detected among the membrane- attached active community in both the mesophilic and thermophilic MFC, likely acted as a barrier for oxygen flowing from the cathodic chamber through the membrane, favoring the strictly anaerobic Received 10th November 2017 exoelectrogenic microorganisms, but competing with them for xylose and its degradation products. This Accepted 8th January 2018 study provides novel information on the active microbial communities populating the anodic chamber of DOI: 10.1039/c7ra12316g mesophilic and thermophilic xylose-fed MFCs, which may help in developing strategies to favor rsc.li/rsc-advances exoelectrogenic microorganisms at the expenses of competing microorganisms. This article is licensed under a 1. Introduction electrons exogenously to the solid anode electrode. Electrons can be transferred to the anode essentially through three Open Access Article. Published on 15 January 2018. Downloaded 9/30/2021 9:34:23 PM. The microbial fuel cell (MFC) is an emerging technology for the mechanisms: short range, long range, and mediated electron direct bioconversion of chemical energy of organic substrates to transfer (for reviews, see Kumar et al.2 and Kalathil et al.3). Some electrical energy. MFCs consist of two electrodes (anode and microorganisms, such as Geobacter sulfurreducens, can transfer cathode) connected through an external electrical circuit. The electrons to a surface directly via redox-active proteins present anode acts as electron acceptor in the bioelectrochemical redox on the outer surface of their cell membrane, such as c-type reactions of microbial metabolism, whereas the cathode acts as cytochromes, or via conductive pili called nanowires.4,5 G. sul- electron donor for biotic or abiotic reactions. The combination furreducens develops multi-layer structured biolms, in which of anodic and cathodic reactions creates a potential difference nanowires connect the different cells, enabling the electron between the electrodes which drives the electrons to migrate transfer to the anode.6 Mediators, in their oxidized form, from the anode to the cathode, thus generating electrical penetrate the microbial cell and become reduced during current (for a review, see Butti et al.1). cellular metabolism. They then diffuse out of the cell and Biological electricity production in MFCs requires microor- release the electrons at the anode, becoming oxidized again and ganisms capable to oxidize the substrates and transfer the thus reusable.5 Some species, such as Pseudomonas, produce mediators such as pyocyanin endogenously.7 Once mediators are produced, also other microorganisms present in the mixed aLaboratory of Chemistry and Bioengineering, Tampere University of Technology, P.O. culture system can use them to transfer the electrons to the Box 541, FI-33101 Tampere, Finland. E-mail: paolo.dessi@tut.; Tel: +358 417239696 anode.8 bMicrobial Communities Laboratory, School of Natural Sciences, National University Pure cultures of electrochemically active microorganisms, of Ireland Galway, University Road, Galway, H91 TK33, Ireland such as Geobacter sp.9–11 and Shewanella sp.,12,13 have shown cUNESCO-IHE, Institute for Water Education, Westvest 7, 2611AX Del,The power production from simple substrates such as volatile fatty Netherlands – † Electronic supplementary information (ESI) available. See DOI: acids and sugars at mesophilic conditions (25 37 C) and – 10.1039/c7ra12316g neutral pH (6.8 7.3). Mixed cultures are more practical for This journal is © The Royal Society of Chemistry 2018 RSC Adv.,2018,8,3069–3080 | 3069 View Article Online RSC Advances Paper wastewater treatment, as they contain a consortium of hydro- growing (i) as anodic biolm, (ii) in suspended form in the lytic, fermentative and electroactive microorganisms able to anodic solution (planktonic), and (iii) as biolm on the produce electricity from complex substrates.9 However, due to membrane of a mesophilic (37 C) and a thermophilic (55 C) the competition for electron donor with non-exoelectrogenic xylose-fed MFC. Both presence and expression of the 16S rRNA microorganisms such as methanogenic archaea,14 power gene were determined with the aim to investigate both the production can remain low, and operational conditions must be composition of the overall microbial community and the active optimized to favor exoelectrogenic microorganisms. Catal subpopulation. Power production, as well as xylose and et al.15 compared electricity production from 12 mono- metabolite concentration proles were also analyzed to deter- saccharides present in lignocellulosic biomass, including mine the possible differences in the electricity production pentoses and hexoses, in a mesophilic (30 C) MFC inoculated pathways at 37 and 55 C. with a mixed culture adapted to acetate. Xylose resulted in the highest potential for electricity production over the other 2. Experimental hexoses and pentoses tested. Thermophilic electricity production could be advantageous 2.1 Source of anodic microorganisms because of the high rate of biochemical reactions, and thus high In order to ensure a large variety of microbial species capable of electron production rates, of thermophilic microorganisms.16 living under a broad temperature range and degrading xylose, MFCs have been operated at temperatures up to 98 C.17 two inocula (15 mL each) were mixed and provided to each However, although over 20 species of microorganisms, mainly MFC. The rst one was activated sludge from a municipal belonging to the Proteobacteria phylum, have been reported to wastewater treatment plant (Viinikanlahti, Tampere, Finland), produce electricity under mesophilic conditions, the number of which has shown potential for anaerobic energy production in known thermophilic exoelectrogenic microorganisms is much the form of dark fermentative hydrogen production at temper- lower.18 To date, only few species have been reported to produce atures up to 55 C.27 The second one was anolyte from a xylose- 28 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. electricity at thermophilic conditions, including Firmicutes such fed MFC operating at 37 C. The volatile solids content was À as Caloramator australicus,18 Thermincola potens,19 Thermincola 10.6 (Æ0.2) and 8.4 (Æ0.5) g L 1 for the activated sludge and the ferriacetica,20 and Thermoanaerobacter pseudethanolicus,21 as anolyte, respectively. The mixture of the two inocula was ushed 22 well as Deferribacteres such as Calditerrivibrio nitroreducens. with N2 for 10 min before introducing into the anode chambers Investigating the composition of the active subpopulation, of the MFCs. rather than the whole microbial community, is crucial in understanding the role of microorganisms in MFCs. DNA-based 2.2 Anolyte composition methods may drive to erroneous conclusions in the detection of The anolyte was prepared according to M¨akinen et al.,29 but the key species in bioreactors.23 Previously performed microbial This article is licensed under a EDTA, yeast extract, and resazurin were not added. The community analyses have, nevertheless, mainly targeted the À substrate was xylose (0.3 or 1 g L 1, as specied in Section 2.4). presence of the 16S rRNA gene (DNA) whereas, to our knowl- Æ ff 19 The pH was kept at 7 ( 0.2) using phosphate bu er. The anolyte edge, only one study has also focused on 16S rRNA gene À conductivity was 14.6 mS cm 1. The composition of the feeding Open Access Article. Published on 15 January 2018. Downloaded 9/30/2021 9:34:23 PM. expression (RNA), which is an indicator of the microbial solution was the same as the anolyte, but with a 10-times higher activity.23 Furthermore, especially in studies on thermophilic xylose concentration. MFCs, microbial community analyses have mainly focused on the anode-attached microbial community, lacking information on the planktonic microbial community. The latter community 2.3 MFC con guration could be involved in electricity generation as well, either The h-type two-chamber MFCs were constructed by connecting directly, by performing mediated electron transfer to the two glass bottles (Adams & Chittenden Scientic Glass, USA) anode24 or indirectly, by converting the substrates to separated