J. Microbiol. Biotechnol. (2016), 26(3), 511–520 http://dx.doi.org/10.4014/jmb.1510.10102 Research Article Review jmb Genomic Barcode-Based Analysis of Exoelectrogens in Wastewater Biofilms Grown on Anode Surfaces S Kerstin Dolch1, Jessica Wuske1, and Johannes Gescher1,2* 1Institute for Applied Biosciences, Department of Applied Biology, Karlsruhe Institute of Technology, Fritz-Haber-Weg 2, D-76131 Karlsruhe, Germany 2Institute for Biological Interfaces, Karlsruhe Institute of Technology, D-76344 Eggenstein-Leopoldshafen, Germany Received: November 2, 2015 Revised: December 4, 2015 The most energy-demanding step of wastewater treatment is the aeration-dependent Accepted: December 10, 2015 elimination of organic carbon. Microbial fuel cells (MFCs) offer an alternative strategy in which carbon elimination is conducted by anaerobic microorganisms that transport respiratory electrons originating from carbon oxidation to an anode. Hence, chemical energy is First published online directly transformed into electrical energy. In this study, the use and stability of barcode- December 23, 2015 containing exoelectrogenic model biofilms under non-axenic wastewater treatment conditions *Corresponding author are described. Genomic barcodes were integrated in Shewanella oneidensis, Geobacter Phone: +49-721-608-41940; sulfurreducens, and G. metallireducens. These barcodes are unique for each strain and allow Fax: +49 721 608 41941; distinction between those cells and naturally occurring wild types as well as quantification of E-mail: [email protected] the amount of cells in a biofilm via multiplex qPCR. MFCs were pre-incubated with these three strains, and after 6 days the anodes were transferred into MFCs containing synthetic wastewater with 1% wastewater sludge. Over time, the system stabilized and the coulomb efficiency was constant. Overall, the initial synthetic biofilm community represented half of the anodic population at the end of the experimental timeline. The part of the community that contained a barcode was dominated by G. sulfurreducens cells (61.5%), while S. oneidensis and S upplementary data for this G. metallireducens cells comprised 10.5% and 17.9%, respectively. To the best of our knowledge, paper are available on-line only at this is the first study to describe the stability of a synthetic exoelectrogenic consortium under http://jmb.or.kr. non-axenic conditions. The observed stability offers new possibilities for the application of pISSN 1017-7825, eISSN 1738-8872 synthetic biofilms and synthetically engineered organisms fed with non-sterile waste streams. Copyright© 2016 by The Korean Society for Microbiology Keywords: Wastewater treatment, microbial fuel cell, Shewanella, Geobacter, barcoding, qPCR and Biotechnology Introduction These organisms catalyze the efficient conversion of chemical energy stored in the organic carbon sources to The application of microbial fuel cells (MFCs) in a electrical energy if the anode is connected to an electrical wastewater treatment plant is one of many relevant techniques load and a cathode. that can be applied to advance the already 100-year-old Previously, it was shown that MFCs used with wastewater wastewater treatment process [23]. Carbon elimination in can produce energy while reducing the amount of organic wastewater treatment plants is conducted via aerobic carbon [4]. The anodic community was analyzed in a few organisms that oxidize the organic carbon sources in a studies. Of the detected organisms, some were closely respiratory step. The wastewater aeration is the most related to microorganisms that were shown to transfer energy-consuming step during the treatment process. To respiratory electrons onto an anode [13]. Deltaproteobacteria reduce costs, an MFC could be implemented in place of the comprising the genus Geobacter seem to play a particularly aerobic oxidation step. Here, microorganisms use a solid key role in many communities, while other reports describe anode as an electron acceptor for anaerobic respiration. wastewater-fed communities on anodes that were dominated A 2016 ⎪ Vol. 26⎪ No. 0 512 Dolch et al. by members of the Bacteroidetes, Betaproteobacteria, and with 1% wastewater sludge. The current density, total Gammaproteobacteria [14]. It is largely unknown how amount of organic carbon (TOC), and coulomb efficiency these organisms contribute to the electron transfer process were measured over time. at the anode. Moreover, the anode serves as a nonspecific surface for biofilm growth that can be occupied by Materials and Methods microorganisms that do not contribute to the transfer of electrons. To improve the performance of an MFC, the Strain Preparations anodic microbial community must be selected and The barcodes were integrated into a non-coding region of the monitored [10]. This will be a major challenge for future genomes of S. oneidensis, G. sulfurreducens, G. metallireducens, and fuel cell research because tools have not been established to E. coli, with a space of at least 100 bp to adjacent coding regions. assemble tailored biofilms under non-axenic conditions or The synthetic sequences were designed with GeneDesign software to exclude the formation of secondary structures and ensure even to understand the functional role of all major binding of qPCR primers to the designated sequences only [25]. contributors of anode biofilm communities [22]. An overview of all strains and plasmids used in this study is Models for extracellular respiratory organisms, also called shown in Tables S1 and S2, and the primers are shown in Table 1. exoelectrogens, are Shewanella spp. and Geobacter spp. Barcoded strains are referred to with a subscript bc on each strain Whereas Shewanella oneidensis is a facultative anaerobic name. An overview of the insertion site of the barcoded strains is microorganism that can use a wide variety of electron shown in Fig. S1. acceptors [31], not all strains of Geobacter can grow in the Integration of the barcode into the S. oneidensis genome was presence of oxygen. Geobacter sulfurreducens can tolerate up conducted using the suicide vector pMQ150. The plasmid was to 10% oxygen [16], whereas Geobacter metallireducens is linearized with BamHI and SalI (New England Biolabs, Frankfurt strongly inhibited by oxygen. However, G. metallireducens am Main, Germany). Primers 1 and 4 were designed with a is extremely versatile in terms of the electron donors it can homology of 35 nt to the ends of the linearized plasmid. Primer use [1]. Many studies have analyzed the abilities of sets 1-2 and 3-4 were used to amplify a region of 500 bp upstream and downstream, respectively, of the insertion site. The two S. oneidensis and G. sulfurreducens in terms of biofilm growth fragments were assembled using a subsequent PCR step with on anodes. Whereas S. oneidensis seems to form rather thin primers 1 and 4. This was possible because primers 2 and 3 films, G. sulfurreducens can form several-micrometer-thick overlapped with 25 nt. These primers further contained the biofilms that are conductive [20]. barcode sequence. The entire fragment was cloned into the linear Hence, the three organisms have characteristics that plasmid according to the isothermal assembly method described would, in combination, form an organism that is extremely by Gibson et al. [8]. Afterwards, the plasmid was transformed in robust and can use a multitude of electron donors and Escherichia coli WM3064 and subsequently transferred into S. oneidensis acceptors. Instead of combining the characteristics in one MR-1 using conjugation; integration was described elsewhere [27]. organism, the combination of their abilities in a synthetic Integration of the barcode into G. sulfurreducens was performed three-organism biofilm would potentially be almost as using 602- and 601-bp-long fragments upstream and downstream efficient. Moreover, all three strains are genetically tractable. of the insertion site, amplified using primer sets 5-6 and 7-8. The Hence, the stable cultivation of synthetically engineered primers 9 and 10 were used for the amplification of a kanamycin resistance cassette from the plasmid pSCVAM. Primers 6 and 9 as Geobacter and Shewanella strains under non-axenic conditions well as 7 and 10 had a 25 bp overlap for assembly in a consecutive makes it possible to use available waste streams for PCR. The overlapping region of primers 7 and 10 was part of the biotechnological processes without prior costly deactivation. barcode sequence. The three PCR fragments were assembled via It was the aim of this study to analyze how stable a PCR and cloned in the plasmid pJET 1.2 using the CloneJET PCR community of model organisms would be under wastewater Cloning Kit according to the manufacturer`s instructions (Fermentas, treatment conditions. To monitor the three exoelectrogens St. Leon-Rot, Germany). The 2.2 kbp insert was amplified with and distinguish them from naturally occurring wild types primers 5 and 8 and transformed into G. sulfurreducens as described in wastewater sludge, barcodes were integrated into their by Coppi et al. [5]. As a minor modification, agar plates containing genomes. A barcode is a synthetic and unique DNA 35 µg/ml instead of 25 µg/ml kanamycin were used. sequence that is integrated in a non-coding region so that Integration into G. metallireducens was similar, but an upstream the phenotype does not change. Microbial electrochemical fragment of 598 nt (primers 11 and 12), the kanamycin resistance systems were started with all three strains at the same time.