Coupling Dark Metabolism to Electricity Generation Using Photosynthetic Cocultures Jonathan P. Badalamenti, Ce ´sar I. Torres, Rosa Krajmalnik-Brown Swette Center for Environmental Biotechnology, The Biodesign Institute, Arizona State University, Tempe, Arizona; telephone: 480-727-9689; fax: 470-727-0889; e-mail: cit@asu.edu; telephone: 480-727-7574; fax(480) 727-0889; e-mail: dr.rosy@asu.edu (RKB) ABSTRACT: We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electro- chemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlor- obium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter . With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S 0 as the electron acceptor to Geobacter . Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current. Biotechnol. Bioeng. 2013;9999: 1–9. ß 2013 Wiley Periodicals, Inc. KEYWORDS: microbial electrochemical cell; fermentation; photosynthesis; glycogen; coculture Introduction Biogeochemical cyclesoccur via energy-conserving microbial transformations requiring cooperation among individual microbes performing specialized functions within diverse communities (Ehrlich and Newman, 2009). However, the unculturability of most microorganisms poses a major challenge in understanding the individual and synergistic roles for key microbial populations in situ. Bottom-up reconstruction of relevant biotransformations using cocul- tures simplifies examination of complex multispecies interactions and can even lead to laboratory culturing of previously unculturable bacteria (Stewart, 2012). Benefits of coculture-based investigations range from alleviation of product inhibition and consumption of dead-end metabo- lites (Jiao et al., 2012), to the exchange of beneficial substrates or growth factors (Yan et al., 2012), maintenance of energy balance (McCarty and Bae, 2011), selective coupling of redox reactions (Cord-Ruwisch et al., 1998), understanding routes of extracellular electron transfer (Stams et al., 2006; Summers et al., 2010), and applications in synthetic biology (Winter- mute and Silver, 2010). In microbial electrochemical cells (MXCs), anode-respir- ing bacteria (ARB) couple the oxidation of organic substrates to the transfer of electrons to an electrode, creating an electrical current (Logan and Rabaey, 2012). Interfacing bacteria with electrodes thus creates opportunities for integrating MXCs with coculture-based studies, since the anode can serve as the sole electron acceptor, and substrate oxidation can be monitored in real time. For example, a coculture of fermentative Clostridium cellulolyticum and anode- respiring Geobacter sulfurreducens cooperatively captured electricity from cellulose (Ren et al., 2007). In a separate study, a photosynthetic MXC utilizing the green alga Chlamydomonas reinhardtii in coculture with G. sulfurreducens generated light-responsive current from organic compounds Correspondence to: C.I. Torres and R. Krajmalnik-Brown Contract grant sponsor: U.S. Environmental Protection Agency STAR Fellowship FP91715201-0 Contract grant sponsor: Swette Center for Environmental Biotechnology at the Biodesign Institute at Arizona State University. Received 2 May 2013; Revision received 5 July 2013; Accepted 17 July 2013 Accepted manuscript online xx Month 2013; Article first published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.25011 ARTICLE ß 2013 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2013 1