Chignell et al. Biotechnol Biofuels (2018) 11:121 https://doi.org/10.1186/s13068-018-1111-2 Biotechnology for Biofuels RESEARCH Open Access Meta‑proteomic analysis of protein expression distinctive to electricity‑generating bioflm communities in air‑cathode microbial fuel cells Jeremy F. Chignell1, Susan K. De Long2 and Kenneth F. Reardon1,3* Abstract Background: Bioelectrochemical systems (BESs) harness electrons from microbial respiration to generate power or chemical products from a variety of organic feedstocks, including lignocellulosic biomass, fermentation byproducts, and wastewater sludge. In some BESs, such as microbial fuel cells (MFCs), bacteria living in a bioflm use the anode as an electron acceptor for electrons harvested from organic materials such as lignocellulosic biomass or waste byprod- ucts, generating energy that may be used by humans. Many BES applications use bacterial bioflm communities, but no studies have investigated protein expression by the anode bioflm community as a whole. Results: To discover functional protein expression during current generation that may be useful for MFC optimiza- tion, a label-free meta-proteomics approach was used to compare protein expression in acetate-fed anode bioflms before and after the onset of robust electricity generation. Meta-proteomic comparisons were integrated with 16S rRNA gene-based community analysis at four developmental stages. The community composition shifted from dominance by aerobic Gammaproteobacteria (90.9 3.3%) during initial bioflm formation to dominance by Deltapro- teobacteria, particularly Geobacter (68.7 3.6%) in mature,± electricity-generating anodes. Community diversity in the intermediate stage, just after robust current± generation began, was double that at the early stage and nearly double that of mature anode communities. Maximum current densities at the intermediate stage, however, were relatively similar (~ 83%) to those achieved by mature-stage bioflms. Meta-proteomic analysis, correlated with population changes, revealed signifcant enrichment of categories specifc to membrane and transport functions among proteins from electricity-producing bioflms. Proteins detected only in electricity-producing bioflms were associated with gluconeogenesis, the glyoxylate cycle, and fatty acid β-oxidation, as well as with denitrifcation and competitive inhibition. Conclusions: The results demonstrate that it is possible for an MFC microbial community to generate robust current densities while exhibiting high taxonomic diversity. Moreover, these data provide evidence to suggest that startup growth of air–cathode MFCs under conditions that promote the establishment of aerobic–anaerobic syntrophy may decrease startup times. This study represents the frst investigation into protein expression of a complex BES anode bioflm community as a whole. The fndings contribute to understanding of the molecular mechanisms at work dur- ing BES startup and suggest options for improvement of BES generation of bioelectricity from renewable biomass. Keywords: Bioelectrochemical, Proteomics, Meta-proteomics, Microbial fuel cell, Metagenomics, Bioflm, Exoelectrogen, Geobacter *Correspondence: [email protected] 1 Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, USA Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chignell et al. Biotechnol Biofuels (2018) 11:121 Page 2 of 17 Background recently [21]. Proteomics, in particular, has been useful In bioelectrochemical systems (BESs), electrochemically for profling protein expression of defned or undefned active microbes convey electrons to or from a conduc- microbial communities [22]. Tese “meta-proteomics” tive electrode [1]. In some BES systems, such as micro- studies have identifed metabolic mechanisms behind bial fuel cells (MFCs), bacteria living in a bioflm use the multispecies fermentation [23], methanogenesis [24], anode as an electron acceptor for electrons harvested or community response to a toxic perturbation [25]. A from organic materials such as lignocellulosic biomass or few studies have used proteomics methods to investi- waste byproducts, and the resulting current is harnessed gate electricity generation by model species or isolates to recover energy during wastewater treatment [2]. Other [26, 27]. Meta-proteomics examinations of BES systems, BES applications include removal of nutrients [3] or met- however, have been limited to a single investigation of als [4], desalination [5], and generation of bioproducts protein expression of biocathode bioflm organisms such as H2 [6], H2O2 [7], or organic molecules from CO2 under optimal and suboptimal conditions [28]. Tis study and sunlight [8]. Successful commercial application of identifed several biocathode proteins associated with an BES technologies will require increases in current gen- optimal reactor potential for use of an electrode as an eration and efciency [9]. More detailed information electron donor to fx CO2. Te question of the proteins regarding the fundamental mechanisms that enable bio- associated with anode mixed-species bioflms that gener- electricity generation will inform strategies for scale-up ate electricity has not yet been addressed. and new applications of BES technology [10]. Te goal of this study was to characterize protein Descriptions of the mechanisms behind electricity expression that is distinctive to a MFC anode commu- generation in BESs have relied primarily on model BES nity when it is generating electricity. Specifcally, a label- genera. Current generation mechanisms described for free meta-proteomics approach was used to compare model BES genera like Shewanella and Geobacter include protein expression in acetate-fed MFC anode bioflms indirect electron transfer via soluble redox compounds before and after the onset of robust current generation. [11], and direct electron transfer via outer membrane Since the types and abundances of proteins expressed cytochromes [12] or pilus-like “nanowires” [13]. Tese depend on the types and abundances of microbial gen- discoveries have informed improvements in BES per- era present in the community, we quantifed changes in formance through design of electrode architecture [14], MFC community structure across developmental stages identifcation of limiting factors during electron transfer in terms of relative abundance of operational taxonomi- [15], and attempts at metabolic engineering of microbes cal units (OTUs). Tis information about community or defned community [16]. Few studies, however, have structure was integrated with meta-proteomics results attempted to determine which of these current gen- in two ways. First, protein expression was normalized to eration mechanisms are most prevalent in mixed cul- abundance levels of individual genera during signifcance ture BES communities or how the interactions between testing for diferential expression of proteins. Second, members of BES consortia afect electricity generation. OTU quantifcation was used as a method of orthogonal So far, those interactions primarily have been described “validation” of meta-proteomics results by comparing in terms of community composition, quantifed as rela- relative abundance of genera based on OTUs with that tive abundance of 16S rRNA genes [17, 18]. Tis kind of based on genera associated with protein identifcations. ecological approach describes compositional changes Compared with using a single method, this sort of mixed of BES community in response to operational changes meta-omics approach ofers the possibility of obtaining a such as the type of substrate [19]. Metabolic syntrophies more complete picture of the activities and interactions among BES community members are thought to explain of the anode community members during MFC startup the generally superior performance of a mixed commu- and electricity production. Such a picture may prove nity compared with pure cultures [20]. However, 16S useful for improving MFC performance through reactor rRNA profling is not well suited to provide insights into design, operating conditions, or community structure interactions among community members. Identifcation modifcation. of the community interactions and mechanisms of cur- rent generation at work during BES bioflm development Methods may suggest strategies to reduce reactor startup time or MFC setup, operation, and harvest increase community resilience to perturbation, thereby The single-chamber, membrane-free, air–cathode reducing operating costs and supporting commercial MFC design used in this study was similar to a previ- scale-up. ous design [29] and is described in detail in Additional Appropriate tools for molecular investigations of file 1. The liquid volume of each MFC was 30 mL, and microbial community function have emerged only the area of the anode and air–cathode was 7.0 cm2. Chignell et al. Biotechnol Biofuels (2018) 11:121 Page 3 of 17 The