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Summary Electrotrophic microorganisms have not been well studied in extreme environments. Here, we report on the nitrate-reducing cathodic microbial biofilm from a haloalkaline environment. The biofilm enriched via electrochemical approach under 9.5 pH and 20 g NaCl/L salinity conditions achieved current density and nitrate reduction efficiency via partial and complete denitrification. Voltammetric characterization of the biocathodes revealed a redox center with 0.294 0.003 ( vs. Ag/AgCl) formal potential putatively involved in the electron uptake process. The lack of soluble redox mediators and hydrogen-driven nitrate reduction suggests direct-contact cathodic electron uptake by the nitrate-reducing microorganisms in the enriched biofilm. 16S-rRNA amplicon sequencing of the cathodic biofilm revealed the presence of unreported Pseudomonas, Natronococcus, and Pseudoalteromonas spp. at , and relative sequence abundances, respectively. The enriched nitrate-reducing microorganisms also reduced nitrate efficiently using soluble electron donors found in the lake sediments, thereby suggesting their role in N-cycling in such environments.

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The extracellular electron transfer (EET)-capable electroactive microorganisms (EAMs) play crucial roles in mineral cycling and interspecies electron transfer in different environments and are used as biocatalysts in microbial electrochemical technologies. Studying EAMs from extreme environments is desired to advance the electromicrobiology discipline, understanding their unique metabolic traits with implications to extreme microbiology, and develop specific bioelectrochemical applications. Here, we present a novel haloalkaliphilic bacterium named Geoalkalibacter halelectricus SAP-1, isolated from a microbial electroactive biofilm enriched from the haloalkaline lake sediments. It is a rod-shaped Gram-negative heterotrophic anaerobe that uses various carbon and energy sources and respires on soluble and insoluble terminal electron acceptors. Besides 16S-rRNA and wholegenome sequence-based phylogeny, the GGDC values of 21.7%, ANI of 78.5%, and 2.77% genomic DNA GC content difference with the closest validly named species Geoalkalibacter ferrihydriticus (DSM 17813 T ) confirmed its novelty. When grown with the solid-state electrode as the only electron acceptor, it produced 460 AE 23 μA/cm 2 bioelectrocatalytic current, thereby confirming its electroactivity. Further electrochemical analysis revealed the presence of membrane redox components with a high formal potential, putatively involved in the direct mode of EET. These are distinct from EET components reported for any known electroactive microorganisms, including well-studied Geobacter spp., Shewanella spp., and Desulfuromonas acetexigens. The capabilities of G. halelectricus SAP-1 to respire on soluble and insoluble electron acceptors including fumarate, SO 4 2À , Fe 3+ , and Mn 4+ suggests its role in cycling these elements in haloalkaline environments.

Summary Electroactive microorganisms (EAMs) are a group of microbes that can access solid extracellular electron donors or acceptors via extracellular electron transfer processes. EAMs are useful in developing various microbial electrochemical technologies. This protocol describes the use of bioelectrochemical systems (BESs) to enrich EAMs at the cathode from an extreme haloalkaline habitat. It also provides information for a detailed characterization of enriched cathodic biofilms via various cross-disciplinary techniques, including electrochemical, analytical, microscopic, and gene sequencing techniques. For complete details on the use and execution of this protocol, please refer to Chaudhary et al. (2021) .