Geoalkalibacter halelectricus: a novel haloalkaliphilic bacterium capable of respiring on insoluble electron acceptors through extracellular electron transfer

Abstract

Abstract: Extracellular electron transfer (EET) is essential to upholding microbial respiration with insoluble electron acceptors or donors in anoxic environments. The EET-capable microorganisms, called electroactive microorganisms, are involved in mineral cycling, corrosion, and interspecies electron transfer processes in different environments and are used in developing bioremediation, biosensing, bioproduction, and bioelectronics applications. Studying these microorganisms from extreme environments is desired to understand their unique metabolic traits and roles in mineral cycling with implications to extreme microbiology and explore them for various bioelectrochemical applications. To this end, this thesis aimed to understand electroactive microorganisms and their EET processes from a haloalkaline environment. First, mixed microbial electroactive biofilms capable of respiring on the solid- state electrode were enriched using an electrochemical cultivation approach from the Lonar lake subsurface sediments. Next, a novel anaerobic bacterium capable of respiring on insoluble electron acceptors such as Fe-oxides and electrodes was isolated. Its novelty was confirmed through various morphological, biochemical, and genomic features, including GGDC, ANI, and % GC difference, along with 16S rRNA and whole genome sequencing-based phylogeny, and it was named Geoalkalibacter halelectricus SAP-1 sp. nov. (=JCM35356 and =MTCC13188). Notably, G. halelectricus not only reduces insoluble Fe(III)-oxide (e.g., hematite) efficiently but also oxidizes Fe(0), suggesting its bi-directional EET (i.e., both outward and inward EET) capabilities to uphold metabolism with insoluble electron acceptor and donor molecules in haloalkaline conditions. The cyclic voltammetry-based interrogations of the G. halelectricus biofilms suggested the direct mode of EET via two unreported membrane redox-moieties with the formal potential of 0.181 and 0.365 V (vs. Ag/AgCl). The complete genome annotation revealed several unreported multi-heme cytochromes as respiratory process components in SAP-1. Based on these inferences, the final part of the thesis focused on elucidating the outer membrane components involved in the EET process of SAP- 1. For this purpose, an outer membrane cytochrome of SAP-1 was selected and expressed in E. coli through heterologous overexpression, followed by its purification. Further detailed characterization of the purified cytochrome would reveal its identity, structure, and role in the EET process. By reporting on a metabolically versatile novel EET-capable haloalkaliphilic microorganism named Geoalkalibacter halelectricus and its promising Fe-cycling activity besides the presence of unreported and unique membrane components, putatively involved in EET-based anaerobic respiration, this thesis work advances the understanding of the diversity and ecology of extreme electroactive microorganisms and opens up exciting research prospects on unraveling novel EET components and mechanisms.