www.nature.com/npjbiofilms ARTICLE OPEN Microbial electroactive biofilms dominated by Geoalkalibacter spp. from a highly saline–alkaline environment ✉ Sukrampal Yadav 1 and Sunil A. Patil 1 Understanding of the extreme microorganisms that possess extracellular electron transfer (EET) capabilities is pivotal to advance electromicrobiology discipline and to develop niche-specific microbial electrochemistry-driven biotechnologies. Here, we report on the microbial electroactive biofilms (EABs) possessing the outward EET capabilities from a haloalkaline environment of the Lonar lake. We used the electrochemical cultivation approach to enrich haloalkaliphilic EABs under 9.5 pH and 20 g/L salinity conditions. The electrodes controlled at 0.2 V vs. Ag/AgCl yielded the best-performing biofilms in terms of maximum bioelectrocatalytic current densities of 548 ± 23 and 437 ± 17 µA/cm2 with acetate and lactate substrates, respectively. Electrochemical characterization of biofilms revealed the presence of two putative redox-active moieties with the mean formal potentials of 0.183 and 0.333 V vs. Ag/ AgCl, which represent the highest values reported to date for the EABs. 16S-rRNA amplicon sequencing of EABs revealed the dominance of unknown Geoalkalibacter sp. at ~80% abundance. Further investigations on the haloalkaliphilic EABs possessing EET components with high formal potentials might offer interesting research prospects in electromicrobiology. npj Biofilms and Microbiomes (2020) 6:38 ; https://doi.org/10.1038/s41522-020-00147-7 1234567890():,; INTRODUCTION electroactivity include harvesting energy (e.g., electricity or Electromicrobiology is a new subdiscipline of (environmental) hydrogen) from waste streams with extreme characteristics, microbiology, which deals with the study of electrochemical developing energy-efficient bioproduction processes, and bior- interactions or extracellular electron transfer (EET) processes emediation of specific pollutants in extreme environments. between microorganisms and the solid-state electron acceptors Researchers have explored mostly normal habitats for the EAMs 4,13 or donors, and their implications in different environments1,2. The so far . The extreme environments remain poorly studied for microorganisms which use EET to achieve their respiratory or such microorganisms, mainly due to difficulties in sampling and metabolic processes are referred to as electroactive microorgan- conducting in situ experiments, as well as lack of appropriate 2,14 isms (EAMs)3,4. These are further subcategorized into exoelectro- enrichment protocols . A few pure culture isolates of the gens and electrotrophs. Exoelectrogens use outward EET to extreme EAMs are known or available4,15,16. Very few studies have reduce the extracellular solid-state electron acceptors, such as reported on the diversity of EAMs from extreme environments. – mineral oxides and electrodes, and achieve their respiration. In These include, for instance, highly saline17 19, extreme acidic20 and contrast, electrotrophs use inward EET to oxidize the solid-state alkaline21, extreme low22 and high temperature19,23, high tem- electron donor sources, in order to maintain their respiratory and perature and pressure24,25, and deep subsurface26 habitats. A metabolic activities. These microorganisms play important roles in combination of some of these extreme conditions also exists in different biogeochemical processes, such as mineral recycling5–7 some environments. Examples include saline-alkaline or haloalka- and interspecies electron transfer8,9, and are used for the line, high temperature–low pH, and extreme saline–high tem- development of various applications ranging from wastewater perature environments that host those microbes, which are treatment and concomitant water reclamation and energy adapted to two different extreme conditions. Exploring such production10,11, bioproduction to bioremediation, and habitats is expected to unravel the unknown microbial diversity biosensing1,12. and metabolic traits that could broaden the understanding of Strengthening of the foundation of electromicrobiology as a different ecological niches for EAMs and may offer opportunities major discipline in microbiology requires a broader and improved for their use under specialized extreme conditions. understanding of EAMs and their EET mechanisms from different The electroactivity of a few microorganisms that can grow environments. In particular, understanding the diversity of under highly saline or alkaline or haloalkaline conditions has been extreme EAMs with different metabolic capabilities holds great studied with the pure culture isolates of Geoalkalibacter ferrihy- potential not only to unravel their ecological significance, but also driticus, Geoalkalibacter subterraneus, and Natrialba magadii to the future development of niche-specific microbial available in the culture repositories13,17,21,27,28. The adaptation electrochemistry-driven biotechnological applications. For and enrichment of the mixed microbial community to develop instance, the use of extreme EAMs is desired to overcome the tolerance to free ammonia under highly saline and alkaline limitations associated with sluggish reaction kinetics and electron conditions has also been reported29. However, the real haloalka- transfer, poor electrolyte conductivity and associated ohmic line habitats have barely been explored for the EAMs. For instance, losses, and low organics removal efficiencies in bioelectrochemical Kumar et al. enriched a mixed microbial community capable of systems operated at normal conditions. Some of the promising producing bioelectrocatalytic current generation from the applications for microorganisms possessing extreme haloalkaline sediments of Texcoco Lake, Mexico, but did not 1Department of Earth and Environmental Sciences, Indian Institute of Science Education and Research Mohali (IISER Mohali), Knowledge City, Sector 81, SAS Nagar, Punjab ✉ 140306, India. email: [email protected] Published in partnership with Nanyang Technological University S. Yadav and S.A. Patil 2 analyze the electroactive community30. To the best of our Electrochemical enrichment or cultivation of the electroactive knowledge, no study has been conducted on a detailed under- microorganisms standing of the diversity of EAMs from the haloalkaline habitat EAMs can grow by linking substrate oxidation reaction to the thus far. reduction of the solid-state electrode in the absence of any other In this study, we present the electrochemical enrichment and electron acceptor. In such cases, microbial substrate oxidation is characterization of the exoelectrogenic microorganisms from the directly related to the bioelectrocatalytic current generation, extreme haloalkaline environment of Lonar Lake. It is the only which can be monitored by the chronoamperometry (CA) haloalkaline hypervelocity impact meteorite Crater Lake formed in technique38. Figure 1 shows the CA profiles, i.e., bioelectrocatalytic basaltic rock in the world (Supplementary Fig. 1). It is known for its current generation by microorganisms at the electrodes polarized high saline (ranging between 5 and 24 g/L) and alkaline at 0.0, 0.2, and 0.4 V. The reactors with working electrodes (9.5–10 pH) environment31,32. The variation in the salinity data in polarized at 0.2 V showed current production within 2 days of the literature is due to variations in the sampling locations and inoculation (Fig. 1c, d), and achieved the maximum current seasons. It has been the hotspot for geochemists, astrobiologists, densities of 548 ± 23 and 437 ± 17 µA/cm2 with acetate and ecologists, and microbiologists due to its unique characteristics. lactate substrates, respectively. In the case of 0 and 0.4 V The lake system has been well explored for the isolation of conditions, the start-up time of noticeable current production was >7 days. Moreover, low current densities of 117 ± 6 and 135 ± different microbial strains and to understand the broad microbial 2 2 diversity33–35, but not for the EAMs. For studying the electro- 12 µA/cm at 0 V (Fig. 1a, b), and 155 ± 31 and 238 ± 26 µA/cm at microbiology of this lake, we used the electrochemical enrichment 0.4 V (Fig. 1e, f) were achieved with acetate and lactate, respectively, by the enriched microorganisms compared to 0.2 V or cultivation approach. It involves the use of polarized electrodes − at different potentials as an analog or proxy to different natural condition. The electrodes polarized at 0.2 V showed no or negligible bioelectrocatalytic current response (Supplementary terminal electron acceptor conditions essential to the microbial Fig. 2). It is most likely due to a very little difference between the respiratory activities. It was followed by the detailed characteriza- reduction potential of the electron donor (i.e., acetate or lactate) tion of the best-performing enriched haloalkaliphilic microbial and the potential that was applied at the electrode, which acted EABs via electrochemical, microscopic, and 16S-rRNA amplicon as the electron acceptor. Similarly, both controls, namely abiotic sequencing techniques. connected and biotic unconnected, exhibited no substrate oxidation and current response (Supplementary Fig. 3). Hence, 1234567890():,; RESULTS the electric current production in all other experimental condi- tions can be attributed to the bioelectrocatalytic activity of the Sediment characteristics enriched haloalkaliphilic microorganisms. The sediment samples obtained during winter and monsoon The low bioelectrocatalytic