Retraction for Yadav et al., "The bidirectional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition".

In our paper, we reported the bidirectional extracellular electron transfer capability of Geoalkalibacter halelectricus based on biochemical (i.e., with insoluble Fe-oxide and Fe(0)) and bioelectrochemical (i.e., with electrodes) experimental approaches. We noticed some issues and limitations of the methods and techniques that were used to analyze Fe species, particularly the reduced Fe ions in insoluble and precipitated Fe(II) minerals that led to incorrect interpretation of the results, specifically the reduction of Fe(III) to Fe(0) in this study. We were made aware of thermodynamic constraints that would make the biological reduction of Fe(III) to Fe(0) implausible. Hence, the conclusion about microbial Fe(III)-oxide reduction to Fe(0) is invalid. We also noticed errors in estimating the protein-based iron reduction/oxidation rates and faradic efficiencies, besides the limitations of the methods used for Fe analysis. For these reasons, we retract this article and sincerely apologize for the inconvenience it may have caused to the readers.

Retracted: The bidirectional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition.

Bidirectional extracellular electron transfer (EET) is crucial to upholding microbial metabolism with insoluble electron acceptors or donors in anoxic environments. Investigating bidirectional EET-capable microorganisms is desired to understand the cell-cell and microbe-mineral interactions and their role in mineral cycling besides leveraging their energy generation and conversion, biosensing, and bio-battery applications. Here, we report on iron cycling by haloalkaliphilic Geoalkalibacter halelectricus via bidirectional EET under haloalkaline conditions. It efficiently reduces Fe3+ oxide (Fe2O3) to Fe0 at a 0.75 ± 0.08 mM/mgprotein/d rate linked to acetate oxidation via outward EET and oxidizes Fe0 to Fe3+ at a 0.24 ± 0.03 mM/mgprotein/d rate via inward EET to reduce fumarate. Bioelectrochemical cultivation confirmed its outward and inward EET capabilities. It produced 895 ± 23 µA/cm2 current by linking acetate oxidation to anode reduction via outward EET and reduced fumarate by drawing electrons from the cathode (‒2.5 ± 0.3 µA/cm2) via inward EET. The cyclic voltammograms of G. halelectricus biofilms revealed redox moieties with different formal potentials, suggesting the involvement of different membrane components in bidirectional EET. The cyclic voltammetry and GC-MS analysis of the cell-free spent medium revealed the lack of soluble redox mediators, suggesting direct electron transfer by G. halelecctricus in achieving bidirectional EET. By reporting on the first haloalkaliphilic bacterium capable of oxidizing and reducing insoluble Fe0 and Fe3+ oxide, respectively, this study advances the limited understanding of the metabolic capabilities of extremophiles to respire on insoluble electron acceptors or donors via bidirectional EET and invokes the possible role of G. halelectricus in iron cycling in barely studied haloalkaline environments. IMPORTANCE Bidirectional extracellular electron transfer (EET) appears to be a key microbial metabolic process in anoxic environments that are depleted in soluble electron donor and acceptor molecules. Though it is an ecologically important and applied microbial phenomenon, it has been reported with a few microorganisms, mostly from nonextreme environments. Moreover, direct electron transfer-based bidirectional EET is studied for very few microorganisms with electrodes in engineered systems and barely with the natural insoluble electron acceptor and donor molecules in anoxic conditions. This study advances the understanding of extremophilic microbial taxa capable of bidirectional EET and its role in barely investigated Fe cycling in highly saline-alkaline environments. It also offers research opportunities for understanding the membrane components involved in the bidirectional EET of G. halelectricus. The high rate of Fe3+ oxide reduction activity by G. halelectricus suggests its possible use as a biocatalyst in the anaerobic iron bioleaching process under neutral-alkaline pH conditions.

Geoalkalibacter halelectricus SAP-1 sp. nov. possessing extracellular electron transfer and mineral-reducing capabilities from a haloalkaline environment.

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 whole-genome 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 17813T ) confirmed its novelty. When grown with the solid-state electrode as the only electron acceptor, it produced 460 ± 23 µA/cm2 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, SO4 2- , Fe3+ , and Mn4+ suggests its role in cycling these elements in haloalkaline environments.

Protocol for bioelectrochemical enrichment, cultivation, and characterization of extreme electroactive microorganisms.

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).

Extremophilic electroactive microorganisms: Promising biocatalysts for bioprocessing applications.

Electroactive microorganisms (EAMs) use extracellular electron transfer (EET) processes to access insoluble electron donors or acceptors in cellular respiration. These are used in developing microbial electrochemical technologies (METs) for biosensing and bioelectronics applications and the valorization of liquid and gaseous wastes. EAMs from extreme environments can be useful to overcome the existing limitations of METs operated with non-extreme microorganisms. Studying extreme EAMs is also necessary to improve understanding of respiratory processes involving EET. This article first discusses the advantages of using extreme EAMs in METs and summarizes the diversity of EAMs from different extreme environments. It is followed by a detailed discussion on their use as biocatalysts in various bioprocessing applications via bioelectrochemical systems. Finally, the challenges associated with operating METs under extreme conditions and promising research opportunities on fundamental and applied aspects of extreme EAMs are presented.

Electrochemical enrichment of haloalkaliphilic nitrate-reducing microbial biofilm at the cathode of bioelectrochemical systems.

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 - 43.5 ± 7.2 µA / cm 2 current density and 49.5 ± 13.2 % nitrate reduction efficiency via partial and complete denitrification. Voltammetric characterization of the biocathodes revealed a redox center with - 0.294 ± 0.003 V (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 31.45 % , 11.82 % , and 9.69 % 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.

Microbial electroactive biofilms dominated by Geoalkalibacter spp. from a highly saline-alkaline environment.

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.