Lawrence Berkeley National Laboratory Recent Work Title Sulfate-Reducing Bacteria That Produce Exopolymers Thrive in the Calcifying Zone of a Hypersaline Cyanobacterial Mat. Permalink https://escholarship.org/uc/item/6jj4748h Journal Frontiers in microbiology, 10(APR) ISSN 1664-302X Authors Spring, Stefan Sorokin, Dimitry Y Verbarg, Susanne et al. Publication Date 2019 DOI 10.3389/fmicb.2019.00862 Peer reviewed eScholarship.org Powered by the California Digital Library University of California fmicb-10-00862 April 17, 2019 Time: 16:24 # 1 ORIGINAL RESEARCH published: 24 April 2019 doi: 10.3389/fmicb.2019.00862 Sulfate-Reducing Bacteria That Produce Exopolymers Thrive in the Calcifying Zone of a Hypersaline Cyanobacterial Mat Stefan Spring1*, Dimitry Y. Sorokin2,3, Susanne Verbarg4, Manfred Rohde5, Tanja Woyke6 and Nikos C. Kyrpides6 1 Department Microorganisms, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, 2 Winogradsky Institute of Microbiology, Research Centre of Biotechnology, Moscow, Russia, 3 Department of Biotechnology, Delft University of Technology, Delft, Netherlands, 4 Department Services Microorganisms, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, 5 Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany, 6 DOE Joint Genome Institute, Walnut Creek, CA, United States Edited by: Jesse G. Dillon, Calcifying microbial mats in hypersaline environments are important model systems for California State University, Long the study of the earliest ecosystems on Earth that started to appear more than three Beach, United States billion years ago and have been preserved in the fossil record as laminated lithified Reviewed by: structures known as stromatolites. It is believed that sulfate-reducing bacteria play a Karim Benzerara, Centre National de la Recherche pivotal role in the lithification process by increasing the saturation index of calcium Scientifique (CNRS), France minerals within the mat. Strain L21-Syr-ABT was isolated from anoxic samples of a Richard Allen White III, RAW Molecular Systems LLC, several centimeters-thick microbialite-forming cyanobacterial mat of a hypersaline lake United States on the Kiritimati Atoll (Kiribati, Central Pacific). The novel isolate was assigned to the Charles K. Lee, family Desulfovibrionaceae within the Deltaproteobacteria. Available 16S rRNA-based University of Waikato, New Zealand population surveys obtained from discrete layers of the mat indicate that the occurrence *Correspondence: T Stefan Spring of a species-level clade represented by strain L21-Syr-AB is restricted to a specific [email protected] layer of the suboxic zone, which is characterized by the presence of aragonitic spherulites. To elucidate a possible function of this sulfate-reducing bacterium in the Specialty section: This article was submitted to mineral formation within the mat a comprehensive phenotypic characterization was Extreme Microbiology, combined with the results of a comparative genome analysis. Among the determined a section of the journal T Frontiers in Microbiology traits of strain L21-Syr-AB , several features were identified that could play a role in Received: 07 December 2018 the precipitation of calcium carbonate: (i) the potential deacetylation of polysaccharides Accepted: 04 April 2019 and consumption of substrates such as lactate and sulfate could mobilize free calcium; Published: 24 April 2019 (ii) under conditions that favor the utilization of formate and hydrogen, the alkalinity Citation: engine within the mat is stimulated, thereby increasing the availability of carbonate; Spring S, Sorokin DY, Verbarg S, Rohde M, Woyke T and Kyrpides NC (iii) the production of extracellular polysaccharides could provide nucleation sites for (2019) Sulfate-Reducing Bacteria calcium mineralization. In addition, our data suggest the proposal of the novel species That Produce Exopolymers Thrive T in the Calcifying Zone of a Hypersaline and genus Desulfohalovibrio reitneri represented by the type strain L21-Syr-AB (=DSM Cyanobacterial Mat. 26903T = JCM 18662T). Front. Microbiol. 10:862. doi: 10.3389/fmicb.2019.00862 Keywords: stromatolites, lithification, biofilm, alkalinity engine, sulfate reduction Frontiers in Microbiology| www.frontiersin.org 1 April 2019| Volume 10| Article 862 fmicb-10-00862 April 17, 2019 Time: 16:24 # 2 Spring et al. Sulfate-Reducing Bacteria Involved in Mineral Precipitation INTRODUCTION influenced formation of dolomite (e.g., Bontognali et al., 2014). Only recently the isolation of a sulfate-reducing bacterium Lithifying microbial mats represent probably the earliest that is specifically associated with the transition zone of a ecosystems on Earth that started to appear more than three lithifying, hypersaline microbial mat was achieved (Spring billion years ago and were preserved in the fossil record et al., 2015a). In this study, we report the phenotypic and as stromatolites (Grotzinger and Knoll, 1999; Nutman et al., genomic characteristics of the novel strain and correlate these 2016). Calcification plays a main role in the lithification traits with a possible function in the biogeochemistry of the process of laminated microbial mats and can be explained by studied microbial mat. two main factors: an alkalinity engine within the mat and the properties of the organic mat matrix creating localized supersaturating conditions for calcium carbonate (Dupraz et al., MATERIALS AND METHODS 2009). The so-called alkalinity engine designates the potential of a microbial community to increase the local pH by its Strains and Cultivation Conditions metabolic activity. An increase of pH can be induced mainly Strain L21-Syr-ABT was isolated from an anaerobic enrichment by photosynthesis or sulfate reduction and may promote culture with syringate as substrate, inoculated with slurries of a mineralization by increasing the availability of carbonate. The cyanobacterial mat sample retrieved from the hypersaline Lake organic mat matrix is composed of extracellular polymeric 21 on the Kiritimati Atoll (Northern Line Islands, Republic substances (EPS) produced by oxygenic photoautotrophic of Kiribati). The location of the sampling site and details microorganisms thriving in the upper layers of the mat. Most of the isolation procedure were described previously (Spring functional groups of freshly produced EPS are acidic and et al., 2015a). For the preparation of media and incubation have the property to bind bivalent cations, especially calcium. under anoxic conditions the anaerobe cultivation technique of Degradation of EPS by heterotrophic bacteria in deeper mat Hungate(1950) with the modifications introduced by Bryant layers can lead to the release of calcium thereby stimulating (1972) was used. For routine cultivation of strain L21-Syr-ABT mineral precipitation. the medium DSM 1526c was used that contained per liter The importance of sulfate reduction in the lithification deionized ultrapure water: 60.0 g NaCl, 6.0 g MgSO4 × 7 of microbial mats has been postulated for a long time (e.g., H2O, 1.5 g KCl, 1.0 g Na2S2O3 × 5 H2O, 1.0 g NH4Cl, Jørgensen and Cohen, 1977; Lyons et al., 1984; Visscher et al., 0.4 g CaCl2 × 2 H2O, 0.4 g K2HPO4, 10.0 ml trace elements 1998) and is mainly based on the following considerations: solution of DSMZ medium 1411, 1.0 g yeast extract, 0.5 mg increase of the calcium carbonate saturation index (SI) resazurin, 10.0 ml vitamins solution of DSMZ medium 141, during growth of sulfate-reducers with hydrogen or formate 1.5 g Na2CO3, 2.5 g sodium pyruvate, and 0.5 g Na2S × 9 (Gallagher et al., 2012), reduction of the sulfate-dependent H2O. The medium was prepared under 80% N2 and 20% inhibition of carbonate precipitation (Wright and Wacey, CO2 gas mixture without the vitamins, carbonate, sodium 2005), release of calcium by the degradation of functional pyruvate and sulfide, which were added to the medium after groups of EPS (Glunk et al., 2011), and provision of autoclaving from sterile anoxic stock solutions. The pH of the nucleation sites promoting mineral precipitation (Braissant completed medium was adjusted to 7.3 – 7.5 and the standard et al., 2007). Indirect evidence for the involvement of sulfate incubation temperature was 35◦C. For testing carbon source reduction in the lithification process was presented in several utilization, sodium pyruvate was omitted and the amount of studies in which the highest sulfate reduction rates were yeast extract was reduced to 0.05 g l−1. Fermentative growth was detected in zones of active calcium mineral precipitation determined in a sulfate-free medium 1526c that was prepared (Visscher et al., 2000; Dupraz et al., 2004; Glunk et al., 2011; without yeast extract and Na2S2O3 × 5 H2O. In addition, Pace et al., 2018). MgSO4 × 7 H2O was replaced with MgCl2 × 6 H2O and Despite their assumed importance, only a few isolates the trace elements solution of DSMZ medium 141 with the of sulfate-reducing bacteria have been obtained from the trace elements of DSMZ medium 3202. Pure chemicals were lithification zones of microbial mats. Previously, the strains obtained from Sigma-Aldrich3 and complex nutrients from H12.1 and H2.3j were isolated from lithifying surface mats of BD Biosciences4. Highborne Cay stromatolites and used in growth experiments For comparison, the type strains Desulfovibrio alkalitolerans to demonstrate the effect of EPS production and substrate DSM 16529T and Desulfovibrio africanus subsp. africanus