Characterizing the Microbial Sulfur Metabolizing Community of the Salton Sea Geothermal System

Abstract

Reduction of sulfur compounds has been proposed to be the earliest form of respiration, appearing 3.5 billion years ago as a mechanism for oxidation-reduction cycling in a nutrient-deficient environment. Today, sulfur-based respiration is common in environments where the availability of electron acceptors is limited, such as deep subsurface environments. The anoxic, high-temperature fluids ejected by the Salton Sea mud volcanoes is one such environment. Located in the Imperial and Riverside counties in Southern California, USA, the Salton Sea Geothermal System is a diverse ecosystem consisting of the endorheic Salton Sea, salt pools, petroleum seeps, and mud volcanoes. Water and various gases are collected in the subsurface of these volcanoes, heated, and bubbled out in a continuous cycle. Here, microbial life is diverse, ranging from thermophiles, halophiles, and obligate and facultative anaerobes. Characterization of the community has been performed via a wide-reaching 16S rRNA community analysis and by an isolate-specific analysis. This dual method allows for both breadth and depth in the characterization of sulfur metabolism in the mud volcanoes. Community analysis has been approached in two ways. In the first method, DNA is extracted from raw mud samples, followed by PCR amplification targeted at the highly-conserved 16S RNA region of the genome, isolation of specific amplicons by cloning into an E. coli vector, and amplicon purification and sequencing. In the second method, the 16S amplicons are sequenced directly using high throughput sequencing techniques. Sequences are checked for quality and high matches are used for alignment and phylogeny building. By using both methods we are able to gain a more complete picture of the entire community present in this deep subsurface environment. In addition to using community analysis, we have isolated sulfur and thiosulfate reducing microbes from this system. One of the isolates was characterized in detail and determined to be a novel anaerobic, hyperthermophilic archaeon that grows only in the presence of thiosulfate and ferric iron as electron acceptors. Genome annotation identified several candidate genes proposed to be involved in hydrocarbon oxidation and sulfur respiration. The isolate is neutrophilic, growing optimally at 90°C and 0–3% w/v NaCl. 16S rRNA sequence analysis places this isolate within the genus Pyrobaculum, a group of hyperthermophilic Crenarchaeota, and a neighbor joining phylogenetic tree from 16S RRNA sequence alignments was constructed. Based on genotypic and phenotypic differences between this isolate and the established members of the Pyrobaculum genus, the isolate is concluded to be a new species, for which the name Pyrobaculum igneolutum is proposed. Characterization of the metabolic schemes of the microbial life responsible for sulfur reduction is of great importance to the elucidation of complex biogeochemical cycling and is of practical relevance to cleaner hydrocarbon oxidation in the oil industry. Support or Funding Information This project is supported by NSF award MCB 1518306. Figure 1Open in figure viewerPowerPoint The 16S neighbor joining phylogenetic tree of the Pyrobaculum genus and related species, with Pyrobaculum igneolutum included. GenBank accession numbers are provided in parentheses. Introns were removed prior to alignment. Each number indicates the bootstrap value from 1000 trials. Bar, 1 substitution per 100 bases. Figure 2Open in figure viewerPowerPoint The effect of (A) pH, temperature, and (C) NaCl on the growth of the isolate.