Sulfur disproportionating microbial communities in a dynamic, microoxic‐sulfidic karst system

Biogeochemical sulfur cycling in sulfidic karst systems is largely driven by abiotic and biological sulfide oxidation, but the fate of elemental sulfur (S0) that accumulates in these systems is not well understood. The Frasassi Cave system (Italy) is intersected by a sulfidic aquifer that mixes with small quantities of oxygen-rich meteoric water, creating Proterozoic-like conditions and supporting a prolific ecosystem driven by sulfur-based chemolithoautotrophy. To better understand the cycling of S0 in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide-oxidizing mats dominated by Beggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related to Sulfurovum, Halothiobacillus, Thiofaba, Thiovirga, Thiobacillus, and Desulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated with Bacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae. Desulfocapsa and Sulfurovum populations accounted for 12%–26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S0 disproportionation in pure culture. Gibbs energy (∆Gr) calculations revealed that S0 disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat-sediment interface showed that Beggiatoa mats consume dissolved sulfide and oxygen, but a net increase in acidity was only observed in the sediments below. Together, these findings suggest that disproportionation is an important sink for S0 generated by microbial sulfide oxidation in this oxygen-limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur-rich environments

Organic stabilization of extracellular elemental sulfur in a Sulfurovum-rich biofilm: a new role for extracellular polymeric substances?

This work shines light on the role of extracellular polymeric substance (EPS) in the formation and preservation of elemental sulfur biominerals produced by sulfur-oxidizing bacteria. We characterized elemental sulfur particles produced within a Sulfurovum-rich biofilm in the Frasassi Cave System (Italy). The particles adopt spherical and bipyramidal morphologies, and display both stable (α-S8) and metastable (β-S8) crystal structures. Elemental sulfur is embedded within a dense matrix of EPS, and the particles are surrounded by organic envelopes rich in amide and carboxylic groups. Organic encapsulation and the presence of metastable crystal structures are consistent with elemental sulfur organomineralization, i.e., the formation and stabilization of elemental sulfur in the presence of organics, a mechanism that has previously been observed in laboratory studies. This research provides new evidence for the important role of microbial EPS in mineral formation in the environment. We hypothesize that the extracellular organics are used by sulfur-oxidizing bacteria for the stabilization of elemental sulfur minerals outside of the cell wall as a store of chemical energy. The stabilization of energy sources (in the form of a solid electron acceptor) in biofilms is a potential new role for microbial EPS that requires further investigation

Molecular characterization of core lipids from halophilic archaea grown under different salinity conditions

Halorhabdus utahensis, Natronomonas pharaonis, Haloferax sulfurifontis and Halobaculum gomorrense were grown at salinity values between 10% and 30% NaCl (w/v). The strains represent four haloarchaeal genera and have a range of salinity optima. Analysis of core membrane lipids of each strain using gas chromatography– mass spectrometry (GC–MS) revealed structures consistent with saturated, unsaturated and polyunsaturated dialkyl glycerol diethers (DGDs) including both phytanyl (C_(20)) and sesterpanyl (C_(25)) isoprenoid chains. In addition, we observed three trends related to salinity: (i) the proportion of unsaturated DGDs increased with increasing NaCl concentration in the medium, (ii) strains with a higher optimal NaCl concentration had a higher proportion of unsaturated DGDs and (iii) C_(25–20) DGDs occurred in the two strains with higher salinity optima, N. pharaonis and H. utahensis. The strong linear correlation between optimal growth salinity and fraction of unsaturated DGDs suggests that membrane lipid unsaturation is an important adaptation to specific salinity niches in archaeal halophiles. In addition, in three of the four strains, the fraction of unsaturated DGDs increased above a salinity threshold or in response to increasing salinity in the medium. Thus, halophilic archaea regulate membrane lipid unsaturation in response to environmental salinity change, regardless of their salinity optima

Metagenomic Evidence for Sulfide Oxidation in Extremely Acidic Cave Biofilms

<div><p>Snottites are extremely acidic (pH 0–2) biofilms that form on the walls and ceilings of hydrogen sulfide-rich caves. Recent work suggests that microbial communities including snottites and related cave wall biofilms accelerate cave formation by oxidizing sulfide to sulfuric acid. Therefore, we used full-cycle rRNA methods and metagenomics to explore the community composition and sulfur metabolism of snottites from the sulfidic Frasassi and Acquasanta cave systems, Italy. Acquasanta snottites were dominated by strains of <i>Acidithiobacillus thiooxidans</i>, with a smaller population of <i>Ferroplasma</i> sp. Frasassi snottites were also dominated by <i>At. thiooxidans</i> but with a more diverse community including relatives of ‘G-plasma’ (Thermoplasmatales), <i>Acidimicrobium</i>, and rare taxa. We identified diverse homologues of sulfide:quinone oxidoreductase (SQR) in the metagenomic datasets. Based on phylogenetic analysis, the numerically dominant <i>At. thiooxidans</i> populations have four different types of SQR, while <i>Ferroplasma</i> has two and <i>Acidimicrobium</i> and G-plasma each have one. No other genetic evidence for sulfur oxidation was detected for either <i>Acidimicrobium</i> or G-plasma, suggesting that they do not generate sulfuric acid. Our results confirm earlier findings that <i>At. thiooxidans</i> is the dominant primary producer and sulfide oxidizer in sulfidic cave snottites.</p> </div

Anaerobic biodegradation of the isoprenoid biomarkers pristane and phytane

Isoprenoids, a diverse class of compounds synthesized by all three domains of life, comprise many of the biomarker compounds used in paleoenvironmental and paleoecological reconstruction of Earth history. These biomarkers include hopanoids, sterols and archaeal membrane lipids. While changes in hydrocarbon profiles in anoxic sediments and oilfields indicate that anaerobic microbial metabolism is involved in the disappearance or alteration of isoprenoids, direct links between specific compounds and their microbial degraders are lacking. Here we describe pristane (Pr) and phytane (Ph) degradation associated with NO^-_3 reduction. We confirmed isoprenoid conversion to CO_2 using ^(13)C-labeled Ph. After 120 days, dissolved inorganic carbon (DIC) produced in incubations grown with ^(13)C-labeled Ph had a δ^(13)C value of +76.7 ± 11.9‰, significantly higher than values for incubations with unlabeled Ph (−35.7 ± 2.0‰) and those without an added carbon substrate (−30.0 ± 2.1‰). Additional incubations, displayed NO^-_3 reduction after amendment with archaeal diphytanyl glycerol diether (DGD) core lipids, but not in those amended with glycerol diphytanyl glycerol tetraether (GDGT) core lipids. Both 16S rRNA clone libraries and whole cell rRNA-targeted fluorescent in situ hybridization (FISH) indicated that the likely Pr and Ph degrading Bacteria were Gamma proteobacteria, with > 99% similarity to Pseudomonas stutzeri

Fate of sulfide in the Frasassi cave system and implications for sulfuric acid speleogenesis

The oxidation of hydrogen sulfide (H2S) has led to the formation of some of the world\u27s largest caves through a process known as sulfuric acid speleogenesis (SAS). Here we present a multi-year study of the large, sulfidic, and actively-forming Frasassi cave system, Italy. We show that despite the presence of abundant sulfide-oxidizing biofilms in Frasassi streams, H2S(g) degassing to the cave atmosphere was the major sink for dissolved sulfide. Degassing rates ranged from 0.9 to 80 μmol m− 2 s− 1, whereas microbial oxidation rates were between 0.15 and 2.0 μmol m− 2 s− 1. Furthermore, microsensor measurements showed that sulfuric acid is not a major end product of microbial sulfide oxidation in the streams. Our results suggest that subaerial SAS will be important for karstification, and more important than subaqueous SAS, wherever ground waters with high sulfide concentrations emerge as flowing streams in contact with cave air

Niche differentiation among sulfur-oxidizing bacterial populations in cave waters

The sulfidic Frasassi cave system affords a unique opportunity to investigate niche relationships among sulfur-oxidizing bacteria, including epsilonproteobacterial clades with no cultivated representatives. Oxygen and sulfide concentrations in the cave waters range over more than two orders of magnitude as a result of seasonally and spatially variable dilution of the sulfidic groundwater. A full-cycle rRNA approach was used to quantify dominant populations in biofilms collected in both diluted and undiluted zones. Sulfide concentration profiles within biofilms were obtained in situ using microelectrode voltammetry. Populations in rock-attached streamers depended on the sulfide/oxygen supply ratio of bulk water (r=0.97; P\u3c0.0001). Filamentous epsilonproteobacteria dominated at high sulfide to oxygen ratios (\u3e150), whereas Thiothrix dominated at low ratios (\u3c75). In contrast, Beggiatoa was the dominant group in biofilms at the sediment–water interface regardless of sulfide and oxygen concentrations or supply ratio. Our results highlight the versatility and ecological success of Beggiatoa in diffusion-controlled niches, and demonstrate that high sulfide/oxygen ratios in turbulent water are important for the growth of filamentous epsilonproteobacteria

Efficient Low-pH Iron Removal by a Microbial Iron Oxide Mound Ecosystem at Scalp Level Run

Acid mine drainage (AMD) is a major environmental problem affecting tens of thousands of kilometers of waterways worldwide. Passive bioremediation of AMD relies on microbial communities to oxidize and remove iron from the system; however, iron oxidation rates in AMD environments are highly variable among sites. At Scalp Level Run (Cambria County, PA), first-order iron oxidation rates are 10 times greater than at other coal-associated iron mounds in the Appalachians. We examined the bacterial community at Scalp Level Run to determine whether a unique community is responsible for the rapid iron oxidation rate. Despite strong geochemical gradients, including a &gt;10-fold change in the concentration of ferrous iron from 57.3 mg/liter at the emergence to 2.5 mg/liter at the base of the coal tailings pile, the bacterial community composition was nearly constant with distance from the spring outflow. Scalp Level Run contains many of the same taxa present in other AMD sites, but the community is dominated by two strains of Ferrovum myxofaciens, a species that is associated with high rates of Fe(II) oxidation in laboratory studies.IMPORTANCE Acid mine drainage pollutes more than 19,300 km of rivers and streams and 72,000 ha of lakes worldwide. Remediation is frequently ineffective and costly, upwards of $100 billion globally and nearly$5 billion in Pennsylvania alone. Microbial Fe(II) oxidation is more efficient than abiotic Fe(II) oxidation at low pH (P. C. Singer and W. Stumm, Science 167:1121-1123, 1970, https://doi.org/10.1126/science.167.3921.1121). Therefore, AMD bioremediation could harness microbial Fe(II) oxidation to fuel more-cost-effective treatments. Advances will require a deeper understanding of the ecology of Fe(II)-oxidizing microbial communities and the factors that control their distribution and rates of Fe(II) oxidation. We investigated bacterial communities that inhabit an AMD site with rapid Fe(II) oxidation and found that they were dominated by two operational taxonomic units (OTUs) of Ferrovum myxofaciens, a taxon associated with high laboratory rates of iron oxidation. This research represents a step forward in identifying taxa that can be used to enhance cost-effective AMD bioremediation

Geochemical Niches of Iron-Oxidizing Acidophiles in Acidic Coal Mine Drainage

A legacy of coal mining in the Appalachians has provided a unique opportunity to study the ecological niches of iron-oxidizing microorganisms. Mine-impacted, anoxic groundwater with high dissolved-metal concentrations emerges at springs and seeps associated with iron oxide mounds and deposits. These deposits are colonized by iron-oxidizing microorganisms that in some cases efficiently remove most of the dissolved iron at low pH, making subsequent treatment of the polluted stream water less expensive. We used full-cycle rRNA methods to describe the composition of sediment communities at two geochemically similar acidic discharges, Upper and Lower Red Eyes in Somerset County, PA, USA. The dominant microorganisms at both discharges were acidophilic Gallionella-like organisms, “Ferrovum” spp., and Acidithiobacillus spp. Archaea and Leptospirillum spp. accounted for less than 2% of cells. The distribution of microorganisms at the two sites could be best explained by a combination of iron(II) concentration and pH. Populations of the Gallionella-like organisms were restricted to locations with pH&gt;3 and iron(II) concentration of &gt;4 mM, while Acidithiobacillus spp. were restricted to pH&lt;3 and iron(II) concentration of &lt;4 mM. Ferrovum spp. were present at low levels in most samples but dominated sediment communities at pH&lt;3 and iron(II) concentration of &gt;4 mM. Our findings offer a predictive framework that could prove useful for describing the distribution of microorganisms in acid mine drainage, based on readily accessible geochemical parameters