Geomicrobiology of sulfuric acid speleogenesis: microbial diversity, nutrient cycling, and controls on cave formation

textMuch of the terrestrial subsurface is inaccessible for study, but caves represent distinctive shallow subsurface habitats where biogeochemical processes can be easily examined. Previously defined speleogenesis models are almost entirely based on abiotic chemical and hydrologic controls, as biological controls on cave formation have not been considered significant. Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, and the sulfuric acid speleogenesis model was introduced in the early 1970s as a cave enlargement process resulting primarily from hydrogen sulfide autoxidation to sulfuric acid and replacement of carbonate by gypsum on subaerially exposed surfaces. The reduced sulfur compounds serve as rich energy sources for microorganisms that colonize the cave in both subaqueous and subaerial environments. Several evolutionary lineages of the class “Epsilonproteobacteria” dominate the microbial diversity of subaqueous mats, and these microbes support the cave ecosystem through sulfur cycling and chemolithoautotrophic carbon fixation. The “Epsilonproteobacteria” occupy microbial mats in additional sulfidic cave and spring habitats, expanding the evolutionary and ecological diversity of these previously unknown organisms. The interior of the Lower Kane Cave microbial mats is devoid of oxygen and this provides habitat for anaerobic metabolic guilds, dominated by sulfate-reducing and fermenting bacteria. These anaerobic groups are responsible for autochthonous hydrogen sulfide and volatile organosulfur gas production. Cycling of carbon and sulfur compounds by the subaqueous microbial communities affects sulfuric acid speleogenesis. Compared to the total flux of sulfide into the cave, little hydrogen sulfide volatilizes into the cave atmosphere or oxidizes abiotically. Instead, the primary loss mechanism is from subaqueous microbial sulfur oxidation. Consequently, despite the cave waters being slightly supersaturated with respect to calcite, the “Epsilonproteobacteria” generate sulfuric acid as a byproduct of their metabolism, locally depress pH, and focus carbonate dissolution. The hydrogen sulfide that volatilizes into the cave air is oxidized at the cave walls where interactions between cave-wall biological and physicochemical factors influence subaerial speleogenesis and low temperature authigenic quartz precipitation. The recognition of the geomicrobiological contributions to subaqueous and subaerial carbonate dissolution fundamentally changes the model for sulfuric acid speleogenesis and the mechanisms for subsurface porosity development.Geological Science

Karst spring microbial diversity differs across an oxygen-sulphide ecocline and reveals potential for novel taxa discovery

Strong geochemical gradients of dissolved oxygen and sulphide establish habitats where specialized bacterial and archaeal taxonomic groups occupy specific redox-sensitive niches, primarily based on metabolic and ecological requirements. In contrast, knowledge of microeukaryote diversity and their ecology in redox-stratified habitats is poor, as species-specific occupation of such geochemical gradients has not been well established. Here we assessed total microbial diversity from rRNA genes retrieved from two morphologically distinct microbial mats formed along an oxygen-sulphide gradient in the outflow channel from the Žveplenica sulphidic karst spring, Slovenia. Microbial mats contained diverse bacteria and archaea associated with chemolithoautotrophic and primary productivity, and overall microeukaryotic diversity was higher under oxygenated conditions. The oxygenated mats were comprised of undescribed and undifferentiated fungi, Annelida, Nematoda, Apicomplexa, and Gastrotricha, some being represented by novel lineages. Under anoxic conditions, diversity was dominated by Ciliophora, Nematoda, and Fungi-Ascomycota, also affiliated with novel lineages. Colonization of the distinct mat types related to ecological tolerance of specific geochemical conditions, and the associations between bacterial and archaeal diversity with distinct microeukaryotes may be related to grazing options and food web structure within the karst system.Key words: karst, spring, sulphide, geochemical gradient, diversity, microeukaryotes.Pestra mikrobna diverziteta vzdolž ekokline kisik-sulfid kraškega izvira odkriva potencial za okritje novih taksonovIzraziti geokemijski gradienti raztopljenega kisika in sulfida so osnova za habitate, kjer v odvisnosti od redoks potenciala, predvsem pa glede na metabolne in ekološke zahteve, specializirane bakterijske in arhejske taksonomske skupine zasedajo občutljive ekološke niše. Nasprotno pa je poznavanje diverzitete mikroevkariontov in njihove ekologije v habitatih, stratificiranih glede na redoks potencial, pomanjkljivo, saj vrstno specifične kolonizacije takšnih gradientnih okolij še nismo dobro proučili. Celotno mikrobno diverziteto smo ovrednotili na podlagi zaporedij rRNA genov, ki so bila pridobljena iz dveh morfološko različnih mikrobnih biofilmov, ki nastajata vzdolž gradienta kisik-sulfid v žveplenem kraškem izviru Žveplenica, Slovenija. Mikrobni biofilmi so vsebovali pestro združbo bakterij in arhej, ki jim pripisujemo kemolitoavtotrofen metabolizem s primarno produkcijo, celotna mikroevkariontska diverziteta pa je bila v oksigeniranih okoljskih razmerah višja. Oksigeniran biofilm so večinoma sestavljale še neopisane in nepoznane glive ter predstavniki skupin Annelida, Nematoda, Apicomplexa in Gastrotricha; nekatere izmed njih pripadajo celo novim linijam. V anoksičnih razmerah so prevladovali predstavniki skupin Ciliophora, Nematoda in Glive-Ascomycota, ki tudi pripadajo novim genetskim linijam. Kolonizacija različnih tipov biofilmov glede na ekološko toleranco specifičnih geokemijskih razmer ter povezava med bakterijsko in arhejsko diverziteto z značilnimi mikroevkariontskimi predstavniki sta lahko povezana z različnimi možnostmi prehranjevanja in s strukturo prehranjevalne verige v kraškem sistemu.Ključne besede: kras, izvir, sulfid, geokemijski gradient, diverziteta, mikroevkarionti.

A unique midgut-associated bacterial community hosted by the cave beetle \u3cem\u3eCansiliella servadeii\u3c/em\u3e (Coleoptera: Leptodirini) reveals parallel phylogenetic divergences from universal gut-specific ancestors

Background Cansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches. Results Abundant microbial cell masses were observed on the external appendages. Cansiliella’s midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals’ digestive traits. Conclusions Results suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions

cave hygropetric beetles and their feeding behaviour a comparative study of cansiliella servadeii and hadesia asamo coleoptera leiodidae cholevinae leptodirini

Several Leptodirini beetles (Leiodidae) are known to dwell in hygropetric habitats where films of water run down the cave walls, but observations of their behaviour are lacking. The ultra- specialised hygropetricolous beetles belonging to the genera Cansiliella and Hadesia are biogeographically and phylogenetically unrelated leptodirines. As the species of the former genus are known to be associated with the moonmilk deposits our study aimed to obtain data on their foraging behaviour, as well as to compare the feeding strategies of both genera. In situ monitoring of C. servadeii from the cave Grotta della Foos (Italy) and H. asamo from Bravenik Cave (Bosnia and Herzegovina), was complemented by video recordings to ensure accurate results. Mouthparts and tarsi of both species were examined using scanning electron microscopy and compared with H. weiratheri from Montenegro to evaluate potential morphological adaptations to the hygropetricolous ecological niches. The three species had significantly different mouthpart morphologies, likely due to differences in semi-aquatic feeding strategies and overall ecology. A series of new observations on site movement and feeding behaviour are presented, compared and discussed. Key words: Coleoptera, ecology, behaviour, moonmilk, cave hygropetric. Primerjava jamskih higropetricnih hroscev in njihovegaprehranjevanja; Cansiliella servadeii in Hadesia asamo (Coleoptera, Leiodidae, Cholevinae, Leptodirini) Stevilni hrosci iz poddružine Leptodirinae (Leiodidae) so prilagojeni na življenje v posebnem habitatu »jamskem higropetriku «, t.j. tankem sloju tekoce vode, ki tece po sigi. Zaenkrat je njihovo vedenje se precejsnja neznanka. Visoko specializirani higropetricni vrsti Cansiliella servadeii in Hadesia asamo sta geografsko loceni in filogenetsko nesorodni. Ker je prva ocitno vezana na depozite mehke sige (t.i. jamsko mleko), smo želeli raziskati iskanje hrane pri tej vrsti ter primerjati strategijo hranjenja obeh vrst. Da bi povecali zanesljivost rezultatov, smo poleg in situ opazovanja C. servadeii iz jame Grotta della Foos (Italija) in H. asamo iz jame Bravenik (Bosna in Hercegovina), njuno vedenje tudi posneli. Z vrsticnim elektronskim mikroskopom smo raziskali obustne okoncine in stopalca obeh vrst in jih primerjali s H. weiratheri iz Crne gore, da bi preverili potencialne morfoloske prilagoditve na specificno ekolosko niso. Bistvene razlike v zgradbi ustnega aparata so najverjetneje posledica razlik v strategiji hranjenja, kot tudi razlicne splosne ekologije omenjenih vrst. V prispevku predstavljamo, primerjamo in razpravljamo o novih opažanjih povezanih z gibanjem in prehranjevanjem higropetricnih hroscev. Kljucne besede: Coleoptera, ekologija, vedenje, jamsko mleko, jamski higropetrik

Microbial Diversity of Cave Ecosystems

The formation of natural caves (speleogenesis) is due to any number of processes that result in the hollowing out of rock, including dissolution, mechanical weathering, volcanic activity, or even the melting of glacial ice. Caves are classified based on the solid rock that they developed within, the proximity to the groundwater table (e.g., above, at, or below it), the speleogenetic history of a feature, and the overall passage morphology and organization (e.g., cave length, passage shape, passage arrangement, passage levels) (Fig. 10.1). Caves are one type of feature that characterizes a karst landscape, which develops in soluble rocks (e.g., limestone, dolomite, gypsum, halite) that roughly coincides with the global distribution of carbonate sedimentary rocks of all geologic ages (e.g., Ford and Williams 2007). Although karst comprises ∼15–20% of the Earth’s ice-free land surface, karst caves are not interconnected, not within the same hydrological drainage basin and definitely not across different drainage basins

Bacterial diversity differences along an epigenic cave stream reveal evidence of community dynamics, succession, and stability

Unchanging physicochemical conditions and nutrient sources over long periods of time in cave and karst subsurface habitats, particularly aquifers, can support stable ecosystems, termed autochthonous microbial endokarst communities (AMEC). AMEC existence is unknown for other karst settings, such as epigenic cave streams. Conceptually, AMEC should not form in streams due to faster turnover rates and seasonal disturbances that have the capacity to transport large quantities of water and sediment and to change allochthonous nutrient and organic matter sources. Our goal was to investigate whether AMEC could form and persist in hydrologically active, epigenic cave streams. We analyzed bacterial diversity from cave water, sediments, and artificial substrates (Bio-Traps®) placed in the cave at upstream and downstream locations. Distinct communities existed for the water, sediments, and Bio-Trap® samplers. Throughout the study period, a subset of community members persisted in the water, regardless of hydrological disturbances. Stable habitat conditions based on flow regimes resulted in more than one contemporaneous, stable community throughout the epigenic cave stream. However, evidence for AMEC was insufficient for the cave water or sediments. Community succession, specifically as predictable exogenous heterotrophic microbial community succession, was evident from decreases in community richness from the Bio-Traps®, a peak in Bio-Trap® community biomass, and from changes in the composition of Bio-Trap® communities. The planktonic community was compositionally similar to Bio-Trap® initial colonizers, but the downstream Bio-Trap® community became more similar to the sediment community at the same location. These results can help in understanding the diversity of planktonic and attached microbial communities from karst, as well as microbial community dynamics, stability, and succession during disturbance or contamination responses over time

Regime shift in sandy beach microbial communities following Deepwater Horizon oil spill remediation efforts.

Sandy beaches support a wide variety of underappreciated biodiversity that is critical to coastal ecosystems. Prior to the 2010 Deepwater Horizon oil spill, the diversity and function of supratidal beach sediment microbial communities along Gulf of Mexico coastlines were not well understood. As such, it was unclear if microbial community compositional changes would occur following exposure to beached oil, if indigenous communities could biodegrade oil, or how cleanup efforts, such as sand washing and sediment redistribution, would impact microbial ecosystem resiliency. Transects perpendicular to the shoreline were sampled from public beaches on Grand Isle, Louisiana, and Dauphin Island, Alabama, over one year. Prior to oil coming onshore, elevated levels of bacteria associated with fecal contamination were detected (e.g., Enterobacteriales and Campylobacterales). Over time, significant shifts within major phyla were identified (e.g., Proteobacteria, Firmicutes, Actinobacteria) and fecal indicator groups were replaced by taxa affiliated with open-ocean and marine systems (e.g., Oceanospirillales, Rhodospirillales, and Rhodobacterales). These new bacterial groups included putative hydrocarbon degraders, similar to those identified near the oil plume offshore. Shifts in the microbial community composition strongly correlated to more poorly sorted sediment and grain size distributional changes. Natural oceanographic processes could not account for the disrupted sediment, especially from the backshore well above the maximum high-tide levels recorded at these sites. Sand washing and tilling occurred on both open beaches from August through at least December 2010, which were mechanisms that could replace fecal indicator groups with open-ocean groups. Consequently, remediation efforts meant to return beaches to pre-spill compositions caused a regime shift that may have added potential ecosystem function, like hydrocarbon degradation, to the sediment. Future research will need to assess the persistence and impact of the newly formed microbial communities to the overall sandy beach ecosystems

Diversity of Uncultured Epsilonproteobacteria from Terrestrial Sulfidic Caves and Springs▿

This study expands the phylogenetic diversity of Epsilonproteobacteria using the 16S rRNA gene framework. Of the 73 lineages defined by sequence similarities at or greater than 99%, most were found at only one site. In contrast, eight lineages were retrieved from sites spanning geographic distances from 1,000 to >10,000 km

Chemolithoautotrophic Organic Matter Contributions to Subterranean Food Webs Dominated by Filter-feeders

IntroductionMost groundwater and cave ecosystems depend on an influx of allochthonous, surface-derived organic matter sourced by diffuse flow through overlying rock and soil or by localized flow from the surface into sinkholes or entrances. The amount of organic matter entering the subsurface is usually low, resulting in oligotrophic conditions and food scarcity that affect community members' dispersal and colonization patterns. In situ, chemolithoautotrophically-produced organic matter has the potential to supplement organic matter pools in the subsurface, especially if the surface and subsurface are hydrologically disconnected. Chemolithoautotrophic contributions are less understood for most groundwater and cave ecosystems, especially from ecosystems dominated by sessile filter-feeders that cannot easily move to search for food. Our study focuses on uncovering the microbiology and organic matter contributions in Croatian Dinaric Karst caves, specifically in the Neretva and Lika River basins, that contain the only subterranean serpulid tube worm, Marifugia cavatica, the only known cave-adapted freshwater bivalves, Congeria kusceri and Congeria jalzici, and stygobitic and stygophilic sponges, Eunapius subterraneus and Ephydatia fluviatilis, respectively.MethodsWe collected surface water, invertebrates, and representative examples of surface organic matter, as well as subsurface water, stygobionts, biofilms, and sediments from Pukotina u Tunelu Polje Jezero in the Neretva River basin and Markov Ponor and Susik Ponor in the Lika River basin. To evaluate microbial communities, 16S rRNA genes were sequenced, analyzed using mothur to obtain operational taxonomic units (OTUs) at 99% sequence similarity, and classified with the SILVA v138.1 reference database. We used the program FAPROTAX and recently published literature to identify putative metabolisms for OTUs, focusing on identifying chemolithoautotrophic functions. We measured stable carbon (δ13C) and nitrogen (δ15N) isotope compositions to assess potential food sources for the stygobionts from surface and subsurface materials.We compared microbial community diversity among caves and sample types using non-metric multidimensional scaling (NMDS) on a Bray-Curtis dissimilarity matrix of rarefied presence/absence data. Analysis of similarity (ANOSIM) on the dissimilarity matrix was used to compare sample type and cave. Welch's t-test was used to compare differences in isotopic composition between surface and caves, and Kruskal-Wallis was used to compare differences among caves. Markov Chain Monte Carlo simulations were employed using mixSIAR v3.1.12, with a chain length of 100000, to calculate the contribution of food sources using a diet tissue discrimination factor of δ13C=1.2±0.39‰ and δ15N=4±0.18‰. All analyses were performed in R using vegan (v. 2.6.4) and stats (v. 4.2) packages.Results and DiscussionMicrobial community composition varied significantly among sample types in each cave (ANOSIM; R=0.74, p<0.005) but weakly among caves and the surface (ANOSIM; R=0.19, p=0.019, Fig. 1a). Putative chemolithoautotrophs included methylotrophs, dissimilatory nitrate-reducers, sulfur-compound-oxidizers, and hydrogen-oxidizers (Fig. 1b). Nitrospirales comprised 2.4% to 10.7% of the biofilms but ≤ 0.2% in water. In Markov Ponor and Susik Ponor, putative methylotrophs (order Methylococcales) comprised 14.0% to 28.3% of the sequence reads in water and 1.2% to 38.7% of reads from biofilms and sediment (Fig. 1b).We hypothesized that isotopic compositions of filter-feeding stygobiont tissues would point to diets that relied on chemolithoautotrophic carbon. δ13C values for dissolved organic carbon in the caves were statistically similar to those of surface water, at -23.9±1.7‰ (n=9), as was dissolved inorganic carbon, at -6.4±2.9‰ (n=8). δ15N values from the cave water ranged from 4.7‰ to 13.9‰, whereas δ15N values from the surface was 9.6‰. Cave biofilm δ13C and δ15N values ranged, respectively, from -29.8‰ to -27.2‰ (average -28.6‰, n=22) and 3.0‰ to 8.2‰ (average 5.8‰, n=20). In contrast, photosynthetic organic matter from the surface ranged from δ13C of -45.7‰ to -17.0‰ and δ15N from 2.4‰ to 6.9‰. Surface invertebrates ranged from δ13C of -32.7‰ to -19.0‰ and δ15N of 1.1‰ to 7.5‰. M. cavatica tissues from the two river basins did not vary significantly, ranging from δ13C of -32‰ to -33‰, but δ15N varied significantly (Welch's t-test p<0.005). The isotopic compositions of Congeria spp. and the sponges varied significantly between the two river basins.Different chemolithoautotrophic pathways have the potential to discriminate against 13C by up to 35‰ or more and 15N from 0‰ to 18‰. Contributions from such fractionation values were evident in biofilm and stygobiont δ13C values compared to surface organic matter δ13C values. Preliminary mixing model results suggest that allochthonous organic matter contributed to most of the stygobionts' diet, likely due to high flow and input rates during the rainy season prior to collection. However, chemolithoautotrophically-produced organic matter could contribute up to 10% of some stygobiont diets, depending on the stygobiont and cave system (Fig. 1c). As such, there is potential that in situ chemolithoautotrophically-produced organic matter could serve as a small dietary buffer for sessile stygobionts during changes in surface conditions that affect the water supply and nutrient input. This research has implications for understanding the microbial ecology and diversity of Croatian karst that support endemic fauna and should motivate efforts to protect the watersheds associated with their habitats

Bray-Curtis similarity distances among groups of samples for sampling times and beach locations.

<p>For Grand Isle, comparisons are (A) between different sampling times and (B) between different locations along the beach profile, and for Dauphin Island comparisons are (C) between different sampling times and (D) between different locations along the beach profile. N.S. = comparisons were not significant. *weakly significant, between <i>p</i>-values 0.051 to 0.1; **significant, between 0.0001 to 0.05; ***highly significant, <0.0001.</p