Caves with dim natural light, and lighted hypogean environments, have been found to host phototrophic microorganisms from various taxonomic groups. These microorganisms group themselves into assemblies known as communities or biofilms, which are associated with rock surfaces. In this work, the phototrophic biofilms that colonise speleothems, walls and floors in three tourist caves (Spain) were studied. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to study these organisms and acquire three-dimensional data on their biofilm structure. CLSM was used in a multi-channel mode whereby the different channels map individual biofilm components. Cyanobacteria, green microalgae, diatoms, mosses and lichens were found to be grouped as biofilms that differed according to the sampling sites. The biofilms were classified into six types regarding their environmental conditions. These types were defined by their constituent organisms, the thickness of their photosynthetic layers and their structure. Light-related stress is associated with lower biofilm thickness and species diversity, as is low humidity, and, in the case of artificially illuminated areas, the duration of light exposure.

Eighteen minerals belonging to eight chemical groups were identified from three caves within Şălitrari Mountain, in the upper Cerna River basin (Romania) by means of scanning electron microscopy, electron microprobe analysis, and X-ray powder diffraction. One passage in the Great Cave from Şălitrari Mountain, the largest cave investigated, exhibits abnormal relative humidity and temperature ranges, allowing for a particular depositional environment. The cave floor is covered by alluvial sediments (ranging from cobble, sand, and clay to silt-sized material), bear bones, bat guano, and rubble. These materials reacted with percolating meteoric water and hydrogen sulfide-rich hypogene hot solutions, precipitating a variety of secondary minerals. Most of these minerals are common in caves (e.g. calcite, gypsum, brushite), however, some of them (alunite, aluminite, and darapskite) require very particular environments in order to form and persist. Cave passage morphologies suggest a complex speleogenetic history that includes changes from phreatic to vadose conditions. The latter was punctuated by a sulfuric acid dissolution/precipitation phase, partly overprinted by present-day vadose processes. The cave morphology and the secondary minerals associated with the alluvial sediments in these caves are used to unravel the region’s speleogenetic history.

In Thornton’s Cave, an estavelle in west-central Florida, SEM, EDS, and XRD data reveal biofilms that are predominantly comprised of FeOOH-encrusted hollow sheaths that are overgrown and intercalated with calcite. Fragments of this crystalline biofilm adhere to the walls and ceiling as water levels vary within the cave. Those on the wall have a ‘cornflake’ appearance and those affixed to the ceiling hang as fibrous membranes. PCR of DNA in the active biofilm, combined with morphologic data from the tubes in SEM micrographs, point to Leptothrix sp., a common Fe-oxidizing bacteria, as the primary organism in the biofilm. Recent discoveries of ‘rusticles’ in other Florida caves suggest that Fe-oxidizing bacteria may reside elsewhere in Florida groundwater and may play a role in the mobility of trace metals in the Upper Florida aquifer.
SEM micrographs from two marble tablets submerged for five months, one exposed to microbial activity and a second isolated from microbial action, revealed no visible etchings or borings and very limited loss of mass. EDS data from the electron micrographs of the unfiltered tablet document the same FeOOH-encrusted hollow sheaths and similar deposits of calcite as seen in the ‘cornflakes’. These results, combined with water chemistry data imply that the biofilm may focus or even promote calcite precipitation during low-water level conditions when CO2 degasses from the cave pools.

A combined atomic force microscope (AFM) and scanning electron microscope (SEM) experiment examining the first steps in limestone weathering and erosion is presented. The experiment
deals with the exposure of polished limestone rock tablets to a Western Mediterranean coastal environment and with the rate and patterns of weathering addressed by roughness
quantification and qualitative assessment of nanoforms. Observations show how rock surface roughness increases at high rates after four and six months of exposure, passing from initial roughness RMS values between 14 and 32 mm to values between 396 to 492 mm. From the qualitative SEM approach, it can be concluded that the roughness increase relates with the widening of the space between rock grains and results in the isolation and detaching of rock grains.

The black limestone from Drenov Grič quarry (Central Slove­nia) is considered one of the most beautiful Slovenian natural stones due to its typical color. The limestone was character­ized from mineralogical, chemical, and petrophysical points of view. Furthermore, deterioration phenomena of the limestone from two monuments exposed to indoor and outdoor environ­ments were studied. In situ investigation of two monuments by means of monument mapping has identified several types of deterioration phenomena, such as granular disintegration, flaking, crumbling, efflorescences, crusts, and the presence of microorganisms. Samples were characterized using Opti­cal Microscopy (OM), Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectroscopy (EDS), X-Ray Powder Diffraction Analysis (XRD), porosity accessible to wa­ter under vacuum, capillary absorption, Mercury porosimetry (MIP), and Ar-sorption. Although very low values of porosity of the fresh stone as well as slow capillary kinetics were de­termined, both monuments showed severe deterioration as a consequence of the transport and precipitation of soluble salts within the stone.

Studies of the influence of microbial communities on calcium carbonate deposits mostly rely on classical or molecular microbiology, isotopic analyses, and microscopy. Using these techniques, it is difficult to infer microbial activities in such deposits. In this context, we used isothermal microcalorimetry, a sensitive and nondestructive tool, to measure microbial activities associated with moonmilk ex-situ. Upon the addition of diluted LB medium and other carbon sources to fresh moonmilk samples, we estimated the number of colony forming units per gram of moonmilk to be 4.8 3 105 6 0.2 3 105. This number was close to the classical plate counts, but one cannot assume that all active cells producing metabolic heat were culturable. Using a similar approach, we estimated the overall growth rate and generation time of the microbial community associated with the moonmilk upon addition of various carbon sources. The range of apparent growth rates of the chemoheterotrophic microbial community observed was between 0.025 and 0.067 h21 and generation times were between 10 and 27 hours. The highest growth rates were observed for citrate and diluted LB medium, while the highest carbon-source consumption rates were observed for low molecular weight organic acids (oxalate and acetate) and glycerol. Considering the rapid degradation of organic acids, glucose, and other carbon sources observed in the moonmilk, it is obvious that upon addition of nutrients during snow melting or rainfall these communities can have high overall activities comparable to those observed in some soils. Such communities can influence the physico-chemical conditions and participate directly or indirectly to the formation of moonmilk.

Volcanic caves have been little studied for their potential as sources of novel microbial species and bioactive compounds with new scaffolds. We present the first study of volcanic cave microbiology from Canada and suggest that this habitat has great potential for the isolation of novel bioactive substances. Sample locations were plotted on a contour map that was compiled in ArcView 3.2. Over 400 bacterial isolates were obtained from the Helmcken Falls cave in Wells Gray Provincial Park, British Columbia. From our preliminary screen, of 400 isolates tested, 1% showed activity against extended spectrum ß-lactamase E. coli, 1.75% against Escherichia coli, 2.25% against Acinetobacter baumannii, and 26.50% against Klebsiella pneumoniae. In addition, 10.25% showed activity against Micrococcus luteus, 2% against methicillin resistant Staphylococcus aureus, 9.25% against Mycobacterium smegmatis, 6.25% Pseudomonas aeruginosa and 7.5% against Candida albicans. Chemical and physical characteristics of three rock wall samples were studied using scanning electron microscopy and f lame atomic absorption spectrometry. Calcium (Ca), iron (Fe), and aluminum (Al) were the most abundant components while magnesium (Mg), sodium (Na), arsenic (As), lead (Pb), chromium (Cr), and barium (Ba) were second most abundant with cadmium (Cd) and potassium (K) were the least abundant in our samples. Scanning electron microscopy (SEM) showed the presence of microscopic life forms in all three rock wall samples. 16S rRNA gene sequencing of 82 isolates revealed that 65 (79.3%) of the strains belong to the Streptomyces genus and 5 (6.1%) were members of Bacillus, Pseudomonas, Nocardia and Erwinia genera. Interestingly, twelve (14.6%) of the 16S rRNA sequences showed similarity to unidentif ied ribosomal RNA sequences in the library databases, the sequences of these isolates need to be further investigated using the EzTaxon-e database (http://eztaxon-e. ezbiocloud.net/) to determine whether or not these are novel species. Nevertheless, this suggests the possibility that they could be unstudied or rare bacteria. The Helmcken Falls cave microbiome possesses a great diversity of microbes with the potential for studies of novel microbial interactions and the isolation of new types of antimicrobial agents.

In the present work, thirteen samples collected from the Grotta Inferiore di Sant’Angelo near the town of Cassano allo Jonio (Calabria region, southern Italy) were analyzed for their mineralogy. The Grotta Inferiore di Sant’Angelo is made up of subhorizontal, interlinked galleries between 400 and 450 meters above sea level. The floor is littered with deposits such as bat-guano, gypsum, and many speleothems that also cover the walls. The samples were identified and characterized by X-ray powder diffraction, scanning electron microscopy with energy dispersive spectrometer, microthermometry, and micro-Raman spectroscopy. The ten primary minerals identified in this study belong to six different groups: carbonate, sulfate, apatite, oxide and hydroxide, halide, and silicate. Clay minerals and eight other detrital minerals were also found: enstatite, rutile, magnesite, pyrite, chrysotile, quartz, dolomite, and chlorite. Characterization of cave minerals could be useful to improve the knowledge of the relation between them and the lithology of the host rocks

A detailed petrographic, structural and morphometric investigation of different types of caves carved in the quartz–sandstones of the “tepui” table mountains in Venezuela has allowed identification of the main speleogenetic factors guiding cave pattern development and the formation of particular features commonly found in these caves, such as funnel-shaped pillars, pendants and floor bumps. Samples of fresh and weathered quartz–sandstone of the Mataui Formation (Roraima Supergroup) were characterised through WDS dispersive X-ray chemical analyses, picnometer measurements, EDAX analyses, SEM and thin-section microscopy. In all the caves two compositionally different strata were identified: almost pure quartz–sandstones, with content of silica over 95% and high primary porosity (around 4%), and phyllosilicate-rich quartz–sandstone, with contents of aluminium over 10% and low primary porosity (lower than 0.5%). Phyllosilicates are mainly pyrophyllite and kaolinite. SEMimages on weathered samples showed clear evidence of dissolution on quartz grains to different degrees of development, depending on the alteration state of the samples. Grain boundary dissolution increases the rock porosity and gradually releases the quartz grains, suggesting that arenisation is a widespread and effective weathering process in these caves. The primary porosity and the degree of fracturing of the quartz–sandstone beds are the main factors controlling the intensity and distribution of the arenisation process. Weathering along iron hydroxide or silt layers, which represent inception horizons, or a strata-bounded fracture network, predisposes the formation of horizontal caves in specific stratigraphic positions. The loose sands produced by arenisation are removed by piping processes, gradually creating anastomosing open-fracture systems and forming braided mazes, geometric networks or main conduit patterns, depending on the local lithological and structural guidance on the weathering process. This study demonstrates that all the typical morphologies documented in these quartz–sandstone caves can be explained as a result of arenisation, which is guided by layers with particular petrographic characteristics (primary porosity, content of phyllosilicates and iron hydroxides), and different degrees of fracturing (strata-bounded fractures or continuous dilational joints).

Karst caves are unique biogeomorphological systems. Cave walls offer habitat for microorganisms which in-turn have a geomorphological role via their involvement in rock weathering, erosion and mineralisation. The attenuation of light with distance into caves is known to affect ecology, but the implications of this for biogeomorphological processes and forms have seldom been examined. Here we describe a semi-quantitative microscopy study comparing the extent, structure, and thickness of biocover and depth of endolithic penetration for samples of rock from the Puerto Princesa Underground River system in Palawan, the Philippines, which is a natural UNESCO World Heritage Site.

Organic growth at the entrance of the cave was abundant (100% occurrence) and complex, dominated by phototrophic organisms (green microalgae, diatoms, cyanobacteria, mosses and lichens). Thickness of this layer was 0.28 ± 0.18 mm with active endolith penetration into the limestone (mean depth = 0.13 ± 0.03 mm). In contrast, phototrophs were rare 50 m into the cave and biofilm cover was significantly thinner (0.01 ± 0.01 mm, p b 0.000) and spatially patchy (33% occurrence). Endolithic penetration here was also shallower (b0.01mm, p b 0.000) and non-uniform. Biofilm was found 250 m into the cave, but with a complete absence of phototrophs and no evidence of endolithic bioerosion.

We attribute these findings to light-induced stress gradients, showing that the influence of light on phototroph abundance has knock-on consequences for the development of limestone morphological features. In marine caves this includes notches, which were most well-developed at the sheltered cave entrance of our study site, and for which variability in formation rates between locations is currently poorly understood.