Comparing bacterial community fingerprints from white colonizations in Altamira Cave (Spain)

Monitoring bacterial communities is critical for assessing biodeterioration among other processes. This study presents a strategy and an example of comparative analysis of bacterial communities developing in a cave environment, Altamira Cave which contains unique paleolithic paintings. The analyzed question was whether white colonizations discovered throughout the cave corresponded to similar or different bacterial communities. Molecular fingerprints were obtained by PCR–DGGE from DNA and RNA and statistically compared. Results based on DNA analysis showed that a similar bacterial community was present in white colonizations throughout the cave. Fingerprints based on RNA confirmed the similarity of the major metabolically active components of these communities. The proposed procedure confirmed that white colonizations in Altamira Cave were a consequence of the development of a single complex bacterial community, and the method proves to be highly useful for comparative analysis of microbial communities, including biodeteriorating processes and any other comparative analysis of bacterial communities.     

Differential Effects of Distinct Bacterial Biofilms in a Cave Environment

Current microbial surveys using molecular methods provide us with critical information on the major components of natural bacterial communities. However, limited investigation has been performed on the influence of bacterial metabolism on the environment. In this study, we analyzed the pH generated by distinct bacterial communities in a cave environment. Different bacterial biofilms developing on the walls of the cave were visually distinguished by their colorations (e.g., white, yellow, and gray) and mineral depositions, and previous studies have reported on their bacterial diversity and distribution. Using pH microelectrodes, we carried out in situ measurements and were able to detect differences among these bacterial biofilms. White biofilms and carbonate depositions resulted in alkaline pH values. Gray biofilms also increased the pH although these values remained lower than in white biofilms. A combination of gray–white biofilms resulted in alkaline pH values with highest values at the white edge of the colonies. Yellow biofilms generated a slightly acid pH. These results suggest that different bacterial communities can lead to distinct effects on their environment, for instance, precipitation or dissolution of carbonates in caves. These results add information about metabolic response to current knowledge from bacterial diversity surveys, providing information on the interaction between complex bacterial communities and the geological substrate.     

Microbial Community Diversity of Moonmilk Deposits at Ballynamintra Cave, Co. Waterford, Ireland

Caves are extreme and specialised habitats for terrestrial life that sometimes contain moonmilk, a fine-grained paste-like secondary mineral deposit that is found in subterranean systems worldwide. While previous studies have investigated the possible role of microorganisms in moonmilk precipitation, the microbial community ecology of moonmilk deposits is poorly understood. Bacterial and fungal community structure associated with four spatially isolated microcrystalline, acicular calcite moonmilk deposits at Ballynamintra Cave (S. Ireland) was investigated during this study. Statistical analyses revealed significant differences in microbial activity, number of bacterial species, bacterial richness and diversity, and fungal diversity (Shannon's diversity) among the moonmilk sites over an area of approximately 2.5 m². However, the number of fungal species and fungal community richness were unaffected by sampling location. SIMPER analysis revealed significant differences in bacterial and fungal community composition among the sampling sites. These data suggest that a rich assemblage of microorganisms exists associated with moonmilk, with some spatial diversity, which may reflect small-scale spatial differences in cave biogeochemistry.     

New Undescribed Lineages of Non-extremophilic Archaea Form a Homogeneous and Dominant Element Within Alpine Moonmilk Microbiomes

The moonmilk deposits within the alpine Hundsalm cave in Austria offered the opportunity to investigate anthropogenically uninfluenced microbiomes. Via cultivation experiments we were able to show that the communities were cold-adapted and oligotrophic. Combined qPCR, DGGE, cloning and sequencing data further highlighted that the archaeal community basically comprises a low number of species, though highly abundant. These organisms are assumed to form new lineages within the Euryarchaeota, while the detected Thaumarchaeota, closely related to ammonium oxidizers, form a second, but minor, abundant group within the moonmilk deposits. Moreover, in terms of abundance the archaeal community clearly outnumbers bacteria (e.g., genera Pseudomonas, Flavobacterium and Rhodococcus) and fungi within the investigated microbiomes. In contrast to the highly complex bacterial and fungal communities, only a low number of archaeal species form a constant and essential element within the moonmilk speleothems and other cave-internal habitats.     

Biogenic Evidences of Moonmilk Deposition in the Mawmluh Cave,Meghalaya, India

Moonmilk, a microcrystalline secondary cave deposit, actively forms on the floor of Krem Mawmluh – a limestone cave in Meghalaya, Northeastern India. Due to the abundance of micrite and calcified microbial filaments, we hypothesize that these deposits form as a result of ongoing microbial interactions. Consistent with this idea, we report electron microscopic and microbiological evidences for the biological origin of moonmilk in Krem Mawmluh. Scanning electron microscopy indicated abundant calcified microbial filaments, needle calcite, fibre calcites (micro-fibre and nano-fibre calcite crystals), biofilm and microbial filaments in the moonmilk. The total viable culturable microbes showed high population densities for microbes in the moonmilk and moonmilk pool waters. In vitro culture experiments, confirmed the capability of many of the isolated strains to precipitate calcite and some of the identified isolates belonged to the Bacillus sp. and Actinomycetes. These results clearly support the biogenic nature of the deposits.     

Active microbial ecosystem in Iron-Age tombs of the Etruscan civilization

Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted ~2500 years ago and are covered with CaCO₃ needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO₃ precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO₃ precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacteroidetes and Proteobacteria); is populated by photosynthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO₃ precipitating on the rock surfaces (confirmation that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or acidophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.     

Mineralogical Footprints of Bacterial Biofilms Associated with Labit Cave,a Part of the Longest Cave System in India

The present study was an attempt to demonstrate the capabilities of the microbial strains from the unexplored Labit cave in India to precipitate calcite providing evidence for biotic processes involved in formation of speleothem deposits. Six calcifying bacterial strains majority belonging to genus Bacillus were isolated from the cave. SEM studies revealed an array of various in vitro crystal polymorphs generated by the isolated bacteria which are similar to microscopic observations on natural formations in speleothems. The EDX spectrum of the precipitated crystals predominately composed of calcium carbonate indicating the relevance of bacterial biofilm in cave geomicrobiology and biogenic evolution of cave formations in the studied cave, which is further supported by XRF analysis and Raman spectroscopy.     

Role of the microbial community in formation of speleothem (moonmilk) in the Snezhnaya carst cave (abkhazia)

The results of investigation of speleothem (moonmilk) from the Snezhnaya cave (West Caucasus, Abkhazia) are reported. The structure of microbial complexes from moonmilk was investigated by quantitative PCR; strains of culturable microorganisms were isolated and their preferred temperature and carbon sources were determined. Among eubacteria, ~34% belonged to the iron bacteria (Rhodoferax). Most bacterial strains were shown to be facultative psychrophiles with the maximum growth rate at 4°C. The microstructure and elemental composition of moonmilk were investigated using a scanning electron microscope (EVO-40HV, Carl Zeiss) and silicon drift X-ray detector X-MAX 80mm². The visually plastic and homogeneous mass of moonmilk was shown to be highly heterogeneous, containing various microstructures. The elemental composition of some nanostructures depended on the structure of bacterial biofilms. Some loci of the biofilm were found to contain up to 46% (wt/wt) of iron oxides. High calcium content, up to 61.5% (wt/wt), was found only in cubic crystalline structures which were not involved in microbiological processes.     

Metabolically active microbial communities of yellow and grey colonizations on the walls of Altamira Cave, Spain

Aims: To determine the major components of total and metabolically active microbial communities of yellow and grey colonizations threatening the conservation of palaeolithic paintings in Altamira Cave (Spain). Methods and Results: Micro‐organisms present in yellow and grey colonizations were determined from DNA analysis with those showing metabolic activity determined from RNA analysis. Microbial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA libraries were constructed from PCR amplified products. Proteobacteria was the most frequent bacterial phylum. Other phyla detected from RNA‐based microbial surveys were Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae and Gemmatimonadetes. The detected metabolically active micro‐organisms represented only a fraction of the total bacterial community present in the studied colonizations as compared from DGGE analysis. Conclusions: The major bacterial participants in the development of yellow and grey colonizations in Altamira Cave were determined using RNA‐based molecular techniques. Micro‐organisms showing undetectable activity represent a potential risk for the conservation of these paintings if environmental conditions experience variations. Significance and Impact of the Study: Caves with palaeolithic paintings are affected by microbial deterioration. Assessing the composition of the microbial communities colonizing these caves represents a first stage to understand and control these colonizations.     

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.     

Microbial Activity in Caves−A Geological Perspective

Caves are commonly the home of diverse microbial biotas, the sites of active mineral precipitation, and/or receptacles for the deposition of sediment. Mineral precipitation is commonly considered to be abiogenic despite the fact that microbes are present in caves, especially in the twilight zone. Detailed analysis of cave substrates from a geological perspective shows that microbes can mediate constructive (microbe calcification, trapping and binding, mediation of crystal growth) and destructive (substrate etching and breakdown) processes. Potentially these processes can significantly influence the formation and preservation of any cave deposit. Preservation of microbes is possible if mineralization takes place while the microbe is alive or shortly after its demise. If not, all record of the microbe will be lost to decay. Even if the microbes are preserved, it may be difficult to determine if they played an active or passive role in the formation of the deposits in which they are entombed. For old cave deposits, such an assessment must rely on spatial relationships and comparison of textures with those known to form as a result of microbial activity. Nevertheless, available evidence indicates that microbes can play a major role in the formation and modification of cave deposits. Equally, however, it is apparent that the full scope and impact of microbial activity on cave deposits has yet to be realized. Recognition of microbial activity in old CaCO 3 cave deposits relies on (1) documentation and recognition of mineralized microbes, (2) recognition of stromatolitic structures that formed through microbial activity, and/or (3) the identification of fabrics/textures that are known to be indicative of microbial activity. All of these criteria fundamentally rely on the interpretation of fabrics preserved in the cave deposits. Virtually all of these interpretations are open to debate.     

Five-Year Monitoring of Bacterial Communities in Dripping Water from the Heshang Cave in Central China: Implication for Paleoclimate Reconstruction and Ecological Functions

Microbial lipids in stalagmites are increasingly used to reconstruct the paleoclimate change, necessitating the investigations on microbial communities in dripping waters. A pilot molecular survey was conducted on bacterial communities of dripping waters at two sites (1D and 3D) in Heshang Cave of Hubei Province in central China for a period of 2008 to 2013. The samples were subjected to genome DNA extraction, 16S rRNA gene amplification, clone library construction and phylogenetic analysis to explore the seasonal variation of bacterial communities and their association with environmental factors including regional air temperature, precipitation, cave temperature, pH, conductivity and dripping rate of the water samples. Seasonal variations were clearly observed in components and diversities of bacterial communities at both sites. Bacterial community was dominated by Gammaproteobacteria in autumn and winter, whereas Betaproteobacteria became dominant in samples collected in summer and spring. Among the environmental factors investigated, regional air temperature was found to have a strong impact on bacterial communities indicated by cluster and redundancy analysis. Moreover the bacterial biodiversity was observed to increase with the temperature rising. Bacteria identified in dripping water were either oligotrophs or able to acquire nutrients from minerals under oligotrophic conditions. They may also be able to induce calcite precipitation in cave systems. Our data shed light on the potential of microbes used as a tool for the reconstruction of paleo-temperature as well as on the ecological functions of bacterial communities in oligotrophic caves.     

Chromium Adsorption by Three Yeast Strains Isolated from Sediments in Morocco

Kartchner Caverns is an oligotrophic subterranean environment that hosts a wide diversity of actively growing calcite speleothems (secondary mineral deposits). In a previous study, we demonstrated that bacterial communities extracted from these surfaces are quite complex and vary between formations. In the current study, we evaluated the influence of several environmental variables on the superficial bacterial community structure of 10 active formations located in close proximity to one another in a small room of Kartchner Caverns State Park, Arizona, USA. Physical (color, dimensions) and chemical (elemental profile and organic carbon concentration) properties, as well as the DGGE-based bacterial community structure of the formations were analyzed. While elemental concentration was found to vary among the formations, the differences in the community structure could not be correlated with concentrations of either organic carbon or any of the elements evaluated. In contrast, the locations of formations within a distinct region of the cave as well as the relative location of specific formations within a single room were found to have a significant influence on the bacterial community structure of the formations evaluated. Interestingly, Canonical Correspondence Analysis suggests an association between the observed drip pathways (drip lines) feeding the formations (as determined by the patterns of soda straws and small stalactites that reveal water flow patterns) and the bacterial community structure of the respective formations. The results presented here indicate that a broad range of formations fed by a diversity of drip sources must be sampled to fully characterize the community composition of bacteria present on the surfaces of calcite formations in carbonate caves.     

Sulphate-reducing bacteria induce low-temperature Ca-dolomite and high Mg-calcite formation

In this study, we demonstrate that sulphate‐reducing bacteria induce anoxic low‐temperature Ca‐dolomite formation both in situ in Lagoa Vermelha and Brejo do Espinho, two neighbouring, dolomite‐precipitating hypersaline lagoons in Brazil, and in laboratory culture experiments. The metabolic activity of sulphate‐reducing bacteria facilitates dolomite formation under anoxic conditions, as demonstrated with experiments using dialysis bags. Overall changes in the chemical conditions of the medium exclusively, without the presence of bacteria, did not result in carbonate precipitation. Only pure cultures of metabolizing sulphate‐reducing bacteria induced Ca‐dolomite and high Mg‐calcite precipitates, indicating that the carbonate nucleation takes place in the locally changed microenvironment around the sulphate‐reducing bacterial cells. Not all pure strains, however, produced Ca‐dolomite under similar conditions, suggesting that the bacterial metabolism, activity and the rate of mineral precipitation have an influence on the type of carbonate formed.     

Metagenomic Analysis from the Interior of a Speleothem in Tjuv-Ante's Cave,Northern Sweden

Speleothems are secondary mineral deposits normally formed by water supersaturated with calcium carbonate percolating into underground caves, and are often associated with low-nutrient and mostly non-phototrophic conditions. Tjuv-Ante’s cave is a shallow-depth cave formed by the action of waves, with granite and dolerite as major components, and opal-A and calcite as part of the speleothems, making it a rare kind of cave. We generated two DNA shotgun sequencing metagenomic datasets from the interior of a speleothem from Tjuv-Ante’s cave representing areas of old and relatively recent speleothem formation. We used these datasets to perform i) an evaluation of the use of these speleothems as past biodiversity archives, ii) functional and taxonomic profiling of the speleothem’s different formation periods, and iii) taxonomic comparison of the metagenomic results to previous microscopic analyses from a nearby speleothem of the same cave. Our analyses confirm the abundance of Actinobacteria and fungi as previously reported by microscopic analyses on this cave, however we also discovered a larger biodiversity. Interestingly, we identified photosynthetic genes, as well as genes related to iron and sulphur metabolism, suggesting the presence of chemoautotrophs. Furthermore, we identified taxa and functions related to biomineralization. However, we could not confidently establish the use of this type of speleothems as biological paleoarchives due to the potential leaching from the outside of the cave and the DNA damage that we propose has been caused by the fungal chemical etching.     

Actinobacteria Isolated from an Underground Lake and Moonmilk Speleothem from the Biggest Conglomeratic Karstic Cave in Siberia as Sources of Novel Biologically Active Compounds

Actinobacteria isolated from unstudied ecosystems are one of the most interesting and promising sources of novel biologically active compounds. Cave ecosystems are unusual and rarely studied. Here, we report the isolation and characterization of ten new actinobacteria strains isolated from an ancient underground lake and moonmilk speleothem from the biggest conglomeratic karstic cave in Siberia with a focus on the biological activity of the obtained strains and the metabolite dereplication of one active strain. Streptomyces genera isolates from moonmilk speleothem demonstrated antibacterial and antifungal activities. Some of the strains were able to inhibit the growth of pathogenic Candida albicans.     

Dissolution of calcite in the twilight zone: bacterial control of dissolution of sinking planktonic carbonates is unlikely

We investigated the ability of bacterial communities to colonize and dissolve two biogenic carbonates (Foraminifera and oyster shells). Bacterial carbonate dissolution in the upper water column is postulated to be driven by metabolic activity of bacteria directly colonising carbonate surfaces and the subsequent development of acidic microenvironments. We employed a combination of microsensor measurements, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and image analysis and molecular documentation of colonising bacteria to monitor microbial processes and document changes in shell surface topography. Bacterial communities rapidly colonised shell surfaces, forming dense biofilms with extracellular polymeric substance (EPS) deposits. Despite this, we found no evidence of bacterially mediated carbonate dissolution. Dissolution was not indicated by Ca²⁺ microprofiles, nor was changes in shell surface structure related to the presence of colonizing bacteria. Given the short time (days) settling carbonate material is actually in the twilight zone (500–1000 m), it is highly unlikely that microbial metabolic activity on directly colonised shells plays a significant role in dissolving settling carbonates in the shallow ocean.     

Comparative bacterial diversity in recent Hawaiian volcanic deposits of different ages

Volcanic activity creates new landforms that can change dramatically over time as a consequence of biotic succession. Nonetheless, volcanic deposits present severe constraints for microbial colonization and activity. We have characterized bacterial diversity on four recent deposits at Kilauea volcano, Hawaii (KVD). Much of the diversity was either closely related to uncultured organisms or distinct from any reported 16S rRNA gene sequences. Diversity indices suggested that diversity was highest in a moderately vegetated 210-year-old ash deposit (1790-KVD), and lowest for a 79-year-old lava flow (1921-KVD). Diversity for a 41-year-old tephra deposit (1959-KVD) and a 300-year-old rainforest (1700-KVD) reached intermediate values. The 1959-KVD and 1790-KVD communities were dominated by Acidobacteria, Alpha- and Gammaproteobacteria, Actinobacteria, Cyanobacteria, and many unclassified phylotypes. The 1921-KVD, an unvegetated low pH deposit, was dominated by unclassified phylotypes. In contrast, 1700-KVD was primarily populated by Alphaproteobacteria with very few unclassified phylotypes. Similar diversity indices and levels of trace gas flux were found for 1959-KVD and 1790-KVD; however, statistical analyses indicated significantly different communities. This study not only showed that microorganisms colonize recent volcanic deposits and are able to establish diverse communities, but also that their composition is governed by variations in local deposit parameters.     

The impact of reduced pH on the microbial community of the coral Acropora eurystoma

Rising concentrations of atmospheric carbon dioxide are acidifying the world''s oceans. Surface seawater pH is 0.1 units lower than pre-industrial values and is predicted to decrease by up to 0.4 units by the end of the century. This change in pH may result in changes in the physiology of ocean organisms, in particular, organisms that build their skeletons/shells from calcium carbonate, such as corals. This physiological change may also affect other members of the coral holobiont, for example, the microbial communities associated with the coral, which in turn may affect the coral physiology and health. In the present study, we examined changes in bacterial communities in the coral mucus, tissue and skeleton following exposure of the coral Acropora eurystoma to two different pH conditions: 7.3 and 8.2 (ambient seawater). The microbial community was different at the two pH values, as determined by denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis. Further analysis of the community in the corals maintained at the lower pH revealed an increase in bacteria associated with diseased and stressed corals, such as Vibrionaceae and Alteromonadaceae. In addition, an increase in the number of potential antibacterial activity was recorded among the bacteria isolated from the coral maintained at pH 7.3. Taken together, our findings highlight the impact that changes in the pH may have on the coral-associated bacterial community and their potential contribution to the coral host.     

Speleothems from Sahastradhara Caves in Siwalik Himalaya,India: Possible Biogenic Inputs

Stalactites and moonmilk from Sahastradhara caves in Siwalik Himalayas were studied to understand the role of microbes in their genesis. Fourier spectroscopy in the moonmilk indicates a complex milieu of organic compounds that is unusual for inorganic formations. Stable C and O isotopes show trends in the moonmilk and stalactite, which suggest biogenic input; the geochemical inference is consistent with evidence from microscopy and laboratory-based microbial cultures. Light microscopy of moonmilk samples show the presence of a number of microbial forms similar to Cyanobacteria, and scanning electron microscope (SEM) images show microbial structures similar to Spirulina. The total number of microbial cells using SYBR Gold is 6.5 × 10⁵ cells, g sed^?1in moonmilk and 3.2 × 10⁵ cells, g sed^?1 in stalactites. FISH indicates approximately 3.5 × 10⁵ cells, g sed^?1 in moonmilk and 2 × 10⁵ cells, g sed^?1 in stalactites. SEM images of the moonmilk indicate a large network of microbial filaments along with minerals, which are identified as calcite based on their x-ray diffraction pattern. In vitro laboratory cultures with pure monogenic strains isolated from the moonmilk and stalactites raise pH in the medium, which facilitate calcite precipitation. The mineral precipitating isolates were identified as: Bacillus pumilis, B. cereus, B. anthracis, B. lentus, B. sphaericus, B. circulans and Actinomycetes. The Sahastradhara moonmilk and statactites are colonized by a diverse microbial community and the isolated bacterial strains induce biomineralization on different nutrient media, supporting their biogenic origin.