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Sulfidic cave walls host abundant, rapidly-growing microbial communities that display a variety of morphologies previously described for vermiculations. Here we present molecular, microscopic, isotopic, and geochemical data describing the geomicrobiology of these biovermiculations from the Frasassi cave system, Italy. The biovermiculations are composed of densely packed prokaryotic and fungal cells in a mineral-organic matrix containing 5 to 25% organic carbon. The carbon and nitrogen isotope compositions of the biovermiculations (d13C 5 235 to 243%, and d15N 5 4 to 227%, respectively) indicate that within sulfidic zones, the organic matter originates from chemolithotrophic bacterial primary productivity. Based on 16S rRNA gene cloning (n567), the biovermiculation community is extremely diverse, including 48 representative phylotypes (.98% identity) from at least 15 major bacterial lineages. Important lineages include the Betaproteobacteria (19.5% of clones), Gammaproteobacteria (18%), Acidobacteria (10.5%), Nitrospirae (7.5%), and Planctomyces (7.5%). The most abundant phylotype, comprising over 10% of the 16S rRNA gene sequences, groups in an unnamed clade within the Gammaproteobacteria. Based on phylogenetic analysis, we have identified potential sulfur- and nitrite-oxidizing bacteria, as well as both auto- and heterotrophic members of the biovermiculation community. Additionally, many of the clones are representatives of deeply branching bacterial lineages with no cultivated representatives. The geochemistry and microbial composition of the biovermiculations suggest that they play a role in acid production and carbonate dissolution, thereby contributing to cave formation.
Groundwater quality was monitored in four Spanish caves using concentrations of nitrate, potassium, phosphorus and in some cases total organic carbon. Three of the caves are located in NW Spain and contain prehistoric cave paintings and hence have special conservation interest. Of these, two are open show caves (Tito Bustillo and Pindal Caves), while the other one (Herrerías Cave) is not managed for tours and is partially closed off to public access. The fourth cave (Las Maravillas Cave) is located in SW Spain and is opened to the public because of its geological features and natural beauty. In this paper, we compare two sampling methodologies used in the four caves. In Pindal and Herrerias Cave high temporal resolution is achieved with a dripwater collector that collects discrete samples every 48 hours. In Tito Bustillo and Las Maravillas Caves a higher spatial resolution is achieved (16 sampling points in each one), but with a frequency of sampling ranging from 15 days to 6 months. Wastewater and livestock waste appear to be the principal sources of contamination to cave waters. Caves with concentrated livestock (stables) or urban and residential wastewater systems directly situated above the cave exhibit the highest level of contamination detected in elevated concentrations of nitrogen species and in some cases depressed oxygen availability in waters.
Nitrogen fixation was measured in situ for the first time by acetylene reduction for a greyish mat composed of Scytonema julianum in cave-like environments. Mat-specific rates (129.9-215.7 nmol C2 H4 m-2 s-1 for daytime fixation and 65.1-120.6 nmol C2 H4 m-2 s-1 for nighttime fixation) recorded in the Vapor cave differed considerably due to the energy reserves stored during photosynthesis being exhausted and used in the dark phase. The most influential environmental parameter for nitrogen fixation in the Vapor cave is temperature in the daytime and nighttime fixations. Nitrogen fixation by cyanobacteria may contribute considerably to the overall nitrogen cycle in harsh environments such as caves. Nitrogenase activity in Scytonema julianum was roughly 30 times higher than that of Scytonema mirabile, which also grew in cave environments, which is due to the characteristics of each site. The entrance of Vapour cave (Spain) faces SE, measures 0.75 x 0.6 m and opens to shafts of a total depth of 80 m. Its dimensions and environmental conditions (relative humidity up to 100%; maximum temperature, 43ºC) imply that it is isolated from external influences, and that the microclimate differs substantially from that experienced externally. Nitrogen fixation, photon flux density, relative humidity and temperature in the Vapor cave were taken hourly over a 24-hour period in winter.
The troglobitic beetle, Cansiliella servadeii percolating water on moonmilk, a speleothem formation in Grotta della Foos, Italy. Results from analyses of stable isotopes of carbon and nitrogen suggest that acquires and assimilates dissolved allochthonous organic carbon, inorganic nitrogen, and possibly phosphorus and other nutrients from the microbial fauna associated with moonmilk.
Recent sediments in Lake Kozjak, in the Plitvice Lakes National Park, Croatia, were used to study the impact of the material delivered
to the lake sediment via Rječica Brook. Sediment cores, top 40 cm, were taken near the mouth of the Rječica Brook at three locations in the lake at different distances from the shore, water depth 2-9 m and analyzed using different isotopic and chemical methods. Sediment depth profiles of the following parameters were obtained: organic and carbonate fraction, C/N analyses of organic fraction, 14C activity (a14C) and d13C values of carbonate (d13Ccarb) and organic fraction (d13Corg) and d15N of total nitrogen. The results were compared with sediment
core collected in the middle of Lake Kozjak, water depth 21 m. Distribution of organic matter, C/N values, d13Corg and d13Ccarb in sediment profiles showed strong influence of terrestrial
organic and carbonate fraction run-off via Rječica Brook which decreased rapidly in the direction of the open lake. The sedimentation rate as well as the ratio of allogenic and authigenic
fraction in sediments was estimated. Lower d13Corg values in surface sediments might be a consequence of the increase in algal productivity but might also be a contribution of fossil fuel combustion. The anthropogenicaly induced 14C in the atmosphere
in 1963/64 is visible though shadowed in sediments.
Andragulla Cave is 2 m high, 6 m wide, and 2 m long (deep). Its lack of depth means that it is not isolated from external influences, and the microclimate is very similar to that experienced externally. The common stress factors on the growth of cyanobacterial communities on walls inside of Andragulla Cave include excess light, dryness, lack of nutrients, and cold temperatures. Nitrogen fixation, photon flux, relative humidity, and temperature in Andragulla Cave were measured hourly over 24 hours in winter. Nitrogen fixation by the reddish-brown mat formed by both cyanobacteria Scytonema mirabile and Gloeocapsa sanguinea in cave-like environments was measured in situ for the first time by acetylene reduction. The mat-specific rates (1.6 to 7.5 nmol C2H4
m22 s21) were lower compared to published values from laboratory measurements of rehydrated samples from different environments. Daytime fixation was slightly higher than nighttime fixation, where nitrogen fixation by Gloeocapsa sanguinea played an important role. The most influential parameters for environmental nitrogen fixation in
Andragulla Cave were photon flux for daytime fixation, temperature for nighttime fixation, and relative humidity for both. Nitrogen fixation by cyanobacteria may be significant in these N-poor ecosystems.
Karst watersheds with significant losing streams represent a particularly
vulnerable setting for groundwater contamination because of the direct connection to surface water. Because of the existing agricultural land-use and future likelihood of urbanization, two losing-stream karst basins were chosen for intensive monitoring in Boone County, Missouri: Hunters Cave and Devils Icebox. Both caves were formed in Burlington Limestone and have similar recharge areas (33 to 34 km2) and land uses. Year-round monitoring was conducted from April 1999 through March 2002 to characterize the water quality of the main cave streams relative to herbicide, nutrient, and sediment contamination. Water sampling entailed grab samples at regular intervals and runoff-event samples collected using automated sampling equipment. Total nitrogen, phosphorus, and sediment concentrations and loads were consistently higher in the Devils Icebox stream compared to Hunters Cave. Median total N fluxes were 96 g km22 d21 at Devils Icebox and 30 g km22 d21 at Hunters Cave, while median total P fluxes were 8.5 g km22 d21 at Devils Icebox and 3.3 g km22 d21 at Hunters Cave. Herbicides or their metabolites were detected in more than 80% of the samples from both cave streams, and herbicide concentrations and areal loss rates were generally similar between the sites. Overall, the greater loads and mass flux of contaminants in the Devils Icebox recharge area compared to Hunters Cave was a result of both greater stream discharge and the occurrence of more cropped fields (94%) on claypan soils with high runoff potential. These claypan soils are known to be especially problematic with respect to surface transport of contaminants. Prevailing land use has significantly degraded the water quality in both recharge areas, but a watershed plan has been developed for the Bonne Femme watershed, which encompasses these two recharge areas. With the baseline data collected in this study, the impact of changing land uses and the implementation of management practices or new ordinances designed to improve water quality can be documented.
Twenty nine phototrophic taxa are reported from a small volcanic cave. These included five cyanobacteria, two of which are heterocystous nitrogen-fixers, and nine diatoms, several of which have previously been reported from caves. Of the five lichen taxa, two are foliose nitrogen-fixers, suggesting that combined nitrogen was in short supply. The aerophytic flora show similarities with communities from limestone caves suggesting that low light and high humidity are of overriding importance for some taxa.
The microbial diversity of moonmilk, a hydrated calcium carbonate speleothem, was evaluated from two Italian caves to provide context for the food web of highly-specialized troglobitic beetles, Cansiliella spp. (Leptodirinae), with distinctive carbon and nitrogen isotope values indicative of a novel food source. The moonmilk and associated percolating waters had low to no extractable chlorophyll, with an average organic C:N ratio of 9, indicating limited allochthonous input and a significant contribution from microbial biomass. The biomass from moonmilk was estimated to be ~104 micro- and meiofaunal individuals per m2 and ~107 microbial cells/ml. Proteobacteria dominated the 16S rRNA gene sequences retrieved from the moonmilk from both caves. The distribution of other proteobacterial classes and phyla in the moonmilk were statistically similar to each other, even though the two caves are geographically separated from each other. Comparing the moonmilk gene sequences to sequences from previously described environmental clones or cultured strains revealed the uniqueness of the moonmilk habitat, as ~15% of all of the moonmilk sequences were more closely related to each other than to sequences retrieved from any other habitat. However, comparative analyses confirmed that as much as ~34% of the clones sequences were also closely related to environmental clones and cultured strains derived from soil and freshwater habitats, which is likely due to the fact that the putative inoculation source for the moonmilk bacterial communities is from overlying soil and percolating fluids from the surface. Prior to our studies of Cansiliella spp., moonmilk has not been considered a food source for cave animals. Our findings provide unique insight into moonmilk microbial diversity that could reveal the underpinnings of the moonmilk carbon and nitrogen cycle that influences the isotopic composition and the morphological adaptations of the troglobitic beetles associated with the moonmilk.
Lentic cave habitatsare almost always heterotrophic habitats where there are food and oxygen input from the surface. This hydrological exchange seems to be the key factor shaping most groundwater communities. Litter processing in cave water environments has not been experimentally studied as much as it has in lotic subterranean systems, although detritus is likely a critical resource for organisms inhabiting shallow groundwater habitats. The present study sought to evaluate the processing rates and the nitrogen and phosphorous dynamics in plant debris deposited in lentic habitats of two Neotropical limestone caves during 99 days. 84–10×10 cm2 litterbags with mesh sizes of 0.04 mm2 and 9 mm2 were used. In each weighed litter bag, 50 green, intact plant leaf disks (± 2.0 gr/bag) were conditioned. At the end of the experiment, the average weight loss was only 17.4%. No macroinvertebrates were found associated to the debris, but significant differences in the processing rate in relation to the cave and mesh size were observed. The weight loss rate of the plant debris was considered slow (average 0.003 K-day). The amount of nitrogen and remaining phosphorous in the plant debris in the two caves showed variations over time with a tendency to increase probably due to the development of microorganisms which assimilate nitrogen and phosphorus. The slow processing rate of the plant debris can be due mainly to the fact that these lentic cave habitats are restrictive to colonization by shredder invertebrates. Furthermore, the abrasive force of the water, which plays an important role in the processing and availability of fragmented debris for colonization by microorganisms, is absent.
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