Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of Lake Baikal, Russia.

One of the fundamental methods for cultivating bacterial strains is conventional plating on solid media, but this method does not reveal the true diversity of the bacterial community. In this study, we develop a new technique and introduce a new device we term, I-tip. The I-tip was developed as an in situ cultivation device that allows microorganisms to enter and natural chemical compounds to diffuse, thereby permitting the microorganisms to grow utilizing chemical compounds in their natural environment. The new method was used to cultivate microorganisms from Baikalian sponges, and the results were compared with conventional plating as well as a pyrosequencing-based molecular survey. The I-tip method produced cultures of 34 species from five major phyla, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, 'missing' only two major phyla detected by pyrosequencing. Meanwhile, standard cultivation produced a smaller collection of 16 species from three major phyla, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, failing to detect over half of the major phyla registered by pyrosequencing. We conclude that the I-tip method can narrow the gap between cultivated and uncultivated species, at least for some of the more challenging microbial communities such as those associated with animal hosts.

Diversity of cyanobacterial species and phylotypes in biofilms from the littoral zone of Lake Baikal.

The majority of naturally occurring biofilms contain numerous microorganisms that have not yet been cultured. Additionally, there is little information available regarding the genetic structure and species diversity of these communities. Therefore, we characterised the species diversity, structure and metagenome of biofilms grown on stones and steel plates in the littoral zone of Lake Baikal (East Siberia, Russia) by applying three different approaches. First, light microscopy enabled identification of the species diversity of biofilm-forming cyanobacteria on different substrates with the dominance of Rivularia rufescens, Tolypothrix limbata, Chamaesiphon fuscus, Ch. subglobosus, and Heteroleibleinia pusilla. Additionally, scanning electron microscopy was used to show the spatial structure of biofilms. Finally, sequence analysis of 30,660 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to Cyanobacteria (8-46% sequences), Proteobacteria (14-43%), and Bacteroidetes (10-41%). Rivularia sp., Pseudanabaena sp., and Chamaesiphon spp. were the dominant cyanobacterial phylotypes.

Stratified distribution of nutrients and extremophile biota within freshwater ice covering the surface of Lake Baikal.

Biological entities and gradients of selected chemicals within the seemingly barren ice layers covering Lake Baikal were investigated. Ice cores 40-68 cm long were obtained from in shore and offshore sites of Southern Lake Baikal during the cold period of a year (March-April) in 2007 and 2008. In microscopic observations of the melted ice, both algae and bacteria were found in considerable numbers (>10(3) cells/L and >10(4) cells/ml, respectively). Among all organisms found, diatom was generally the most predominant taxon in the ice. Interestingly, both planktonic and benthic algae were present in considerable numbers (2-4×10(4) cells/L). Dominant phototrophic picoplankton were comprised of small green algae of various taxa and cyanobacteria of Synechococcus and Cyanobium. The bacterial community consisted mostly of short rod and cocci cells, either free-living or aggregated. Large numbers of yeast-like cells and actinomycete mycelium were also observed. Concentrations of silica, phosphorus, and nitrate were low by an order of magnitude where biota was abundant. The profile of the ice could be interpreted as vertical stratification of nutrients and biomass due to biological activities. Therefore, the organisms in the ice were regarded to maintain high activity while thriving under freezing conditions. Based on the results, it was concluded that the freshwater ice covering the surface of Lake Baikal is considerably populated by extremophilic microorganisms that actively metabolize and form a detritus food chain in the unique large freshwater ecosystem of Lake Baikal.

Activity of bacteria in water of hot springs from Southern and Central Kamchatskaya geothermal provinces, Kamchatka Peninsula, Russia.

The hot-spring waters of numerous hot springs at the Kamchatka Peninsula, Russia differ in their chemical characteristics and elemental composition. Total bacterial abundance (TBA) as well as enzymatically active and respiring bacteria was enumerated in water samples collected from the Nalychevskie, Oksinskie, Apapelskie, and Dachnye hot springs. 5-Carboxyfluorescein diacetate activity was detected in all water samples and comprised 29-65% of the TBA as determined by 4',6-diamidino-2-phenylindol staining. The respiratory activity of iron-oxidizing bacteria was assayed by 5-cyano-2,3-ditolyltetrazolium chloride reduction. Respiring cells accounted for 9-14% of the TBA, indicating a positive correlation with the number of iron-oxidizing bacteria from the hot-spring biomats. Enumeration of heterotrophic bacteria revealed a high-density bacterial population only in the water of the Apapelskie hot spring, which has a temperature of 36 degrees C. Therefore, it appears that heterotrophic and iron-oxidizing bacteria inhabiting the hot-spring waters are essential for the geochemical processes occurring in hydrothermal systems.

Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia.

The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperature of 55-56 degrees C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-microm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH.

Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia / Biominerales de Fe-Si de las fuentes termales de Vilyuchiskie, en la península de Kamchatka (Rusia)

The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperatureof 55–56°C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-rayspectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-μm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH (AU)

Se describe el estudio de la estructura micromorfológica de los tapetes microbianos (biomats) de las fuentes termales del sistema hidrotermal de Vilyunchinskaya, en la península de Kamchatka (Rusia). Las fuentes hidrotermales de Vilyichinskie tenían una temperatura de descarga de 55–56ºCy sus aguas son del tipo Na-Ca-HCO3, ricas en ácidos silícico y bórico. El aguay los tapetes microbianos tenían una alta concentración de Fe, Mn, Sr y As. La enumeración de la abundancia total de bacterias (ATB) mostró una baja densidad de poblaciones bacterianas. Sin embargo, en el agua de las fuentes termales las fracciones de bacterias metabólicamente activas y de bacterias respiradoras oxidadoras de Fe eran elevadas, del 68 y el 21% de ATB, respectivamente. El estudio mediante el microscopio electrónico de barrido equipado con un espectrofotómetro de rayos X dispersor de energía (SEM-EDX) mostró que en los tapetes microbianos marrones predominaban las bacterias unicelulares en forma de bacilo de unos 5 μm. Las cápsulas minerales de estas bacterias contenían gran cantidad de Fe y Si. La observación con microscopía electrónica de transmisión reveló la presencia de partículas extracelulares e intracelulares. De los tapetes microbianos se aislaron bacterias oxidadoras de Fe mediante placas de agar con medio selectivo. Por tanto, se puede concluir que los microorganismos de los tapetes microbianos de las fuentes termales de Vilyuchinskie son esenciales para el depósito de minerales de hierro a pH neutro (AU)