M. C. Portillo

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).

Amplification by PCR Artificially Reduces the Proportion of the Rare Biosphere in Microbial Communities

The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the ‘rare biosphere’. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature.

Is the availability of different nutrients a critical factor for the impact of bacteria on subterraneous carbon budgets

Bacteria thriving in underground systems, such as karsts, adapt to use a variety of nutrients. Most of these nutrients derive from superficial processes. This study shows that bacteria are able to differentially induce carbonate precipitation or dissolution depending on the availability of nutrients for growth. Different bacterial strains isolated from caves, representing the most common components of these microbial communities, were cultured with different carbon and nitrogen sources (e.g., acetate, glucose, peptone, humic acids) and induced changes in pH were measured during growth. Carbonate can either precipitate or dissolve during bacterial growth. The induction of carbonate precipitates or their dissolution as a function of consumption of specific carbon sources revealed the existence of an active nutrient cycling process in karsts and links nutrients and environmental conditions to the existence of a highly significant carbon sink in subterraneous environments.

Differential microbial communities in hot spring mats from Western Thailand.

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.

Assessment of Bacterial and Fungal Growth on Natural Substrates: Consequences for Preserving Caves with Prehistoric Paintings

The most representative bacterium (Pseudonocardia sp.) and fungus (Fusarium sp.) from the microbial communities of a cave containing paleolithic paintings were isolated and their growth on natural substrates assessed. Growth was tested at the in situ and optimal, laboratory growth temperature. Development was analyzed with and without supplemented nutrients (glucose, ammonium, phosphate, peptone). Results showed that the assayed bacterium on natural substrate was able to develop best at in situ temperature and the addition of organic nutrients and/or phosphate enhanced its growth. The growth of the assayed fungus, however, was limited by low temperature and the availability of ammonium. These results confirm a differential behavior of microorganisms between the laboratory and the natural environments and could explain previous invasion of fungi reported for some caves with prehistoric paintings.

Moonmilk Deposits Originate from Specific Bacterial Communities in Altamira Cave (Spain)

The influence of bacterial communities on the formation of carbonate deposits such as moonmilk was investigated in Altamira Cave (Spain). The study focuses on the relationship between the bacterial communities at moonmilk deposits and those forming white colonizations, which develop sporadically throughout the cave. Using molecular fingerprinting of the metabolically active bacterial communities detected through RNA analyses, the development of white colonizations and moonmilk deposits showed similar bacterial profiles. White colonizations were able to raise the pH as a result of their metabolism (reaching in situ pH values above 8.5), which was proportional to the nutrient supply. Bacterial activity was analyzed by nanorespirometry showing higher metabolic activity from bacterial colonizations than uncolonized areas. Once carbonate deposits were formed, bacterial activity decreased drastically (down to 5.7% of the white colonization activity). This study reports on a specific type of bacterial community leading to moonmilk deposit formation in a cave environment as a result of bacterial metabolism. The consequence of this process is a macroscopic phenomenon of visible carbonate depositions and accumulation in cave environments.

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow‐pigmented, oxalotrophic bacteria

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1(T), YOx(T) and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1(T), YOx(T) and NS13 belong to the Betaproteobacteria, most closely related to Oxalicibacterium flavum TA17(T) (97.2-99.7% sequence similarity). The major whole-cell fatty acids were C(16:0), C(16:1)omega7c and C(17:0) cyclo. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1(T) and YOx(T) from O. flavum TA17(T) and from each other. Therefore, it is concluded that the strains OD1(T) and YOx(T) represent novel species within the genus Oxalicibacterium, for which the names Oxalicibacterium horti sp. nov. (type strain OD1(T)=DSM 21640(T)=NBRC 13594(T)) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx(T)=DSM 21641(T)=CCM 2767(T)) are proposed.

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.

Feasibility of sunflower oil cake degradation with three different anaerobic consortia

Sunflower oil cake (SuOC) is the solid by-product from the sunflower oil extraction process and an important pollutant waste because of its high organic content. For the anaerobic digestion of SuOC three different industrial reactors were compared as inoculum sources. This was done using a biochemical methane production (BMP) test. Inoculum I was a granular biomass from an industrial reactor treating soft-drink wastewaters. Inoculum II was a flocculent biomass from a full-scale reactor treating biosolids generated in an urban wastewater treatment plant. Inoculum III was a granular biomass from an industrial reactor treating brewery wastes. The highest kinetic constant for methane production was achieved using inoculum II. The inoculum sources were analyzed through PCR amplification of 16S rRNA genes and fingerprinting before (t = 0) and after the BMP test (t = 12 days). No significant differences were found in the bacterial community fingerprints between the beginning and the end of the experiments. The bacterial and archaeal communities of inoculum II were further analyzed. The main bacteria found in this inoculum belong to Alphaproteobacteria and Chloroflexi. Of the Archaea detected, Methanomicrobiales and Methanosarcinales made up practically the whole archaeal community. The results showed the importance of selecting an appropriate inoculum in short term processes due to the fact that the major microbial constituents in the initial consortia remained stable throughout anaerobic digestion.

Fervidobacterium thailandense sp. nov., an extremely thermophilic bacterium isolated from a hot spring

Strain FC2004T, a strictly anaerobic, extremely thermophilic heterotroph, was isolated from a hot spring in Thailand. Typical cells of strain FC2004T were rod shaped (0.5-0.6×1.1-2.5 µm) with an outer membrane swelling out over an end. Filaments (10-30 µm long) and membrane-bound spheroids containing two or more cells inside (3-8 µm in diameter) were observed. The temperature range for growth was 60-88°C (optimum 78-80°C), pH range was 6.5-8.5 (optimum pH 7.5) and NaCl concentration range was 0 to <5 g l-1 (optimum 0.5 g l-1). S0 stimulated growth yield. S2O32- and NO3- did not influence growth. Glucose, maltose, sucrose, fructose, cellobiose, CM-cellulose and starch were utilized for growth. The membrane was composed mainly of the saturated fatty acids C16:0 and C18:0. The DNA G+C content was 45.8 mol%. The 16S rRNA gene sequence of strain FC2004T revealed highest similarity to species of the genus Fervidobacterium: F. pennivorans DSM 9078T (97-96 %), F. islandicum AW-1 (96 %), F. changbaicum CBS-1T (96 %), F. islandicum H21T (95 %), F. nodosum Rt17-B1T (95 %), F. riparium 1445tT (95 %) and F. gondwanense AB39T (93 %). Phylogenetic analysis of 16S rRNA gene sequences and average nucleotide identity analysis suggested that strain FC2004T represented a novel species within the genus Fervidobacterium, for which the name Fervidobacterium thailandense sp. nov. is proposed. The type strain is FC2004T (=JCM 18757T=ATCC BAA-2483T).