Phenotypic study of bacteria associated with the caribbean sclerosponge, Ceratoporella nicholsoni.

Heterotrophic bacteria associated with the Caribbean sclerosponge, Ceratoporella nicholsoni (Hickson), were found to occur extracellularly and were confined to the mesohyl regions of the sponge tissue. Physiological, metabolic, and morphological attributes of the culturable bacteria associated with the sponge were recorded by using numerical taxonomy methods for the analysis of 158 phenotypic attributes. Morphometric methods were used to determine the proportion of the total sponge-associated bacteria that were culturable by the methods employed, with the results ranging from 3 to 11% of the total bacteria inhabiting the sponge. Approximately 78% of the culturable bacteria clustered into four groups or phena, representing two previously undescribed Vibrio spp., an Aeromonas sp., and a coryneform- or actinomycete-like sp. Most of the bacteria were facultative anaerobes, fermenting sucrose and fucose but unusual in an inability to ferment glucose. This study was the first comprehensive study of heterotrophic bacteria associated with a sponge from the Caribbean basin, a region reputed to contain the most prolific sponge populations, with respect to biomass and diversity. The possible significance of these associations is discussed.     

Microbial symbionts of the Australian Great Barrier Reef sponge, Candidaspongia flabellata

Microbial symbionts of the newly described rare, biochemically active Dictyoceratid sponge, Candidaspongia flabellata (Very White Fan) found in the Australian Great Barrier Reef, are being studied in detail. The chemistry of this sponge species is distinctive, and includes a previously undescribed compound, fanolide as well as homosesterterpene and bishomoscalarane secondary metabolites (Bergquist et al., 1999). Current research is focused on assessing the diversity of the microbial community associated with this sponge. The entire culturable community of this sponge has been studied in detail. A total of 228 bacteria, 25 fungi, 3 actinomycetes and 9 cyanobacteria were isolated from 10 individuals of this sponge. Eight eubacteria (designated AB001–AB008), along with seven cyanobacteria were consistently found associated with C. flabellata and absent from the surrounding water column, suggesting that these bacteria have a specific association with the sponge. Partial 16S ribosomal RNA gene sequencing of these isolates was done for phylogenetic characterisation. Electron microscopy was also used to confirm the presence of many morphotypes of bacteria and indicated spatial arrangements of particular morphotypes.     

Bacteria associated with an encrusting sponge (Terpios hoshinota) and the corals partially covered by the sponge

Terpios hoshinota, a dark encrusting sponge, is known to be a competitor for space in coral reef environments, and facilitates the death of corals. Although numerous cyanobacteria have been detected in the sponge, little is known of the sponge-associated bacterial community. This study examined the sponge-associated bacterial community and the difference between the bacterial communities in the sponge and the coral partially covered by the sponge by analysis of 16S rRNA gene sequences of samples isolated from the sponge covering the corals Favia complanata, Isopora palifera, Millepora sp., Montipora efflorescens and Porites lutea. The sponge-associated bacterial community was mainly (61-98%) composed of cyanobacteria, with approximately 15% of these alphaproteobacteria and gammaproteobacteria, although the proportions varied in different sponge samples. The dominant cyanobacteria group was an isolated group closely related to Prochloron sp. The comparison of the bacterial communities isolated from sponge-free and the sponge-covered P. lutea showed that covering by the sponge caused changes in the coral-associated bacterial communities, with the presence of bacteria similar to those detected in black-band disease, suggesting the sponge might benefit from the presence of bacteria associated with unhealthy coral, particularly in the parts of the coral closest to the margin of the sponge.     

Interactions of Botryococcus braunii Cultures with Bacterial Biofilms

Unicellular microalgae generally grow in the presence of bacteria, particularly when they are farmed massively. This study analyzes the bacteria associated with mass culture of Botryococcus braunii: both the planktonic bacteria in the water column and those forming biofilms adhered to the surface of the microalgal cells (∼10⁷–10⁸ culturable cells per gram microalgae). Furthermore, we identified the culturable bacteria forming a biofilm in the microalgal cells by 16S rDNA sequencing. At least eight different culturable species of bacteria were detected in the biofilm and were evaluated for the presence of quorum-sensing signals in these bacteria. Few studies have considered the implications of this phenomenon as regards the interaction between bacteria and microalgae. Production of C4-AHL and C6-AHL were detected in two species, Pseudomonas sp. and Rhizobium sp., which are present in the bacterial biofilm associated with B. braunii. This type of signal was not detected in the planktonic bacteria isolated from the water. We also noted that the bacterium, Rhizobium sp., acted as a probiotic bacterium and significantly encouraged the growth of B. braunii. A direct application of these beneficial bacteria associated with B. braunii could be, to use them like inoculants for large-scale microalgal cultures. They could optimize biomass production by enhancing growth, particularly in this microalga that has a low growth rate.     

Antimicrobial activity and diversity of bacteria associated with Taiwanese marine sponge Theonella swinhoei

Marine sponges often rely on other epiphytes for protection from harmful predators. To understand the diversity and antimicrobial activity present among epiphytic bacteria isolated from marine sponge. We used both the 16S rRNA tag pyrosequencing method and the culture-based method to investigate the bacterial communities of Theonella swinhoei collected off the shore of southern Taiwan. Eight-hundred and eighteen operational taxonomic units (OTUs; 97% sequence similarity) were identified from 23,700 sponge-derived sequence tags. The bacteria associated with T. swinhoei were found to be highly diverse—as many as 12 different phyla of bacteria were identified. However, in terms of population evenness, the community was dominated by two phyla—Acidobacteria (71.54%) and Chloroflexi (19.60%). A total of 700 bacterial strains were isolated and cultured from samples of the sponge T. swinhoei. Within these culturable strains, only 12% were Actinomycetes. Despite the low percentage of Actinobacteria from the samples, among the 51 strains of culturable bacteria that showed high antimicrobial activity, a great majority (62%) were Actinomycetes (30 strains of Streptomyces and 1 strain each of Micromonospora and Brevibacterium). The remaining isolates that produced antimicrobial compounds were Gammaproteobacteria (10 strains of Pseudoalteromonas) and Firmicutes (8 and 1 strains of Bacillus and Paenibacillus, respectively). We speculated that many more Actinomycetes are yet to be isolated from T. swinhoei microbiota. Advanced techniques, such as high-throughput culture and culturome, should allow the isolation and purification of these medically important groups of bacteria from sponge.     

Bacteria Associated with the Bleached and Cave Coral Oculina patagonica

The relative abundance of bacteria in the mucus and tissues of Oculina patagonica taken from bleached and cave (azooxanthellae) corals was determined by analyses of the 16S rRNA genes from cloned libraries of extracted DNA and from isolated colonies. The results were compared to previously published data on healthy O. patagonica. The bacterial community of bleached, cave, and healthy corals were completely different from each other. A tight cluster (>99.5% identity) of bacteria, showing 100% identity to Acinetobacter species, dominated bleached corals, comprising 25% of the 316 clones sequenced. The dominant bacterial cluster found in cave corals, representing 29% of the 97 clones sequenced, showed 98% identity to an uncultured bacterium from the Great Barrier Reef. Vibrio splendidus was the most dominant species in healthy O. patagonica. The culturable bacteria represented 0.1–1.0% of the total bacteria (SYBR Gold staining) of the corals. The most abundant culturable bacteria in bleached, cave, and healthy corals were clusters that most closely matched Microbulbifer sp., an α-proteobacterium previously isolated from healthy corals and an α-protobacterium (AB026194), respectively. Three generalizations emerge from this study on O. patagonica: (1) More bacteria are associated with coral tissue than mucus; (2) tissue and mucus populations are different; (3) bacterial populations associated with corals change dramatically when corals lack their symbiotic zooxanthellae, either as a result of the bleaching disease or when growing in the absence of light.     

Allogenic succession of marine bacterial communities in response to pharmaceutical waste

Vibrio spp. predominated in the culturable bacterial community of surface waters of the Puerto Rico Trench at the site of disposal for nearly ten years of pharmaceutical wastes. In this area and surrounding waters as far as 1000 km north of the dumpsite and south into the Caribbean Sea, Vibrio spp. comprised up to 100% of the culturable bacteria, with Acinetobacter spp. being the second most prevalent group. Pseudomonas spp., reported to be common in these waters a decade earlier, were virtually absent from all samples examined during a three year study involving 9 cruises. Staphylococcus spp. were also found in water samples collected within the dumpsite. Using cultures isolated from surface water samples collected at the dumpsite, laboratory experiments confirmed that pharmaceutical waste can enrich for Vibrio spp., in preference to Pseudomonas spp., with growth of the strains proportional to the amount of waste added.     

Allogenic succession of marine bacterial communities in response to pharmaceutical waste

Vibrio spp. predominated in the culturable bacterial community of surface waters of the Puerto Rico Trench at the site of disposal for nearly ten years of pharmaceutical wastes. In this area and surrounding waters as far as 1000 km north of the dumpsite and south into the Caribbean Sea, Vibrio spp. comprised up to 100% of the culturable bacteria, with Acinetobacter spp. being the second most prevalent group. Pseudomonas spp., reported to be common in these waters a decade earlier, were virtually absent from all samples examined during a three year study involving 9 cruises. Staphylococcus spp. were also found in water samples collected within the dumpsite. Using cultures isolated from surface water samples collected at the dumpsite, laboratory experiments confirmed that pharmaceutical waste can enrich for Vibrio spp., in preference to Pseudomonas spp., with growth of the strains proportional to the amount of waste added.     

Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges.     

Bioremediation of bacteria pollution using the marine sponge Hymeniacidon perlevis in the intensive mariculture water system of turbot Scophthalmus maximus

Sessile filter‐feeding marine sponges (Porifera) have been reported to possess high efficiency in removing bacteria pollution from natural or aquaculture seawater. However, no investigation has been carried out thus far in a true mariculture farm water system. Therefore this study sought to investigate the ability of the marine sponge Hymeniacidon perlevis to bioremediate the bacteria pollution in the intensive aquaculture water system of turbot Scophthalmus maximus. Sponge specimens were hung in fish culture effluent at different temperature to investigate the optimal temperature condition for bacteria removal by H. perlevis. Turbots S. maximus were co‐cultured with sponge H. perlevis in 1.5 m³ of water system at 15–18°C for 6 weeks to control the growth of bacteria. It was found that H. perlevis was able to remove pathogenic bacteria efficiently at 10–20°C, with a maximal removal of 71.4–78.8% of fecal coliform, 73.9–98.7% of pathogenic vibrio, and 75.0–83.7% of total culturable bacteria from fish‐culture effluent at 15°C; H. perlevis continuously showed good bioremediation of bacteria pollution in the S. maximus culture water system, achieving removal of 60.0–90.2% of fecal coliform, 37.6–81.6% of pathogenic vibrio, and 45.1–83.9% of total culturable bacteria. The results demonstrate that H. perlevis is an effective bioremediator of bacteria pollution in the turbot S. maximus culture farm water system. Biotechnol. Bioeng. 2010;105: 59–68. © 2009 Wiley Periodicals, Inc.     

Diversity of bacteria and polyketide synthase associated with marine sponge Haliclona sp.

The microbial community associated with a marine sponge (Haliclona sp.) collected from Tateyama city, Japan was studied using 16S rRNA gene clone libraries. Two DNA templates were prepared using methods recommended for Gram-positive and Gram-negative bacteria in the Qiagen kit manual. From each DNA template, two 16S rRNA genes were PCR amplified, using the combination of universal bacterial primer 27f and primers 1385r and 1492r, respectively. A total of 347 clones were sequenced and compared with those available in DNA data banks. These sequences were members of ten bacterial phyla. Interestingly, more than 30 % of the clones represent novel sequences. A comparison of these sequences with sequences in a library prepared from DNA extracted from the surrounding water shows minimum DNA contamination. Taxonomically, the highest diversity was detected in the clone library prepared using a combination of primers 27f and 1492r and DNA isolated using the Gram-positive bacteria protocol. The potential of Haliclona sp.-associated bacteria to produce secondary metabolites was studied by cloning and sequencing the polyketide synthase (PKS, type 1) gene using the same DNA samples. Analysis of partial sequences derived from the sponge metagenome revealed 27 unique ketosynthase domains of PKS type I. This study suggests strongly that this Haliclona sp. plays host to diverse novel bacteria with a potential to produce novel polyketides.     

Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, <Emphasis Type="Italic">Vetulina</Emphasis> sp

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Pathogenic bacteria associated with lesions and thallus bleaching symptoms in the Japanese kelp Laminaria religiosa Miyabe (Laminariales,Phaeophyceae)

During early Spring (April–May) when the seawater salinity drops suddenly and the seawater temperature increases drastically, severe lesions and thallus bleaching were observed in the Laminaria religiosa population at Oshoro Bay, Otaru, Hokkaido, Japan. The healthy and diseased kelp blades were collected and subjected to enumeration of total number of culturable bacteria and bacterial species. Bacterial enumerations were done using 3 different media formulations; high-nutrient media (Media 1), low-nutrient media (Media 2) and modified low-nutrient media with 5% kelp extract (Media 3). Seven bacterial species were isolated from the healthy kelp. These were Alcaligenes aquamarinas, Alteromonas sp., Azomonas agilis, Azotobacter beijerinckii, Escherichia coli, Halobacterium sp. and Halococcus sp. All 7 bacterial species were isolated on Media 2 and Media 3, but only 5 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 2050 CFU/cm² on Media 3. Eight species of bacteria were isolated from the diseased kelp thallus with the addition of Erwinia amylovora. All 8 bacteria grew on Media 2 and Media 3, but only 6 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 5830 CFU/cm² on Media 3. However, only 3 species were isolated from the lesioned area. The most abundant species was Alteromonas sp. followed by Halococcus sp. and Alcaligenes aquamarinas. The surface bacteria showed best growth on Media 3. Scanning Electron Microscopic images of the healthy and diseased thallus gave distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population. In an effort to identify the symptoms causative organism, the isolated bacterial species were cultured and used to test Koch's postulates. Out of the 8 species, only Alteromonas sp. induced lesions on the axenic kelp blades. The inoculated bacteria were also re-isolated without any significant contamination. Hence, Alteromonas sp. is suggested as the possible disease causing organism.     

Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms

Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir. The present investigation was aimed to determine the role of cyanobacteria in the persistence of V. cholerae O139 in microcosms. An environmental isolate of V. cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study. Survival of culturable V. cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium. Viable but nonculturable V. cholerae O139 were detected using a fluorescent antibody technique. Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria. The viable but nonculturable V. cholerae O139 could be detected in association with cyanobacteria for up to 15 months. These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V. cholerae O139 in an aquatic environment.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.     

Changes in Microbial Communities,Including both Uncultured and Culturable Bacteria,with Mid-Ocean Ballast-Water Exchange during a Voyage from Japan to Australia

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships'' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.     

Diverse UV-B Resistance of Culturable Bacterial Community from High-Altitude Wetland Water

Isolation of most ultraviolet B (UV-B)-resistant culturable bacteria that occur in the habitat of Laguna Azul, a high-altitude wetland [4554 m above sea level (asl)] from the Northwestern Argentinean Andes, was carried out by culture-based methods. Water from this environment was exposed to UV-B radiation under laboratory conditions during 36 h, at an irradiance of 4.94 W/m². It was found that the total number of bacteria in water samples decreased; however, most of the community survived long-term irradiation (312 nm) (53.3 kJ/m²). The percentage of bacteria belonging to dominant species did not vary significantly, depending on the number of UV irradiation doses. The most resistant microbes in the culturable community were Gram-positive pigmented species (Bacillus megaterium [endospores and/or vegetative cells], Staphylococcus saprophyticus, and Nocardia sp.). Only one Gram-negative bacterium could be cultivated (Acinetobacter johnsonii). Nocardia sp. that survived doses of 3201 kJ/m² were the most resistant bacteria to UV-B treatment. This study is the first report on UV-B resistance of a microbial community isolated from high-altitude extreme environments, and proposes a method for direct isolation of UV-B-resistant bacteria from extreme irradiated environments. This article is dedicated to the memory of Carolina Colin.