Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 microm3) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5 degrees C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-microm, 0.2-microm, and even 0.1-microm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-microm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.     

祁连山高山植物根际土放线菌生物多样性

从祁连山老虎沟不同海拔位点的15种植物根际土中培养得到78株特异表型放线菌,并结合菌体形态、生理代谢特征、抗菌活性及16S rDNA序列对其生理及系统发育多样性进行了研究。结果表明,分离菌株分属于链霉菌属(Streptomyces spp.)(73株)、诺卡氏菌属(Nocardia spp.)(4株),另有1株与GenBank中同源性最高的菌株Micromonospora saelicesensis相似性达92%,为1潜在新种。链霉菌属为主要类群,占分离菌株的93.6%,该属菌株在5个海拔位点的15种植物根际土中均有分布,但存在海拔位点、植物种类的差异性和特异性;诺卡氏菌属的菌株仅见于海拔2200 m的猪毛菜、海拔2800 m的钉柱萎陵菜和3800 m处的甘肃蚤缀根际土中;1潜在新种分离自海拔2200 m处的沙生针茅根际土。次级代谢物产生和拮抗性筛选研究结果表明:H2O2酶、脂酶2(Tween-40)、脲酶、蛋白酶、脂酶3(Tween-80)、淀粉酶、H2S、脂酶1(Tween-20)、可溶性色素及有机酸这10类次级代谢物产生菌分别占供试菌株的89.7%、82.1%、70.5%、62.8%、53.8%、52.6%、48.7%、44.9%、32.1%和17.9%,其中,淀粉酶、脂酶1、色素和有机酸仅由链霉菌产生;有29株放线菌对参试人类病原菌具有抑制作用,占供试菌株的37.2%,分布于5个海拔位点的12种植物根际土,其中,从药用植物甘肃黄芪和四裂红景天根际土中分离到的抗性菌株占拮抗性放线菌总数的60%。研究表明,高山地区植物根际土放线菌资源丰富,菌株生理功能多样,是新放线菌种和生物活性物质的重要资源库。     

Phylogenetic diversity of acidophilic sporoactinobacteria isolated from various soils

Spore forming actinobacteria (sporoactinobacteria) isolated from soils with an acidic pH in Pinus thunbergii forests and coal mine waste were subjected to taxonomic characterization. For the isolation of acidophilic actinobacteria, acidified starch casein agar (pH adjusted to 4-5) was used. The numbers of actinobacteria growing in acidic media were between 3.2 x 10(4) and 8.0 x 10(6) CFU/g soil. Forty three acidophilic actinobacterial strains were isolated and their 16S rDNA sequences were determined. The isolates were divided into eight distinctive phylogenetic clusters within the variation encompassed by the family Streptomycetaceae. Four clusters among them were assigned to the genus Streptacidiphilus, whereas the remaining four were assigned to Streptomyces. The clusters belonging to either Streptomyces or Streptacidiphilus did not form monophyletic clade. The growth pH profiles indicated that the representative isolates grew best between pH 5 and 6. It is evident from this study that acidity has played a critical role in the differentiation of the family Streptomycetaceae, and also that different mechanisms might have resulted in the evolution of two groups, Streptacidiphilus (strict acidophiles) and neutrotolerant acidophilic Streptomyces. The effect of geographic separation was clearly seen among the Streptacidiphilus isolates, which may be a key factor in speciation of the genus.     

Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4 x 10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.     

Bacterial diversity in malan ice core from the Tibetan Plateau

Three ice core samples were collected from the Malan ice core drilled from the Tibetan Plateau, and three 16S rDNA clone libraries by direct amplification from the ice-melted water were established. Ninety-four clones containing bacterial 16S rDNA inserts were selected. According to restriction fragment-length polymorphism analysis, 11 clones were unique in the library from which they were obtained and used for partial sequence and phylogenetic analysis, and compared with 8 reported sequences from the same ice core at depth 70 m. Differences among the samples were apparent in clone libraries. The phylotypes were dominated by the Proteobacteria group, Acinetobacter sp. and Cytophaga-Flavobacterium-Bacteroides (CFB) group. They accounted for 92.5% (Proteobacteria), 100% (Acinetobacter sp.), 34.4% (CFB) and 100% (beta-Proteobacteria) in the clone libraries from the samples at ice depths 35, 64, 70, and 82 m, respectively. The Acinetobacter sp. was only found in the deposition at ice depth 82 m and closely clustered with gamma-Proteobateria. Two members (Malan A-21 and 101) of alpha-Proteobacteria from the sample of 35 m and two (Malan B-26 and 48) of beta-Proteobacteria of 64 m were loosely clustered (< 95% similarity) with known bacteria, represented new genera in ice bacteria.     

Phylogenetic and phenotypic diversity of 4-chlorobenzoate-degrading bacteria isolated from soils

Twenty numerically dominant 4-chlorobenzoate (4-CBA)-degrading bacteria were isolated from agricultural soils. The isolates were able to utilize 4-CBA as a sole source of carbon and energy. A total of 65% of the isolates was identified to the species level by fatty acid methyl ester (FAME) analysis, and the isolates were strains of Micrococcus, Pseudomonas, Oerskovia, Cellulomonas, and Arthrobacter species. The chromosomal DNA patterns of the isolates obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from each other. Most of the isolates grew rapidly in 4-CBA medium, but their substrate utilization capabilities were generally restricted. Plasmid DNAs were detected from 55% of the isolates, and one strain, HR7, was shown to have self-transmissible, 4-CBA degradative plasmids. 4-CBA degradative enzymes were inducible by the presence of 4-CBA and most of the isolates appeared to mineralize it through 4-hydroxybenzoate rather than 4-chlorocatechol.     

Genomic insights revealed physiological diversity and industrial potential for Glaciimonas sp. PCH181 isolated from Satrundi glacier in Pangi-Chamba Himalaya

Himalayan niches provide unprecedented opportunities for finding novel microbes of commercial importance. The present study investigated the genome sequence of Glaciimonas sp. PCH181 isolated from the glacial stream of Indian trans-Himalaya. The draft genome sequence has six contigs with 5.3 Mb size, 51.1% G + C content, and possesses 4876 genes. Phylogenomic analysis revealed PCH181 as a putative novel bacterium in the genus Glaciimonas. Genomic insight showed Glaciimonas sp. PCH181 enriched with genes for diverse physiology, cold/stress adaptation, and industrial potential. The presence of genes for CO₂ fixation and hydrogen metabolism suggested for chemolithoautotrophy. However, genes for sugars and organic acids usage showed heterotrophy and validated by physiological experiments. Genes for the metabolism of phenol (up to 500 ppm) and biosynthesis of polyhydroxyalkanoate (25% of dry cell mass) were also verified. Collectively, we present the first whole genome sequencing in the genus Glaciimonas, a taxonomically, physiologically, and industrially noteworthy bacterium.     

Phylogenetic diversity of Fusarium incarnatum-equiseti species complex isolated from Spanish wheat

Wheat is the most important cereal grown in the European Union and Spain is its fifth largest wheat producer. There is little information about Fusarium species associated with wheat in Spain. Phylogenetic diversity of 51 strains belonging to Fusarium incarnatum-equiseti species complex (FIESC) isolated from Spanish wheat was investigated using partial sequences of the translation elongation factor gene (EF-1α). Maximum-parsimony and Bayesian analysis of aligned DNA sequences resolved 18 haplotypes and 7 phylogenetic species. Strains morphologically identified as F. equiseti belonged to two different phylogenetic species, FIESC-5 and FIESC-14. Some correlation between phylogenetic species and geographical region was found. The present results highlight the potential contribution of FIESC to the mycotoxin contamination of Spanish wheat.     

Phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from deep-sea microorganisms

The phylogenetic diversity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, E.C. 4.1.1.39) large-subunit genes of deep-sea microorganisms was analyzed. Bulk genomic DNA was isolated from seven samples, including samples from the Mid-Atlantic Ridge and various deep-sea habitats around Japan. The kinds of samples were hydrothermal vent water and chimney fragment; reducing sediments from a bathyal seep, a hadal seep, and a presumed seep; and symbiont-bearing tissues of the vent mussel, Bathymodiolus sp., and the seep vestimentiferan tubeworm, Lamellibrachia sp. The RuBisCO genes that encode both form I and form II large subunits (cbbL and cbbM) were amplified by PCR from the seven deep-sea sample DNA populations, cloned, and sequenced. From each sample, 50 cbbL clones and 50 cbbM clones, if amplified, were recovered and sequenced to group them into operational taxonomic units (OTUs). A total of 29 OTUs were recorded from the 300 total cbbL clones, and a total of 24 OTUs were recorded from the 250 total cbbM clones. All the current OTUs have the characteristic RuBisCO amino acid motif sequences that exist in other RuBisCOs. The recorded OTUs were related to different RuBisCO groups of proteobacteria, cyanobacteria, and eukarya. The diversity of the RuBisCO genes may be correlated with certain characteristics of the microbial habitats. The RuBisCO sequences from the symbiont-bearing tissues showed a phylogenetic relationship with those from the ambient bacteria. Also, the RuBisCO sequences of known species of thiobacilli and those from widely distributed marine habitats were closely related to each other. This suggests that the Thiobacillus-related RuBisCO may be distributed globally and contribute to the primary production in the deep sea.     

工业废水中阿特拉津降解细菌的遗传和生理多样性

【目的】通过遗传学和生理学实验,揭示分离自工业废水的阿特拉津降解细菌具有遗传和生理多样性,为阐明阿特拉津生物降解的分子机理和阿特拉津降解细菌在污染环境生物修复中的应用提供新见解。【方法】用普通PCR方法检测菌株的阿特拉津降解基因,分析其降解基因组成;用基因组重复序列PCR技术(rep-PCR)分析降解菌株的基因组类型;用Western blot方法检测菌株阿特拉津降解途径的第一个酶三嗪水解酶(TrzN);用不同氮源(阿特拉津、莠灭净、扑草净、西玛津、氰草净、阿特拉通和氰尿酸)和碳源(蔗糖、葡萄糖、麦芽糖、乳糖、柠檬酸钠、乙酸钠和琥珀酸钠)培养降解菌株,通过检测培养液的OD600值,证明菌株能够利用的氮源和碳源种类。【结果】对分离自工业废水的27个阿特拉津降解菌株所进行的阿特拉津降解基因PCR检测表明,其降解基因组成分别为trzN-atzBC、trzN-atzABC和atzADEF;通过rep-PCR实验将27个阿特拉津降解菌株分为7个群;Western blot结果表明,27个菌株中有24个含有三嗪水解酶TrzN;氮源利用实验表明,2个菌株能够利用所有7种氮源生长,其余25个菌株只能利用其中的2-6种;碳源利用实验表明,10个菌株能够利用所有7种碳源生长,其余17个菌株只能利用其中的3-6种。【结论】分离自某工业废水的27株阿特拉津降解功能菌存在相当广泛的遗传和生理学上的多样性,trzN-atzABC降解基因组成为首次发现。     

Variation with depth of the abundance,diversity and pigmentation of culturable bacteria in a deep ice core from the Yuzhufeng Glacier,Tibetan Plateau

It has been suggested that the cryosphere is a new biome uniquely dominated by microorganisms, although the ecological characteristics of these cold-adapted bacteria are not well understood. We investigated the vertical variation with depth of the proportion of pigmented bacteria recovered from an ice core drilled in the Yuzhufeng Glacier, Tibetan Plateau. A total of 25,449 colonies were obtained from 1250 ice core sections. Colonies grew on only one-third of the inoculated Petri dishes, indicating that although the ice core harbored abundant culturable bacteria, bacteria could not be isolated from every section. Four phyla and 19 genera were obtained; Proteobacteria formed the dominant cluster, followed by Actinobacteria, Bacteroidetes and Firmicutes. The proportion of pigmented bacteria increased with depth from 79 to 95% and yellow-colored colonies predominated throughout the ice core, making up 47% of all the colonies. Pigments including α- and β-carotene, diatoxanthin, peridinin, zea/lutein, butanoyloxy, fucoxanthin and fucoxanthin were detected in representative colonies with α-carotene being the dominant carotenoid. To the best of our knowledge, this is the highest resolution study of culturable bacteria in a deep ice core reported to date.

     

Phylogenetic diversity of aerobic saprotrophic bacteria isolated from the Daqing oil field

A diverse and active microbial community in the stratal waters of the Daqing oil field (China), which is exploited with the use of water-flooding, was found to contain aerobic chemoheterotrophic bacteria (including hydrocarbon-oxidizing ones) and anaerobic fermentative, sulfate-reducing, and methanogenic bacteria. The aerobic bacteria were most abundant in the near-bottom zones of injection wells. Twenty pure cultures of aerobic saprotrophic bacteria were isolated from the stratal waters. Under laboratory conditions, they grew at temperatures, pH, and salinity values typical of the stratal water from which they were isolated. These isolates were found to be able to utilize crude oil and a wide range of hydrocarbons, fatty acids, and alcohols. Phylogenetic analysis carried out with the use of complete 16S rRNA sequences showed that the isolates could be divided into three major groups: gram-positive bacteria with a high and a low G + C content of DNA and gram-negative bacteria of the gamma-subclass of the Proteobacteria. Gram-positive isolates belonged to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, and Clavibacter. Gram-negative isolates belonged to the genera Pseudomonas and Acinetobacter. In their 16S rRNA sequences, many isolates were similar to the known microbial species and some probably represented new species.     

Phylogenetic diversity of microorganisms associated with the deep-water sponge Baikalospongia intermedia

The diversity of bacteria associated with deep-water sponge Baikalospongia intermedia was evaluated by sequence analysis of 16S rRNA genes from two sponge samples collected in Lake Baikal from depths of 550 and 1204 m. A total of 64 operational taxonomic units, belonging to nine bacterial phyla, Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Planctomycetes, Cloroflexi, Verrucomicrobia, Acidobacteria, Chlorobi, and Nitrospirae, including candidate phylum WS5, were identified. Phylogenetic analysis showed that the examined communities contained phylotypes exhibiting homology to uncultured bacteria from different lake ecosystems, freshwater sediments, soil and geological formations. Moreover, a number of phylotypes were relative to psychrophilic, methane-oxidizing, sulfate-reducing bacteria, and to microorganisms resistant to the influence of heavy metals. It is noted that the unusual habitation conditions of deep-water sponges contribute to the taxonomic diversity of associated bacteria and have an influence on the presence of functionally important microorganisms in bacterial communities.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

安徽定远盐矿可培养嗜盐微生物多样性

【背景】嗜盐微生物多生活于高盐环境,具有独特的生理代谢特征,是一类重要的极端环境微生物资源。【目的】为更好地认识我国陆相盐矿的嗜盐微生物多样性组成,更好地开发利用嗜盐微生物资源积累丰富的微生物菌种。【方法】对安徽定远盐矿盐芯样品进行嗜盐微生物的纯培养分离,并对所分离菌株进行基于16SrRNA基因的测序和序列相似性分析,并对所分离菌株进行物种多样性分析。在此基础上,对代表菌株进行菌落形态和耐盐度及酶活测定。【结果】通过纯培养共分离获得了嗜盐微生物264株,其中嗜盐古菌150株,占56.8%;嗜盐细菌114株,占43.2%。嗜盐古菌物种分别来自于Halorubrum、 Halopenitus、 Haloterrigena、 Natrinema、 Natronoarchaeum和Natronomonas等6个属;嗜盐细菌物种分别来自于Pseudomonas、Aliifodinibius、Halobacillus、Halomonas和Halospina等5个属。通过代表菌株的酶活平板检测,发现产胞外蛋白酶菌株1株,酯酶1株,淀粉酶2株;能液化明胶菌株2株。在物种多样性组成方面,发现嗜盐古菌的物种多样性指数高于嗜盐细菌。【结论】本研究对我国安徽定远陆相盐矿的可培养嗜盐微生物多样性进行探究,积累了丰富的嗜盐微生物菌株资源。     

Phylogenetic diversity and antimicrobial activities of bryozoan-associated bacteria isolated from Mediterranean and Baltic Sea habitats

To date, only a small number of investigations covering microbe–bryozoa associations have been carried out. Most of them have focused on a few bryozoan species and none have covered the antibacterial activities of associated bacteria. In the current study, the proportion and phylogenetic classification of Bryozoan-associated bacteria with antimicrobial properties were investigated. Twenty-one specimens of 14 different bryozoan species were collected from several sites in the Baltic and the Mediterranean Sea. A total of 340 associated bacteria were isolated, and 101 displayed antibiotic activities. While antibiosis was predominantly directed against Gram-positive test strains, 16S rRNA gene sequencing revealed affiliation of the isolates to Gram-negative classes (Flavobacteria, Alpha- and Gammaproteobacteria). One isolate was related to the Gram-positive Actinobacteria. The sequences were grouped into 27 phylotypes on the basis of similarity values ≥99.5%. A host-specific affiliation was not revealed as members of the same phylotype were derived from different bryozoan species. Site-specific patterns, however, were demonstrated. Strains of the genera Sphingomonas and Alteromonas were exclusively isolated from Mediterranean sites, whereas Shewanella, Marinomonas and Vibrio-related isolates were only from Baltic sites. Although Pseudoalteromonas affiliated strains were found in both habitats, they were separated into respective phylotypes. Isolates with 16S rDNA similarity values <98%, which could possibly represent new species, belonged to the genera Shewanella, Pseudoalteromonas and Tenacibaculum.     

Quantification of viable endospores from a Greenland ice core

Endospores (i.e., bacterial spores) embedded in polar ices present an opportunity to investigate the most durable form of life in an ideal medium for maintaining long-term viability. However, little is known about the endospore distribution and viability in polar ices. We have determined germinable endospore concentrations of bacterial spores capable of germination in a Greenland ice core (GISP2 94 m, ID# G2-271) using two complementary endospore viability assays (EVA), recently developed in our laboratory. These assays are based on bulk spectroscopic analysis (i.e., spectroEVA), and direct microscopic enumeration (i.e., microEVA) of ice core concentrates. Both assays detect dipicolinic acid (DPA) release during l-alanine induced germination via terbium ion (Tb3+)-DPA luminescence. Using spectroEVA, the germinable and total bacterial spore concentrations were found to be 295+/-19 spores mL(-1) and 369+/-36 spores mL(-1), respectively, (i.e., 80% of the endospores were capable of germination). Using microEVA, the germinating endospore concentration was found to be 27+/-2 spores mL(-1). The total cell concentration, as determined by DAPI stain fluorescence microscopy, was 7.0 x 10(3)+/-6.7 x 10(2) cells mL(-1). Culturing attempts yielded 2 CFU mL(-1) (4 degrees C). We conclude that endospores capable of germination in the GISP2 ice cores are readily determined using novel endospore viability assays.