Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

Abstract

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 m 3 ) that may represent intrinsically small organisms or starved, minute forms of normalsized 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°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-m, 0.2-m, and even 0.1-m 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-m-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.

Fluxes of microbes, organic aerosols, dust, sea-salt Na ions, non-sea-salt Ca ions, and methanesulfonate onto Greenland and Antarctic ice

“…Miteva and Brenchley (2005) and Tung et al (2005Tung et al ( , 2006 found from Scanning Electron Microscopy that bacteria in GISP2 ice have sizes typically 0.2 to 0.5 µm, and Priscu (private communication, 2007) found from flow cytometry that, in the WAIS Divide ice core, both biotic particles (those containing DNA that stains with a dye such as Syto 60) and abiotic particles have sizes peaked at 0.3 µm. By contrast, studies of aerosol fluorescence spectra have generally been made on concentrates of atmospheric and marine aerosols (Yamashita and Tanoue, 2003;Coble, 1996) rather sharp emission peak at 353 nm, whereas the emission peak for protein-bound Trp is ∼330 nm, the downshift being the result of binding into proteins.…”

Abundance, viability and diversity of the indigenous microbial populations at different depths of the NEEM Greenland ice core

“…Over 10% of viable bacterial spores were enumerated in GISP2 ice (Yung et al 2007). Systematic studies of 3043-m-deep silty GISP2 ice detected an abundant and diverse microbial population and recovered isolates including ultrasmall and novel microorganisms (Sheridan et al 2003;Miteva et al 2004;Miteva & Brenchley 2005;LovelandCurtze et al 2008LovelandCurtze et al , 2010. Similar viable microbial populations were found in the basal ice of other Greenland glaciers (Yde et al 2010;Stibal et al 2012).…”

Subglacial and Proglacial Ecosystem Responses to Climate Change

“…Sheridan et al (2003) found 6.1 x 10 7 and 9.1 x 10 7 cells ml -1 in two duplicate samples, while Tung et al (2006) counted about 10 11 cells g -1 of sediment. Isolated anaerobic and aerobic enrichment culture studies show a diverse community represented by several major phylogenetic groups: Ǐ-, ǐ-and Ǒ-Proteobacteria, Thermus-Deinococcus, Bacteroides, Eubacterium, Clostridium, Fusobacterium and Actinobacteria (Sheridan et al, 2003;Miteva et al, 2004;Miteva and Brenchley, 2005). The CH 4 concentration in basal ice at GRIP and GISP2 ice cores is much higher (6,000 and 12,000 ppm, respectively) than atmospheric concentrations (1.8 ppm) (Tison et al, 1998;Price and Sowers, 2004), and Tung et al (2005Tung et al ( , 2006 have detected methanogens that are likely to be responsible for producing excess CH 4 concentrations.…”