Isolating and studying environmental ultrasmall microorganisms with possible novel metabolic activities is important because it may help define their functional diversity and ecological role. This study was based on a previous observation that very small cells dominated the microbial population found in a deep Greenland ice core (22
). It has been suggested that ultrasmall microbial cells are capable of occupying the narrow liquid veins in glacier ice and possibly metabolizing at very low rates (25
). Because small cells may be well adapted to the multiple stress conditions in glacier ice, the ultrasmall isolates obtained from glacier ice will be valuable for future experiments designed to test their survival and metabolic capabilities in ice and the factors needed for their recovery and cultivation.
In this study we examined a variety of methods for analyzing ultrasmall cell populations and for cultivating isolates. We found that carefully controlled flow cytometry and SEM analyses confirmed the presence of numerous small cells capable of passing through 0.2-μm, as well as 0.1-μm, filters. Thus, the traditional method of collecting cells by filtering and examining only those captured on the filters excludes many ultrasmall cells and small spores. Occasionally, cells larger than the filter pore sizes were observed in some filtrates. These cells either may have passed through the filters because of variations in pore sizes or flexibility of the cells during filtration (12
) or may have originally been smaller, starved dormant cells that increased their size following cultivation. According to DeLong (8
), this physiological strategy appears to be common, but its actual distribution among microbial phyla is poorly understood. It is still unknown what fraction of the naturally occurring small cells represents physiologically induced dwarf forms versus stable diminutive phenotypes. Limited studies of several cultivated, intrinsically small bacteria suggest that they keep their small size independent of the growth conditions. It also is assumed that these organisms are slowly but constantly growing and thus can become a dominant population in their habitat. This is true for Sphingopyxis alaskensis
), but it is not clear whether similar characteristics will be found for other cultured ultrasmall organisms, such as our isolates.
Other reports indicated that a large portion of the nonculturable cells in environmental samples actually corresponds to the ultrasmall cells (17
). Strategies and procedures for cultivating these organisms differ, but general principles are to mimic the natural environment, use filtration or extinctive dilutions in very-low-nutrient media, and use gradual adaptation to laboratory cultivation conditions (5
). Our enrichment strategy combined a selective filtration step with low-temperature incubation in oligotrophic, anaerobic media for several months. The 0.2-μm filtration excluded the larger cells and allowed only the ultrasmall and very thin filamentous cells to be transferred to the next round of enrichment, thus preventing overgrowth by larger, fast-growing organisms and the possible accumulation of substances inhibitory to the smaller cells. The anaerobic conditions possibly prevented oxidative stress and allowed cells that could have been damaged or dormant to resuscitate. Finally, the long-term incubations at low temperature and in low-nutrient media mimicked some conditions in the glacier environment.
One goal of this work was to answer the question whether isolates obtained after filtration and cultivation differed morphologically and phylogenetically from those not specifically enriched for ultrasmall cells. Our comparisons of the numbers and diversity of isolates from filtered and nonfiltered melted ice and filtered and nonfiltered, low-temperature cultures lead to several conclusions. First, we found that the filtration step increased the number of colonies obtained from both the melted ice and liquid cultures. The increase in culturability of cells directly from filtered melted ice to 2 to 6% may be caused partially by the physical separation and detachment of cell aggregates or by the removal of inhibitory substances during filtration. In addition, there was a significant increase in the number of isolates (1,290) obtained from the filtration enrichments compared to the number of isolates (447) obtained from unfiltered cultures. It is possible that the successive rounds of filtration and incubation provided the acclimation and recovery conditions that ultrasmall cells required to form colonies.
A second conclusion is that the filtration-cultivation steps not only increased the number of colonies but could also alter the diversity of isolates. For example, members of the high-G+C group were isolated mostly from the directly plated melted ice and its filtrates, whereas Proteobacteria
were dominant among isolates from enrichment cultures. A third observation is that the proportions of 0.2-μm-filterable cells in filtered and nonfiltered liquid cultures differed after short incubations but these differences diminished after several months. This is consistent with our report (22
) that long-term cultivation in liquid oligotrophic media at low temperature significantly improved the recovery and/or growth of previously nonculturable organisms, including ultrasmall isolates. These results suggest that the time of incubation is an important factor that may allow damaged or dormant cells to resuscitate and that after sufficient time, ultrasmall cells can become dominant. Other authors also pointed out that long incubation periods at low temperature during subcultivation of ultrasmall environmental microorganisms may initiate an unknown mechanism allowing colony formation on rich media (3
An important question is whether the small-celled population in a specific environment is composed of only a few species or whether it is highly diverse. Different studies have shown that direct cultivation of 0.2-μm-filterable cells from different environments resulted in obtaining remarkably low numbers of isolates of restricted diversity (39
). Our results also showed that despite the relative increase in culturability from filtered melted ice, the isolates obtained directly from the melted ice had limited diversity. The dominance of Arthrobacter
spp. may be explained by pleomorphism (rod-coccus cycle) and their ability to reduce their cell sizes up to 10 times in response to starvation (17
). Some isolates (SO3-1, SO3-5, SO3-12, SO3-16, and SO3-17a), however, were distantly related to known Arthrobacter
species and may represent novel species that form intrinsically smaller cells.
Based on the phylogenetic analysis of the isolates obtained from filtered and nonfiltered cultures, we conclude that diversity increased after the long-term cultivation, and a large number of ultrasmall cells were found in all major phylogenetic groups. Some isolates were related to organisms known for their small sizes, such as Microbacterium
. Representatives of the genus Sphingomonas
were the most numerous Proteobacteria
from all filtered and nonfiltered group 1 and 2 cultures. However, the increased number of isolates obtained from the filtered enrichments suggests that the filtration-cultivation procedure provided favorable growth conditions for this bacterial group, including previously uncultured organisms and some that are related to known ultramicrobacteria, such as UMB 13 and UMB 38. Other Proteobacteria
isolates were related to Acinetobacter
, the characterized species of which also undergo cell size reduction in response to starvation (14
). Interestingly, phylogenetically novel ultrasmall bacteria belonging to the Cytophaga-Flavobacteria-Bacteroides
group were isolated from short-term enrichments in different media, suggesting that their cells may have predominated prior to being outcompeted or have been relatively fast recovering and/or growing during the initial incubation.
The cultivation of spore-forming representatives is interesting because we had previously noted that the absence of spore-forming isolates was surprising (22
), since spores could survive for thousands of years. One explanation had been the inability of spores in the sample to germinate. In contrast, the filtration and liquid incubation in the present investigation resulted in the isolation of a significant number of diverse spore-forming bacilli after 18 months of incubation. Because little is known about spore germination from environmental samples (24
), it is not clear whether the filtration step enhanced germination, perhaps by eliminating inhibitory factors or other competing cells, or whether the longer incubation permitted germination and subsequent cell growth. Several isolates, distantly related to Bacillus mucilaginosus
(9.5%), possessed small cells and spores with similar small sizes and deserve further attention as a possible novel ultrasmall-cell taxon at the family or order level.
An important result from this work is that our procedure of successive filtration-cultivation steps led to a significant increase in the total number and diversity of cultured isolates with a parallel increase of the small-celled organisms compared to direct plating of filtered melted ice. The extended low-temperature cultivation in liquid oligotrophic medium also appeared to be a factor in the recovery of ultrasmall microorganisms and should be considered in the development of strategies for their isolation. In conclusion, the filtration-cultivation procedure combined with long cultivation times allowed the enrichment of ultrasmall microbes from the deep Greenland glacier ice core sample, improved their culturability, and resulted in the isolation of diverse ultramicrobacteria, including phylogenetically novel ones. Further characterization of our collection of ultrasmall isolates may provide insight into novel metabolic properties and the mechanisms for long-term survival under extreme cold conditions.