Microbes From 120,000-year-old Ice Sample Show Life's Tenacity

According to Penn State's Vanya I. Miteva, research associate, and Jean E. Brenchley, professor of microbiology and biotechnology, the majority of the microbes they discovered in an ice-core sample taken from the glacier were less than 1 micron in size -- smaller than most commonly known bacteria, which range from 1 to 10 microns. In addition, a large portion of the cells appeared to be even smaller and passed through filters with 0.2-micron pores.

The scientists are interested in understanding how microbial life can be preserved in polar ice sheets for hundreds of thousands of years under stresses that include subzero temperatures, desiccation, high pressures, and low oxygen and nutrient concentrations. Because the ice was mixed with the ancient permafrost at the bottom of the glacier, the microbes could have been trapped there for perhaps millions of years.

"We are particularly interested in the formation of ultra-small cells as one possible stress-survival mechanism, whether they are starved minute forms of known normal-sized microbes or intrinsically dwarf novel organisms, and also whether these cells are able to carry on metabolic processes while they are so highly stressed," Miteva says.

Physiological changes that accompany the reduction of a cell's size may allow it to become dormant or to maintain extremely low activity with minimal energy.

"Many of these ice-core microbes are related to a variety of ultra-small microorganisms from other cold environments that have been shown to use different carbon and energy sources and to be resistant to drying, starvation, radiation, and other stress factors. Their modern relatives include the model ultra-micro bacterium Sphingopyxis alaskensis, which is abundant in cold Alaskan waters," Brenchley reports. She and Miteva are in the process of closely examining all the microbes they found in order to determine the identities and diversity of the species and to look for ones with novel functions.

The researchers used a variety of methods including repeated sample filtrations, electron microscopy and a modified technique of flow cytometry to quickly reveal the number of cells and to estimate their different sizes, DNA content, and other characteristics. Miteva and Brenchley discovered cells with many different shapes and sizes, including a large percentage that were even smaller than filter-pore sizes of only 0.2 microns.

"It appears that these ultra-small microbes often are missed in research studies because they pass through the finest filters commonly used to collect cells for analysis," Miteva says.

Scientists believe these dwarf cells belong to the "uncultured majority" because they are among the 99 percent of all microbes on Earth that never have been isolated and cultured for study. Obtaining such "isolates" is necessary in order to describe a new organism, study its cell size, examine its physiology, and assess its ecological role.

"We now know just the tip of the iceberg of all the microbes that exist on Earth, and it generally is believed that a large portion of these unknown microbes are very small in size," Miteva says.

"A major challenge is to develop novel approaches for growing some of these previously unculturable organisms," Brenchley says. "At present, no single established protocol exists and little is known about the recovery of these stressed and possibly damaged cells from a frozen environment that subjects them to severe conditions for long periods."

Some of the cells that Miteva and Brenchley were successful in cultivating required special conditions and up to six months to form initial colonies. The researchers discovered that these colonies grew more rapidly during further cultivation and that most continued to form predominantly small cells.

"Our study of the abundance, viability, and identity of the ultra-small cells existing in the Greenland ice is relevant to discovering how small life-forms can be; how cells survive being small, cold and hungry; and what new tricks we need to develop in order to cultivate these small cells," Miteva says. "This study is part of the continuing quest by microbiologists to overcome the current limitations of our methods and to answer the big question, 'What new microbes are out there and what are they doing?'"

This research was supported by the Department of Energy (Grant DE-FG02-93ER20117) and the Penn State Astrobiology Center (NASA-Ames Cooperative Agreement No. NCC2-1057).

More information and photos are available on the Web at http://www.science.psu.edu/alert/Brenchley5-2004.htm