Ötzi’s frozen remains may harbour metabolically active microbes

Some of the microbes lingering on the 5300-year-old remains of “Ötzi the Iceman” may still be metabolically active, despite being kept in icy conservation conditions.

Ötzi’s mummified body was discovered in 1991 thawing out of an Alpine glacier close to the border of Austria and Italy. He is estimated to have lived at some point between 3350 and 3120 BC, and in the 35 years since he was found, studies of his remains have revealed a treasure trove of information, including that he was probably dark-skinned and balding, had numerous tattoos and had a wound in his shoulder from an arrow, suggesting he was murdered.

Ötzi is now kept at the South Tyrol Museum of Archaeology in Bolzano, Italy, in conditions designed to mimic some of those inside the glacier where he was found: a temperature of -6°C (21°F) and a relative humidity of 99 per cent.

Frank Maixner at Eurac Research’s Institute for Mummy Studies in Bolzano and his colleagues have analysed the bacteria and fungi found in skin swabs, tissue fragments and internal thawed water samples from the mummified remains taken in 1992, 2010 and 2019 and compared them with soil and ice samples collected from the discovery site in the 1990s.

On Ötzi, they found both ancient and modern-day microbes, some of which may be metabolically active. “We can really distinguish between the Iceman’s endogenous gut bacteria and microbes that joined from the environment as soon as he died,” says Maixner.

The team’s metagenomic analysis of internal tissues revealed specialist bacteria that thrive without oxygen inside the mammalian gut, including species of Treponema and Kineothrix. Based on the level of damage to the DNA of these bacteria, which accumulates over time, the bacteria probably lived inside Ötzi when he was alive.

There was a wide variety of microbes present , as seen in other prehistoric gut microbial communities, which may reflect the more varied diet of Copper Age humans compared with modern Western societies, says Maixner.

They also found Pseudomonas bacteria – commonly found in soil and water – in all samples. The DNA damage of these bacteria indicates that they probably belong to an ancient community from the discovery site, says Maixner.

On the external Ötzi samples, the team uncovered cold-loving, or psychrophilic, yeasts, including Phenoliferia, Glaciozyma, Goffeauzyma and Mrakia.

DNA damage indicated that these were also ancient microbes, but the abundance of Glaciozyma increased between 2010 and 2019, and it became the dominant strain, while the level of DNA damage dropped. This suggests it may be metabolically active, or able to replicate under the conservation conditions.

“I think it’s good evidence that Glaciozyma colonised the mummy post mortem. They seem to be growing for some reason,” says Nikolay Oskolkov at Lund University in Sweden, but he would like to see more data points to show that the results aren’t a result of experimental procedure.

The growing abundance of this yeast is potentially very interesting, says Damla Kaptan at the University of Stavanger in Norway. But we can’t be sure it is active until we also look for RNA that would be produced from the yeast DNA, which would indicate that its genes are turned on. “There is still the possibility that the yeast remained dormant or became active to some extent during thawing,” she says.

Some of these yeasts encode enzymes for breaking down protein and collagen, so they could harm the mummy, but the team saw no evidence of damage.

The team also found that some of the microbes contain genes required for degrading the toxic compound phenol. Maixner suggests this may be a side effect of researchers in the 1990s treating the mummy to kill off fungi. “When the Iceman was found, there was already active mould formation and they treated him with a phenol-containing substance,” he says. “Perhaps the microbiome was enriched by the treatment.”

Maixner says the study shows that Ötzi isn’t a biologically frozen time capsule, but a complex ecosystem shaped by the succession of his gut microbes after he died, the infiltration of organisms from the glacier over thousands of years and then three decades of conservation. “As these microorganisms have been on the mummy since the beginning, should they be considered as part of the remains?” he says.

He recommends regular genomic surveillance, including looking for other signs of activity like RNA and metabolites produced by microbes, to check if the microbial communities have really awoken from dormancy and are degrading the mummy’s tissues. If so, scientists might need to consider whether conservation conditions should be changed, perhaps by lowering the temperature or humidity to further restrict microbial activity.