A millilitre of water extracted from a gas wellhead in Antrim County, Michigan, contains between 4,200 and 6,800 fungal cells. This count, mentioned in The ISME Journal, derives from formation water obtained from operational shale-gas wells located at depths ranging from 247 to 556 metres. Samples of these waters, subjected to separate filtration, resulted in 205 pure fungal cultures that group into 67 unique categories, with 13 of these below the sequence-similarity threshold commonly utilized to classify a fungus as belonging to a recognized species.
The presence of fungi in the deep subsurface isn’t a novel finding. The introduction of the paper notes that an increasing number of studies have identified them, and the conclusions reaffirm their presence and diversity “as previously documented.” What the authors claim to have been unable to locate is an earlier count: they state they could not find past estimations of fungal biomass in subsurface waters.
Devonian mud
The environment in which these fungi exist was formed during the Upper Devonian, approximately between 382.7 and 358.9 million years ago, as planktonic algae and wood settled into sediment. It remains present in a rock containing up to 25 percent organic matter by weight.
The Antrim Shale is among the most significant sources of biogenic methane in the global subsurface, which explains the presence of wells in this area. Methane constituted 86 to 97 percent of the gas that surfaced alongside the water, and the carbon isotopes indicate that the majority of it is microbially derived, originating from fossil carbon rather than being thermally released.
That represents the raw material. The inquiry posed by the study is who occupies the niche.
The water arrived later, and not collectively
Over the past two million years or so, ice sheets have advanced and retreated across the Michigan Basin, fracturing the rock and allowing meltwater to penetrate deeper. Three of the six wells sampled display isotopic characteristics consistent with late Pleistocene glacial meltwater.
The remaining three do not share this characteristic. The two with the highest salinity appear as remnant basinal brine, ancient seawater that has been concentrated through evaporation, while one is associated with contemporary local rainfall, which the authors interpret as recent infiltration through a more permeable limestone layer below.
The chemistry reflects this distinction. The shallowest well, Conant at 247 metres, yields fresh water with 448 milligrams per litre of total dissolved solids. In contrast, the deepest well, Mancelona West at 556 metres, produces brine at 112,000.
These wells have been operating daily for at least ten years since their drilling or last intervention, each generating more than a million litres cumulatively. The research team collected 80 litres from each wellhead into sterile containers.
Counting cells required two stains and a microscope, using water fixed in glutaraldehyde and filtered onto black polycarbonate membranes. Total cells, stained with DAPI, varied between 41,000 to 69,000 per millilitre. Fungal cells, stained with calcofluor white, provided the count of 4,200 to 6,800, with more than 99 percent of the fungal biomass existing as single cells.
Where the one-fifth biomass figure originates
The study’s own section heading states that fungi make up one-fifth of the biomass in the Antrim Shale. Three different figures are considered behind that statement, which do not align, and only two are based on independent measurements.
When comparing the two microscopy ranges, fungi correspond to approximately one counted cell per ten. This calculation is derived from our analysis; the paper illustrates the enumeration ratio in a figure without specifying an exact number in the text.
A second ratio is derived from quantitative PCR targeting marker genes, and it is significantly lower. Using this method, the fungal to bacterial cell ratio ranged from 1 in 7,028 to 1 in 713, with a median of 1 in 2,572. The authors acknowledge that molecular techniques such as qPCR typically yield lower relative values for fungal abundance.
The third figure is the primary one, and it is not a third separate measurement: it is the qPCR ratio reformulated in terms of carbon. Cell counts were converted to carbon, applying 10 femtograms of carbon per bacterial cell and 6,469 femtograms per fungal cell, resulting in a fungal to bacterial carbon ratio ranging from 1:10.86 at one extreme to 1:1.10 at the other, with a median of 1:4.7. The more favorable 1:1.10 end carries a standard error of 1:71, which indicates a lack of precision. The qPCR figures present a similar issue, with standard errors of 1:6,669 on the 1 in 7,028 and 1:1,107 on the 1 in 713.
The conversion factor is the