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Aims: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water, Methods and Results: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338/EUB338-II/EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77-96%) bacteria and 11% (range 3-22%) identified as Archaea., Conclusions: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water., Significance and Impact of the Study: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.

The recently developed CARD-FISH protocol was refined for the detection of marine Archaea by replacing the lysozyme permeabilization treatment with proteinase K. This modification resulted in about twofold-higher detection rates for Archaea in deep waters. Using this method in combination with microautoradiography, we found that Archaea are more abundant than Bacteria (42% versus 32% of 4',6'-diamidino-2-phenylindole counts) in the deep waters of the North Atlantic and that a larger fraction of Archaea than of Bacteria takes up l-aspartic acid (19% versus 10%).