Deep Ocean | Amano Lab | Hokkaido University

Single-cell heterotrophic activity in deep-ocean prokaryotic communities quantified by BONCAT and microautoradiography

Thu, 01 Jan 2026 00:00:00 +0000

Prokaryotes play a central role in marine biogeochemical cycles, yet quantifying their activity requires sensitive methods, particularly in the deep ocean where their biomass and metabolic rates are low. One widely used method to determine single-cell activity of prokaryotes is bioorthogonal non-canonical amino acid tagging (BONCAT), which offers a non-radioactive approach to measure protein synthesis. However, direct comparisons between BONCAT and radioisotope-based techniques across ocean depth gradients remain limited, particularly for low-activity prokaryotic communities. To address this knowledge gap, we applied BONCAT to quantify single-cell heterotrophic activity in prokaryotic communities from surface to bathypelagic depths (1000–4000 m) in the Southern Ocean near the Kerguelen Islands. Employing picolyl azide-based copper-catalysed click chemistry, we compared BONCAT (L-homopropargylglycine [HPG] incorporation) with microautoradiography (3H-methionine uptake). BONCAT consistently detected active cells throughout the water column, with HPG-derived total fluorescence intensity closely correlating with both microautoradiography (R2 = 0.91, P < .001) and bulk methionine incorporation (R2 = 0.94, P < .001). This strong relationship between BONCAT and microautoradiography was maintained into the upper bathypelagic depths, where detecting single-cell activity becomes challenging. Our results demonstrate that BONCAT provides estimates of single-cell heterotrophic activity consistent with microautoradiography in deep-ocean samples, supporting its application as a non-radioactive alternative in low-activity environments.

Comparison of picolyl azide-based BONCAT and microautoradiography for assessing the heterotrophic prokaryotic activity in the deep ocean

Mon, 20 Oct 2025 00:00:00 +0000

Prokaryotes play a central role in marine biogeochemical cycles, yet quantifying their activity requires sensitive methods due to low biomass and metabolic rates, particularly in the deep ocean. One recent method to determine single-cell activity of prokaryotes is bioorthogonal non-canonical amino acid tagging (BONCAT), which offers a non-radioactive approach to measure protein synthesis. However, direct comparisons between BONCAT and radioisotope-based techniques across ocean depth gradients remain limited, particularly for low-activity prokaryotic communities. To address this knowledge gap, we tested an optimised BONCAT protocol using picolyl azide fluorophores (BONCAT-pic) to assess single-cell heterotrophic activity in prokaryotic communities from surface to bathypelagic depths (1000–4000 m) in the Southern Ocean near the Kerguelen Islands. The method was first optimised using aged coastal and open-ocean seawater, and then compared to microautoradiography with 3 H-methionine uptake. Statistical analysis shows that BONCAT-pic significantly improved detection sensitivity compared to standard azide reagents. BONCAT-pic consistently detected active cells in profiles over the open ocean water column, with cell proportions and fluorescence signals closely correlating with both microautoradiography (R 2 = 0.9, p < 0.001) and bulk methionine incorporation (R 2 = 0.6, p < 0.001). Our results demonstrate that BONCAT-pic is a reliable, fluorescence-based method for quantifying heterotrophic activity at the single-cell level, extending its applicability to prokaryotic communities in the deep ocean.

Limited carbon cycling due to high-pressure effects on the deep-sea microbiome

Mon, 28 Nov 2022 00:00:00 +0000

Deep-sea microbial communities are exposed to high-pressure conditions, which has a variable impact on prokaryotes depending on whether they are piezophilic (that is, pressure-loving), piezotolerant or piezosensitive. While it has been suggested that elevated pressures lead to higher community-level metabolic rates, the response of these deep-sea microbial communities to the high-pressure conditions of the deep sea is poorly understood. Based on microbial activity measurements in the major oceanic basins using an in situ microbial incubator, we show that the bulk heterotrophic activity of prokaryotic communities becomes increasingly inhibited at higher hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was about one-third of that measured in the same community at atmospheric pressure conditions. In the bathypelagic zone—between 1,000 and 4,000 m depth—~85% of the prokaryotic community was piezotolerant and ~5% of the prokaryotic community was piezophilic. Despite piezosensitive-like prokaryotes comprising only ~10% (mainly members of Bacteroidetes, Alteromonas ) of the deep-sea prokaryotic community, the more than 100-fold metabolic activity increase of these piezosensitive prokaryotes upon depressurization leads to high apparent bulk metabolic activity. Overall, the heterotrophic prokaryotic activity in the deep sea is likely to be substantially lower than hitherto assumed, with major impacts on the oceanic carbon cycling.