Eva Sintes | 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.

Anaplerotic processes are key contributors to dark carbon fixation in the ocean

Wed, 25 Sep 2024 00:00:00 +0000

Abstract Anaplerotic carbon fixation is ubiquitous in heterotrophic organisms including those inhabiting the ocean1. Despite its prevalence, the drivers of this process and its significance in ocean carbon cycling remain poorly understood2,3. Here we combined global ocean metatranscriptomic analysis, laboratory experiments on a bacterial model strain, and microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) on marine microbial communities, to uncover the global prevalence of anaplerotic processes in oceanic dark dissolved inorganic carbon (DIC) fixation. Metatranscriptomic analysis revealed high expression levels of key anaplerotic genes, especially in mesopelagic waters, comparable to those of photo- and chemolithoautotrophic DIC fixation genes. Alteromonas emerged as the main contributor to anaplerotic DIC fixation gene expression, highlighting its role in DIC assimilation in the global ocean. Laboratory incubations with a marine Alteromonas representative confirmed their capability to fix DIC, which varied with organic matter availability and temperature. MICRO-CARD-FISH on oceanic samples revealed that Alteromonas contributed 0–40% (14 ± 16%, mean ± s.d.) to the dark DIC fixation in the pelagic ocean. Considering that Alteromonas is an obligate heterotroph lacking chemoautotrophic DIC fixation genes, its contribution to DIC fixation should be attributed to anaplerotic processes. Based on these results, we estimated a contribution of anaplerotic processes to dark DIC fixation of 0–0.5 C Pg y-1 in the global dark ocean. Yet, since Alteromonas is not the only taxon performing anaplerotic DIC fixation, our results represent a baseline conservative estimate. Collectively, our findings place anaplerotic DIC fixation as a relevant processes in the oceanic carbon cycling.

A device for assessing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI)

Wed, 14 Dec 2022 00:00:00 +0000

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high‐pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H‐leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep‐sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep‐sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep‐sea biotic processes.

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.

Impact of hydrostatic pressure on organic carbon cycling of the deep-sea microbiome

Thu, 31 Mar 2022 00:00:00 +0000

Deep-sea microbial communities are exposed to high hydrostatic pressure. While some of these deep-sea prokaryotes are adapted to high-pressure conditions, the contribution of piezophilic (i.e., pressure-loving) and piezotolerant prokaryotes to the total deep-sea prokaryotic community remains unknown. Here we show that the metabolic activity of prokaryotic communities is increasingly inhibited with increasing hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in sit u hydrostatic pressure was only about one-third of that measured on the same community at atmospheric pressure conditions. Only ∼5% of the bathypelagic prokaryotic community are piezophilic while ∼85% of the deep-sea prokaryotes are piezotolerant. A small fraction (∼10%) of the deep-sea prokaryotes is piezosensitive (mainly members of Bacteroidetes, Alteromonas) exhibiting specific survival strategies at meso- and bathypelagic depths. These piezosensitive bacteria elevated their activity by more than 100-fold upon depressurization. Hence, the consistently higher bulk metabolic activity of the deep-sea prokaryotic community measured upon depressurization is due to a rather small fraction of the prokaryotic community. Overall, the heterotrophic prokaryotic activity in the deep-sea is substantially lower than hitherto assumed with major impacts on the oceanic carbon cycling.

Mesozooplankton taurine production and prokaryotic uptake in the northern Adriatic Sea

Thu, 25 Jun 2020 00:00:00 +0000

Dissolved free taurine, an important osmolyte in phytoplankton and metazoans, has been shown to be a significant carbon and energy source for prokaryotes in the North Atlantic throughout the water column. However, the extent of the coupling between taurine production and consumption over a seasonal cycle has not been examined yet. We determined taurine production by abundant crustacean zooplankton and its role as a carbon and energy source for several prokaryotic taxa in the northern Adriatic Sea over a seasonal cycle. Taurine concentrations were generally in the low nanomolar range, reaching a maximum of 22 nmol L−1 in fall during a Pseudonitzschia bloom and coinciding with the highest zooplankton taurine release rates. Taurine accounted for up to 5% of the carbon, 11% of the nitrogen, and up to 71% of the sulfur requirements of heterotrophic prokaryotes. Members of the Roseobacter clade, Alteromonas, Thaumarchaeota, and Euryarchaeota exhibited higher cell‐specific taurine assimilation rates than SAR11 cells. However, cell‐specific taurine and leucine assimilation were highly variable in all taxa, suggesting species and/or ecotype specific utilization patterns of taurine and dissolved free amino acids. Copepods were able to cover the bulk taurine requirements of the prokaryotic communities in fall and winter and partly in the spring–summer period. Overall, our study emphasizes the significance of taurine as a carbon and energy source for the prokaryotic community in the northern Adriatic Sea and the importance of crustacean zooplankton as a significant source of taurine and other organic compounds for the heterotrophic prokaryotic community.