In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Abstract

Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2‐para (iodo‐phenyl)‐3(nitrophenyl)‐5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2–6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

Local differences in phytoplankton-bacterioplankton coupling in the coastal upwelling off Galicia (NW Spain)

“…Four 100 ml dark bottles were filled from each sampling depth. Formaldehyde-killed controls (2% v/v final concentration) were performed in order to account for any abiotic reduction of INT (Martínez-García et al 2009); however, formaldehyde-sensitive abiotic reduction (i.e. some cell-free enzymes) was not accounted for in the killed controls.…”

“…Samples were spiked with INT (final concentration 0.2 mM) and incubated in the same incubator used for primary production for 1-1.5 h. After incubation, samples were sequentially filtered through 0.8 µm and 0.2 µm pore size polycarbonate filters. The reduced INT (INT formazan) was extracted from the filters with propanol as described in Martínez-García et al (2009). Respiration of free-living heterotrophic bacteria (i.e.…”

“…INT (2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride) reduction rate was used as estimator of microbial CR using the in vivo INT reduction method (Martínez-García et al 2009). Four 100 ml dark bottles were filled from each sampling depth.…”

Heterotrophic nanoflagellate grazing facilitates subarctic Atlantic spring bloom development

“…Nitrogen is the limiting nutrient in the Arctic (Tremblay et al, 2015). It has been argued that increased temperatures via increased ice melt and runoff will lead to increased thermal and haline stratification of the upper Arctic Ocean and thermal stratification will lead to more shallow mixing in the Subarctic, hence lower nutrient supply (Carmack and McLaughlin, 2011;Martínez-garcía et al, 2009;Schmittner, 2005). Further, the Arctic sea ice changing may lead to an increased inflow of Atlantic water into the Arctic Ocean (Itkin et al, 2014;Schauer et al, 2004;Zhang et al, 1998).…”

Lagrangian study of microbial plankton respiration in the subtropical North Atlantic Ocean: bacterial contribution and short-term temporal variability

“…In order to avoid such long incubations times, we modified the electron transport system (ETS) enzymatic assay in a way that plankton samples are incubated with 2-para(iodo-phenyl)-3(nitrophenyl)-5(phenyl)tetrazolium chloride (INT) over relatively short time periods (3 to 5.5 h in oligotrophic waters). The amount of reduced INT salt (INTFormazan, INT-F), which is proportional to the amount of oxygen consumption, is subsequently quantified spectrophotometrically (see details in Martínez-García et al 2009). In addition, this technique allows post-incubation fractionation into different size classes, thus avoiding the disruption of the microbial community.…”