Respiration from activity of respiratory chain: enzymatic assays

• Page authors: PRIMO
• Responsible curator: Hagen Buck-Wiese

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ETS activity
Approach: enzymatic assay (tetrazolium reduction)
Context: incubation, lab
Spatial scale: point sample
Temporal scale: minutes
Units: µmol O2 L-1 h-1 (converted from INT-formazan)
Community captured: bulk
Co-measurements: INT-formazan absorbance (485 nm), incubation time, volume, temperature, R/ETS calibration

Method Overview

The Electron Transport System (ETS) assay estimates the maximum respiratory capacity of a microbial community by exploiting the ability of ETS enzymes — principally NADH and NADPH dehydrogenases — to reduce artificial electron acceptors. In the classic assay, disrupted cell preparations are supplied with saturating concentrations of NADH and NADPH as electron donors, and a tetrazolium salt (most commonly INT, iodonitrotetrazolium chloride) as the terminal electron acceptor. INT is reduced to a brightly coloured, insoluble formazan (INT-F) whose absorbance at 485 nm is proportional to ETS activity.

An in vivo variant (in vivo ETS) adds only the tetrazolium salt to intact cells, avoiding the homogenisation step and enabling smaller sample volumes. ETS activity is converted to an equivalent oxygen consumption rate via an empirically derived R/ETS ratio — the ratio of actual respiration (R) to ETS potential activity — which must be determined for each system.

Scale of measurement

As a ship- or lab-based incubation processed from a discrete water sample, the method provides a single point in space. Incubations are short (minutes), which minimises bottle effects; however, the measurement reflects the potential respiratory capacity at saturating substrate concentrations, not the actual in situ rate.

Data generated

The assay yields ETS activity expressed as µmol INT-F produced L^-1 h^-1, which is converted to potential oxygen consumption rate (µmol O₂ L^-1 h^-1) using a calibration factor. This represents the maximum respiratory capacity of the community, which can be used to constrain in situ respiration rates when the R/ETS ratio is known.

Units & currency

Units are µmol O₂ L^-1 h^-1, derived by conversion from INT-formazan measurements. The primary currency measured is tetrazolium formazan fluorescence, which is converted to oxygen equivalents.

Sample size

Typical samples are > 1 L for the classic ETS assay; the in vivo ETS variant can work with < 1 L.

Limitations

Rates measured are potential rates at saturating substrate concentrations (V_max), not in situ rates. The method assumes that all organisms take up INT and are not inhibited by its toxicity, and that INT reduction occurs exclusively via ETS activity rather than other reductive pathways. The empirical R/ETS ratio must be determined for each study environment, as it varies with temperature, community composition, and physiological state, introducing a major source of uncertainty when transferring calibrations across systems.

Example Applications & Protocols

Classic examples

• Martínez-García et al. (2009) In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Recent applications

• Osma et al. (2016) Predicting in vivo oxygen consumption rate from ETS activity and bisubstrate enzyme kinetics in cultured marine zooplankton

Common calculations/conversions

• O₂ consumption (µmol L^-1 h^-1) = ETS activity (µmol INT-F L^-1 h^-1) × R/ETS; the R/ETS ratio typically ranges from 0.09 to 0.5 in marine environments.