View source for Respiration from activity of respiratory chain: enzymatic assays

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* [[Page authors|Page authors]]: [[PRIMO]]
* [[Responsible curator|Responsible curator]]:  [[User:Hagi BucknWise|Hagen Buck-Wiese]]
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{| class="model-ib" style="float:right; margin-left:1em; margin-bottom:1em;"
! ETS activity
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| '''Approach:''' enzymatic assay (tetrazolium reduction)
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| '''Context:''' incubation, lab
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| '''Spatial scale:''' point sample
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| '''Temporal scale:''' minutes
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| '''Units:''' µmol O<sub>2</sub> L<sup>-1</sup> h<sup>-1</sup> (converted from INT-formazan)
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| '''Community captured:''' bulk
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| '''Co-measurements:''' INT-formazan absorbance (485 nm), incubation time, volume, temperature, R/ETS calibration
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== 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<ref name="MartínezGarcía2009">Martínez-García, S., Fernández, E., Aranguren-Gassis, M., & Teira, E. (2009). In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities. ''Limnology and Oceanography: Methods'', 7(6), 459–469. https://doi.org/10.4319/lom.2009.7.459</ref>.

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<ref name="Osma2016">Osma, N., Fernández-Urruzola, I., Gómez, M., Montesdeoca-Esponda, S., & Packard, T. T. (2016). Predicting in vivo oxygen consumption rate from ETS activity and bisubstrate enzyme kinetics in cultured marine zooplankton. ''Marine Biology'', 163, 146. https://doi.org/10.1007/s00227-016-2923-x</ref>.

=== 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<sup>-1</sup> h<sup>-1</sup>, which is converted to potential oxygen consumption rate (µmol O<sub>2</sub> L<sup>-1</sup> h<sup>-1</sup>) 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 O<sub>2</sub> L<sup>-1</sup> h<sup>-1</sup>, 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.

=== Repositories & databases ===

== Limitations ==

Rates measured are potential rates at saturating substrate concentrations (V<sub>max</sub>), 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'' <ref name="MartínezGarcía2009" />

=== Recent applications ===
* Osma et al. (2016) ''Predicting in vivo oxygen consumption rate from ETS activity and bisubstrate enzyme kinetics in cultured marine zooplankton'' <ref name="Osma2016" />

=== Common calculations/conversions ===
* O<sub>2</sub> consumption (µmol L<sup>-1</sup> h<sup>-1</sup>) = ETS activity (µmol INT-F L<sup>-1</sup> h<sup>-1</sup>) × R/ETS; the R/ETS ratio typically ranges from 0.09 to 0.5 in marine environments.

== References ==

[[Category:Main Pages|Model types]]