Advanced Respirometry with Chemical Optical Sensor Spots

Non-invasive Tracking of Oxygen, Carbon Dioxide and pH as Indicator for Nitrification Activity

Eva M. Gilbert, Susanne Lackner
Karlsruhe Institute of Technology (KIT), Engler-Bunte Institute, Water-Chemistry and Water Technology, Karlsruhe, Germany

Respirometry is an indirect way of measuring microbial activity, such as nitrification. The oxygen uptake is proportional to the substrate consumption - given by the stoichiometry of the process. Combining the oxygen uptake with the release (heterotrophic) or uptake (autotrophic) of carbon dioxide and pH changes extends the applicability of these respirometry assays. The measurement of combined processes becomes feasible as tracking pH and carbon dioxide allows allocation of the oxygen uptake to different processes on a pro-rata basis illustrated here for autotrophic nitrification.

Nitrification is a key process in the natural nitrogen cycle and is used in biological wastewater treatment. It describes two successive biochemical reactions which are performed by different groups of bacteria: The first step is the oxidation of NH4+ to NO2- (see Eq. 1), the so-called nitritation, which is carried out by ammonium oxidizing bacteria (AOB). The conversion of each mole NH4+ requires 1.5 moles O2 and additionally releases two hydrogen ions which lowers the pH. For the further oxidation of NO2- to
NO3- (see Eq. 2), the so-called nitratation, which is carried out by nitrite oxidizing bacteria (NOB), only 0.5 moles O2 per mole NO2- are required and pH does not change.

NH4+ + 1.5 O2 -> NO2- + H2O + 2 H+ (Eq. 1)
NO2- + 0.5 O2 -> NO3-                             (Eq. 2)

Both processes are carried out by autotrophic bacteria; these are bacteria that do not require organic carbon - they use CO2 as carbon source for their cell structure. In such systems it is important to trace the concentrations of O2 and CO2 as substrates and the pH value as a key variable to ensure optimal high microbial activity. If O2 or CO2 are limited or the pH value drops too much, the bacteria will reduce activity and the nitrogen conversion will decrease. Non-invasive methods to track these parameters can help studying the effects of substrate limitation and pH value on the nitrification rate without disturbing an experiment. In so-called respirometry batch experiments, conversion rates can be estimated out of the O2 uptake rates. As long as only one process occurs (ensured by inhibiting the other process), the calculation based on the stoichiometry is quite accurate. But this does not reflect the interaction of AOB and NOB. Tracking not only O2 but pH and CO2 as well might enable respirometric batch experiments with both processes simultaneously, as the change in CO2 relative to the pH could indicate which process occurs to what extent. In this study we applied chemical optical sensor spots, which allow non-invasive monitoring of oxygen, CO2 and pH in the cultures, to assess these processes.

Materials & Methods

The suitability of PreSens sensor spots for pH, CO2 and O2 (SP-HP5, SP-CD1, SP-PSt3) to study nitritation and nitratation in batch cultures was tested with biomass from two lab scale enrichment reactors, one AOB and one NOB. A defined volume of biomass was taken from the reactor, washed with tap water and then re-suspended in mineral growth medium and aerated. The batch run was started by spiking ammonium (AOB) or nitrite (NOB) to the batch vessel while monitoring pH, CO2 and O2. Sensor spots were integrated in the test vessels and read out non-invasively with the respective fiber optic transmitters (pH-1 mini, Fibox 3, pCO2 mini). No inhibitors were added, as the bacteria originated from enrichments and therefore no NOB activity was expected in AOB runs.


Extended respirometric assays for testing sole AOB and sole NOB activity were conducted with evaluation of O2, pH and CO2. The result of such runs in shown in Fig. 2 and 3. The oxygen content decreased in both assays, AOB and NOB, due to the oxygen consumption of the bacteria during conversion of ammonium and nitrite, respectively (Fig. 2A + 3A). The nitritation step (AOB activity) produces H+ thereby decreasing the pH which can be seen in Fig. 2B. The CO2 content increases with the decrease in pH based on the dissociation equilibrium. In case of nitratation (NOB activity) the pH stays constant (Fig. 3B) while tracking the consumption of CO2 by NOBs during nitrite oxidation revealed a slow decrease in CO2 concentration.


The PreSens sensor spots for pH, CO2 and O2 were successfully used to study nitrification kinetics in extended respirometry assays. The results showed that additional information is gained by combining measurements of all three parameters in one single vessel. A distinctly different behavior of pH and CO2 during AOB and NOB batch runs was documented which enables quantification of the relative share of NOB activity in O2 depletion during mixed batch runs (AOB and NOB). As both natural systems and wastewater treatment reactors always contain AOB and NOB, extended respirometry assays with monitoring pH, CO2 and O2 are a useful tool for quick evaluation of biomass activity. Only non-invasive measurements - as with the chemical optical sensor spots - make such extended respirometry assays possible. In classical respirometric assays only O2 is monitored as additional electrodes or sampling would disturb the reading too much. Furthermore, the sensor spot technology decreases the required reaction volume significantly and therefore opens more options and higher flexibility to study nitrification kinetics.


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