# ABACO: A New Model of Microalgae-Bacteria Consortia for Biological Treatment of Wastewaters
## 2 Materials and Methods
### Respiratory: Measurements of the Photosynthesis and Respiration Rates  
In order to validate experimentally the percentage of each microbial population proposed in the biological model, respirometric measurements were performed when at steady state. The percentages of microalgae and bacteria in the culture were estimated as functions of the microalgae net photosynthesis rate, the heterotrophic respiration rate, and the nitriving respiration rate, respectively. These measurements were performed with handmade photo-respirometer equipment. This equipment is described in detail in [11]. The method allows one to determine the photosynthesis and respiration rates through the variations in dissolved oxygen concentrations in microalgae-bacteria cultures, as described in detail in [11].  
For evaluating the microalgae net photosynthesis rate of each microalgae-bacteria culture, a sample of the culture was exposed to four light-dark cycles of four minutes each to measure and register the variation in dissolved oxygen. During the light phases, the photosynthetic microalgae generated dissolved oxygen, and this dissolved oxygen was consumed by the endogenous respiration during the dark periods. Thus, the microalgae net photosynthesis rate was calculated as the difference between the slope of the oxygen production during the light period and the slope of the oxygen consumption during the dark period. Subsequently, another sample of the culture was used to determine the heterotrophic respiration rate. For this purpose, 0.8 mL of sodium acetate (30 g/L) was added to the sample and it was exposed to four light-dark cycles of 4 min each. The respiration rate of the heterotrophic bacteria was calculated as the slope of the oxygen consumption with sodium acetate minus the slope of the oxygen consumption during the dark period in the endogenous culture. By following the same method, another sample was used to measure the nitriving respiration rate of the culture. However, the nitriving activity was determined using 0.8 mL of ammonium chloride (3 g/L) instead of sodium acetate. The respiration rate of the nitrifying bacteria was calculated as the slope of the oxygen consumption with ammonium chloride minus the slope of the oxygen consumption during the dark period in the endogenous culture [11].  
Finally, in order to correct the influence of oxygen desorption on the photo-respirometric measurements, the oxygen mass transfer coefficient (K\({}_{\text{L}}\)a) was calculated. This coefficient was measured in the system according to Equation (1).  
\[\frac{\text{d}X_{\text{O}_{2}}}{\text{dt}}=K_{\text{L}}a\left(X_{\text{O}_{2} }^{*}-X_{\text{O}_{2}}\right) \tag{1}\]  
where \(\frac{\text{d}X_{\text{O}_{2}}}{\text{dt}}\) is the oxygen accumulation expressed as the derivate of X\({}_{\text{O}_{2}}\) (mg/L) concentration over time, K\({}_{\text{L}}\)a is the global oxygen mass transfer coefficient (h\({}^{-1}\)), and X\({}_{\text{O}_{2}}^{*}\) is the oxygen saturation concentration in the liquid. Further detailed descriptions of the equipment, the standard protocol, and the metabolic rate calculations are in [11].