An alternative way to remove CO2 from biogas is the use of photosynthetic microorganisms, such as microalgae. This can be achieved by the operation of an open photobioreactor, connected with a mass transfer column, such as a counterflow column. This technology provides up-graded biogas with high quality. The microalgal uptake of CO2 from the biogas in counterflow columns generates pH changes in microalgae culture. To clarify the potential effect of these dynamic pH conditions in the culture, the effect of pH change on the photosynthetic activity and PSII quantum yield was studied for microalgae Chlorella sorokiniana. Thus, assays were carried out, where the pH drop reported in the counterflow columns was replicated in batch microalgae culture through HCl addition and CO2 injection, moving the culture pH from 7.0 to 5.0 and from 7.0 to 5.8, respectively. Moreover, the effect of light/darkness on photosynthetic activity was tested when the pH decreased. The results obtained in this research showed that the photosynthetic activity decreased for the light conditions when the pH was shifted by HCl addition and CO2 injection. Despite this, the value of the PSII quantum yield remained at 0.6–0.7, which means that the microalgae culture did not suffer a negative effect on the photosynthetic system of cells because a high value of PSII efficiency remained. In the same way, the results indicated that when the pH change was corrected, the photosynthetic activity recovered. Moreover, the apparent affinity constant for dissolved inorganic carbon (KDIC) was 0.9 µM at pH 5 and 112.0 µM at pH 7, which suggests that the preferred carbon source for C.sorokniana is CO2. Finally, all the results obtained indicated that the pH drop in the counter-flow column for biogas upgrading did not cause permanent damage to the photosynthetic system, and the decrease in the photosynthetic activity as a result of the pH drop can be recovered when the pH is corrected.