## 3 Results and Discussion
### Impact of Recycling on Water Composition  
To investigate the effect of the improved water recycling strategy--applying sulfate supplementation and using urea as the nitrogen source--on the water composition, a detailed analysis of ion concentrations, dissolved organic carbon and carotenoid concentrations was performed. Because no sulfate was supplemented in the first six recycling steps of the process series using KNO\({}_{3}\), apart from the ASW additions that were needed to compensate water losses during cell harvest, sulfate concentration steeply declined until being completely consumed after three cultivations. Additionally,  
Figure 3: Mean cell dry weight (CDW) concentration (**a**) and areal biomass productivity (**b**) with water recycling in 8 consecutive batch processes with _M. salina_ in thin-layer cascade photobioreactors. Water recycling was performed without regular sulfur supplementation and with KNO\({}_{3}\) as nitrogen source (black), or with sulfur supplementation and urea as nitrogen source (red). Shaded areas represent \(\pm\)1 standard deviation of the respective consecutive processes. the accumulation of K\({}^{+}\) led to an increase in salinity which had to be mitigated by a reduction in NaCl in the fresh ASW for water compensation. This, in turn, decreased Cl\({}^{+}\) (and probably Na\({}^{+}\)) concentration over time from 18.2 to 7.7 g L\({}^{-1}\) after the seventh water recycling. However, the water analysis of the improved process series with urea and sulfate supplementation confirmed that none of the measured salt species (Na\({}^{+}\), Cl\({}^{-}\), SO\({}_{4}\)\({}^{2-}\), Ca\({}^{2+}\), Mg\({}^{2+}\) and K\({}^{+}\)) was depleted over the series of eight batch processes while Mg\({}^{2+}\) was the only component showing a slight accumulation from 0.72 g L\({}^{-1}\) in fresh ASW to a maximum of 1.46 g L\({}^{-1}\) after five recycling steps (Figure 4, Table A1). Since reference seawater has a similar Mg\({}^{2+}\) concentration of 1.31 g L\({}^{-1}\)[33], it is unlikely that this increase would be adverse. The accumulation of K\({}^{+}\) ions up to 9.9 g L\({}^{-1}\), which was seen in the first recycling series, was effectively prevented by the replacement of KNO\({}_{3}\) with urea as nitrogen source. Since water recycling leads to an accumulation of organic matter due to algal cell lysis and secretion of organic substances, e.g., exopolysaccharides or small organic molecules [25; 26], the concentration of dissolved organic carbon was measured after inoculation and before harvest of each cultivation (Figure 5a). Though DOC was partially removed with each cell harvest, it increased from 0.28 g L\({}^{-1}\) at the start of the first process with fresh ASW to a maximum of 1.94 g L\({}^{-1}\) at the end of a cultivation with six times recycled water. Therefore, DOC species might have served as a heterotrophic substrate for algal growth in recycled water, leading to an increased biomass productivity. This hypothesis is supported by a 34% increase in the yield coefficient for urea (as an indicator for overall nutrient consumption) from 4.2 g CDW g\({}^{-1}\) urea in fresh ASW to a maximum of 6.4 g CDW g\({}^{-1}\) urea in three  
Figure 4: Ion concentrations in the growth medium over 8 serial batch processes with _M. salina_ in thin-layer cascade photobioreactors. Left side: Batch processes with water recycling without regular sulfate supplementation and with KNO\({}_{3}\) as nitrogen source. K\({}^{+}\) and Mg\({}^{2+}\) were only determined in the 6 th and 7 th recycling step. Na\({}^{+}\) and Ca\({}^{2+}\) were not determined. Sulfate measurements were connected linearly to improve clarity. At the start of the 4 th and 7 th recycling step, additional sulfate was added (see Section 3.1). Right side: Batch processes with water recycling with sulfate supplementation and with urea as nitrogen source.