## 3 Results and Discussion  
### Microalgae Processes with Water Recycling  
Batch processes with _Microchloropsis salina_ were performed in 8 m\({}^{2}\) open TLC photobioreactors at a physically simulated Mediterranean summer climate. Cells were harvested by centrifugation at the end of each process after 12 to 14 days. The supernatant was supplemented with a concentrated feed medium solution and used as recycled growth medium for subsequent cultivations. In the first process series, with a total of seven consecutive batch processes with medium recycling, KNO\({}_{3}\) was employed as the standard nitrogen source. Using fresh ASW medium, an overall biomass productivity of 15.6 g m\({}^{-2}\) d\({}^{-1}\) (2.3 g L\({}^{-1}\) d\({}^{-1}\)) was achieved after 14 days at a CDW of 31.2 g L\({}^{-1}\) (Figure 1). While single reuse of growth medium had no negative effect on growth rate and process performance, the second and third recycling resulted in a stationary CDW of 22.8 \(\pm\) 0.7 and 13.8 \(\pm\) 0.5 g L\({}^{-1}\), respectively, thereby reducing the productivity at the end of each process to 10.7 and 6.2 g m\({}^{-2}\) d\({}^{-1}\), respectively. This observation was additionally emphasized by a decrease in the specific growth rates during the first 5 days of each cultivation from 0.63 \(\pm\) 0.15 d\({}^{-1}\) with fresh ASW to 0.38 \(\pm\) 0.06 d\({}^{-1}\) with three times recycled water (Figure 2a). The batch process with four times reused water showed a slight recovery to a productivity of 10.0 g m\({}^{-2}\) d\({}^{-1}\); due to an increased volume loss during the preceding cell harvest, the addition of 15 L of fresh ASW was required instead of the usual 10 L ASW. A sixth reuse of water, however, followed the negative trend in microalgal growth with increasing number of medium recycling steps, reaching only 5.3 g L\({}^{-1}\) CDW and a productivity of 2.2 g m\({}^{-2}\) d\({}^{-1}\) after 14 days. The analysis of salt concentrations showed an accumulation of potassium ions from 0.4 g L\({}^{-1}\) in fresh ASW to 9.9 g L\({}^{-1}\) at the end of the sixth water recycling process, which could be attributed to the regular addition of KNO\({}_{3}\) as nitrogen source in the concentrated feed medium. Though potassium facilitates important physiological functions in ion and especially phosphate transport, osmosis and enzyme activity [29; 30], this increase in K\({}^{+}\) concentration by a factor of nearly 25 might cause ion imbalances and possibly has a detrimental effect on algal growth [31]. Furthermore, a sulfate depletion was observed, which may also decrease algae growth efficiency since a low sulfate availability--being an ubiquitous element of amino acids, vitamins and cofactors--is generally associated with a decreased photosynthetic activity and protein synthesis [32]. In that context, sulfate was only partially replenished by the addition of 10 L fresh ASW at the start of each batch process, as it was not included in the feed solution. Supplementing 2.6 g L\({}^{-1}\) MgSO\({}_{4}\) - 7 H\({}_{2}\)O after the seventh water recycling, however, resulted in a partial recovery of CDW concentration and productivity to 19.2 g L\({}^{-1}\) and 9.5 g m\({}^{-2}\) d\({}^{-1}\), respectively (Figure A1), indicating that a sulfate limitation was one but not the only factor limiting algal growth and productivity in recycled water. The mean CDW concentration and overall biomass productivity at the end of eight consecutive batch processes were 19.0 \(\pm\) 8.5 g L\({}^{-1}\) and 9.1 \(\pm\) 4.2 g m\({}^{-2}\) d\({}^{-1}\), respectively (Figure 3). The specific growth rate during the first 5 days of each process decreased from 0.63 \(\pm\) 0.15 d\({}^{-1}\) with fresh ASW to a minimum of 0.18 \(\pm\) 0.07 d\({}^{-1}\) after the sixth water recycling (Figure 2). Figure 1: Consecutive batch processes applying water recycling after harvesting of M. salina in thin-layer cascade reactors at physically simulated climate conditions. Left side: Water recycling without sulfate supplementation and with KNO3 as nitrogen source.