## 3 Results
### Pulse-Based Fed-Batch Cultivation  
While the batch cultivations aimed to initially evaluate the relationship of the PDW spectroscopy signals with process relevant characteristics, a pulse-based fed-batch cultivation was conducted to: (i) show the feasibility to control the process by monitoring the process with PDW spectroscopy; (ii) confirm signal relationships; and (iii) validate the reproducibility during biological duplicate cultivations with independent seed trains. The process intention was implemented without difficulty: a pulse of either rapeseed oil or a nutrient bolus (see dashed lines in Figure 3 and details in the legend), respectively, was added to the culture when the \(\mu_{\mathrm{s}}\)' signal showed no further changes and indicated either carbon or nitrogen limitation. It is worth mentioning, that no signal deflections were observed whenever oil pulses were added to the bioreactor, which would account for an effect of the added oil on \(\mu_{\mathrm{s}}\)' or \(\mu_{\mathrm{a}}\). Within the first 8 h, the intracellular PHB content decreased from 36 to 14 wt% (Figure 3). An increase of the CDW, RCDW, \(\mu_{\mathrm{s}}\)' and \(\mu_{\mathrm{a}}\) was observed in the first 6 h of the cultivation. Subsequently, only minor increases of the CDW and RCDW of about 0.7 g-L-1 were detected until 8 h, whereas the PDW spectroscopy signals did not further increase during this period. After addition of the first pulse (0.5% (w-v-1) rapeseed oil), \(\mu_{\mathrm{s}}\)' and \(\mu_{\mathrm{a}}\) resumed to increase until reaching constant levels at 11.5 h until the next pulse addition (1% (w-v-1) rapeseed oil). The increase of \(\mu_{\mathrm{a}}\) stopped at 16.5 h, whereas \(\mu_{\mathrm{s}}\)' increased further until 18 h. Nitrogen depletion was detected at 17.5 h (Figure S4, Supplementary Materials). The PHB content had already increased to 30 wt% at this time point and further increased to 38 wt% before addition of the next pulse. The CDW increased up to 23 g-L-1, whereas the RCDW  
Figure 2: Batch cultivations for polyhydroxybutyrate (PHB) production by _R. eutropha_ H16. Batch 1 contained 3% (w-v-1) rapeseed oil and 2.25 g-L-1 urea (75 mM nitrogen), Batch 2 contained 4% (w-v-1) rapeseed oil and 4.5 g-L-1 urea, and Batch 3 contained 4% (w-v-1) rapeseed oil and 2.25 g-L-1 urea. Ammonia content (grey diamonds, mM), PHB content (green squares, wt%), cell dry weight (CDW) (filled circles, g-L-1), residual cell dry weight (RCDW) (empty circles, g-L-1), reduced scattering coefficient \(\mu_{\mathrm{s}}\)’ (red line, mm-1), and absorption coefficient \(\mu_{\mathrm{a}}\) (blue line, \(\times 10^{-3}\) mm-1) at 638 nm are shown. Error bars indicate minimum and maximum values of technical duplicates. stopped at a value of 14 g\(\cdot\)L\({}^{-1}\) at 20.5 h. Addition of the next rapeseed oil pulse (2% (w\(\cdot\)v\({}^{-1}\))) at 21 h did not trigger a significant change of the \(\mu_{\mathrm{s}}\)' signal and of the RCDW. In contrast, a sharp increase of \(\mu_{\mathrm{s}}\)' resulted from the rapeseed oil addition and the CDW increased up to 43 g\(\cdot\)L\({}^{-1}\) at 31 h containing 66 wt% PHB. At 31.6 h, a bolus containing urea as a nitrogen source was supplemented to the culture. The addition resulted in a dilution of the culture, which was seen in a step decrease of both PDW spectroscopy signals at this time point. Subsequently, \(\mu_{\mathrm{s}}\)' decreased until 42 h and stayed constant until the next pulse addition. In contrast, \(\mu_{\mathrm{a}}\) resumed to increase until 45 h. The CDW decreased during this period to 24 g\(\cdot\)L\({}^{-1}\), resulting from intracellular PHB degradation. The PHB content decreased to 30 wt%. At the same time, the RCDW increased to 17 g\(\cdot\)L\({}^{-1}\). The addition of the next pulse (4% (w\(\cdot\)v\({}^{-1}\)) rapeseed oil) resulted in resumed growth on rapeseed oil as the primary carbon source instead of degrading the intracellular carbon storage. The PDW spectroscopy signals increased after the rapeseed oil supplementation.