## 4 Discussion  
The purpose of this study was to evaluate the potential of PDW spectroscopy for monitoring plant oil-based _R. eutropha_ cultivations. The batch (Figure 2) and pulse-based fed-batch (Figure 3) cultivations showed that the reduced scattering coefficient \(\mu_{\text{s}}\)' correlates strongly with the CDW and the absorption coefficient \(\mu_{\text{a}}\) with the RCDW (Figure 4). These results demonstrate that PDW spectroscopy is a valuable tool for _in-line_ monitoring of the CDW, RCDW, and PHB accumulation. To the best of our knowledge, the results of this study are the first data showing _in-line_ quantification of PHB. The lack of such _in-line_ or _on-line_ monitoring devices for an adaptive control of the production process was recently emphasized by [PERSON] et al. [3]. Previously, [PERSON] et al. reported the possibility to use a NIR transflectance probe for an _in-line_ quantification of PHB. However, the authors showed only _at-line_ data quantifying the PHB and CDW concentration (up to 9.3 g:L\({}^{-1}\) and 13.7 g:L\({}^{-1}\), respectively) during batch cultivations [34]. During the initial batch cultivations, PHB yields of 0.56-0.66 gPHb-5 GON\({}^{-1}\) were obtained (Figure 2), which are similar to previously reported yields for _R. eutropha_ H16 growth on palm oil [35]. During the high-cell-density fed-batch cultivation 106 g:L\({}^{-1}\) CDW (72 wt% PHB) with a space time yield of 1.65 gPHb:L\({}^{-1}\)-h\({}^{-1}\) were reached, which is comparable to other published high-cell-density plant oil cultivations [21; 24; 25; 29; 36; 37]. In the pulse experiment, a nitrogen bolus was added (32 h) after the PHB production phase to trigger PHB degradation, as described previously [38]. The PDW spectroscopy signal \(\mu_{\text{s}}\)' decreased with the declining CDW while \(\mu_{\text{a}}\) increased with an increasing RCDW (Figure 3). Currently, we do not understand why the strength of the signal was not proportional with the determined _off-line_ value changes after that time point (32 h). For this reason, these measurement points were not used for the linear correlation. A potential hypothesis could be an unequal distribution of PHA granules during PHA mobilization, as it was reported for _Pseudomonas putida_[39], which might have an effect on scattering and absorption coefficients during PHA degradation. A reduction of the \(\mu_{\text{s}}\)' signal after 35 h was observed during the high-cell-density fed-batch cultivation, whereas \(\mu_{\text{a}}\) stayed constant during that period (Figure 5). The decrease of \(\mu_{\text{s}}\)' instead of a leveling off of the signal contradicts that a signal saturation effect was observed. Additionally, scattering coefficients in suspensions with particle contents of up to 40% (v\(\cdot\)v\({}^{-1}\)) were measured successfully with this technology [9; 10]. Heavy foaming, which occurred after 35 h, could be the reason for the observed signal reductions. The surplus of foam was constantly forced into the liquid phase, which increased the overall gas hold-up and total reaction volume in the system. This additional gas volume results in a dilution of the system, which could explain the \(\mu_{\text{s}}\)' decrease (35-45 h) even though the culture continued to accumulate PHB (Figure 5). The foaming occurred after the end of the continuous rapeseed oil feed at 35 h. Before 35 h, the added oil functioned as a natural antifoam agent by decreasing the surface tension of the culture broth. During plant oil cultivations foaming occurs through the emulsification process. _R. eutropha_ emulsifies plant oils before uptake, which is catalyzed by extracellular lipases. The lipases cleave the triacylglycerlecrols in diacylglycerols, monoacylglycerols, glycerol, and free fatty acids (FFAs) [23; 43; 44], which causes heavy foaming during aerated bioreactor cultivations. Nevertheless, \(\mu_{\text{s}}\)' stayed constant after the CDW did not further increase at 45 h, which could indicate the perfect time point for harvesting in an industrial process.