Comparison of the STR cultivation with the CELL-tainer

In the course of the project the CELL-tainer was scaled up and a prototype with a working volume of 150 L already exist (Junne et al. 2013). Since this SUB was the most promising one, among the tested bioreactors, a final comparison was carried out between a conventional STR made of glass and the CELL-tainer. In this comparison the optimized medium, which will be discussed in chapter 4.4, is applied. In order to evaluate the influence on the cell morphology, the flow cytometry was applied throughout the process. In order to obtain identical conditions in both devices special expansion blocks were applied to use the CELL-tainer with 1 L working volume, the same as in the STR.

4.3.4.1 Fed-batch cultivation in STR with glucose as sole carbon source

In Fig. 35 the results of this process with glucose as a sole carbon source is shown for the DCW, cell number, DHA content and process parameter are presented.

Fig. 35: Fed-batch cultivation in STR with 1 L working volume and glucose as sole carbon source. Medium:

25 g L^-1 glucose, 10 g L^-1 yeast extract, SOW, 0.75 g L¹ Na2HPO4, 0.2 mg L^-1 thiamine hydrochloride, 0.006 mg L^-1 biotin, feed: 500 g L^-1 glucose + 13 g L^-1 ammonium sulfate. C:

µ, r_P and q_P calculated from the fitted curves shown in A and B.

The cell density increased until 100 h to a maximal DCW of 49 g L^-1. This was the moment of the highest oxygen consumption as well. The DHA content increased further in the stationary phase until the end of the cultivation to a maximum value of 1.8 g L^-1, which

Results: Comparison of different types of single-use bioreactors (SUBs)

corresponded to 3.9 % of the DCW (Fig. 35 A and B). The production rate increased already in the growth phase up to 22.5 mg L^-1 h^-1 after 75 h. A second local maximum is reached in the production phase, (10.8 mg L^-1 h^-1). The specific production rate decreased over the whole cultivation time (Fig. 35 C). The set point of the pH was adjusted during the cultivation due to the small pH drift (Fig. 35 D).

Flow cytometry measurements were performed every day. Fig. 36 provides an overview of the results with unstained and BOX stained cells in the course of the cultivation.

Fig. 36: Results of the flow cytometry measurements during the cultivation with glucose as main carbon source in 1 L STR. 1: Density plot of the measurement of the unstained cells (a) and the BOX stained cells (b).

Percentages of the subcultures are marked in the graph. 2: Medians of the flow cytometric signals for a) unstained cells, b) percentage of PI stained cells, cells stained with c) Box and with d) Nile red.

It is obvious that the SSC, which is related to the cell granularity, decreased during the cultivation, whereas the FSC, which represents the cell size, stayed constant. When the medians for the cell granularity are compared, it can be seen that the value was lower in the beginning, reached its maximum after 50 h and then decreased constantly (Fig. 36 1 (a) and 2 (a)).

Cells with a low cell membrane potential appeared after 18 h, and their amount decreased in the further course of the cultivation. When the growth rate was decelerated, this second population of cells with a low membrane potential disappeared completely. Cells with totally collapsed cell membrane potential were visible in the right corner after 89 h. The amount increased until the end of the cultivation. They also contribute to the increased median of the BOX signal (Fig. 36 1 (b) and 2 (c)). In the second half of the cultivation, the percentage of

Results: Comparison of different types of single-use bioreactors (SUBs)

PI stained cells increased, too, suggesting a higher amount of non-viable cells. However, the percentage is small, maybe due to the stirrers, which probably destroy non-viable cells.

In Fig. 36 2 (d) the cytometric signal for the Nile red fluorescence is shown. The amount of neutral lipids, mainly associated with storage lipids, increased slightly in the stationary phase, whereas the amount of polar lipids (red fluorescence) remained almost constant. In Fig. 36 2 (b), the slight increase in DHA is visible, which correlates with the increase of the yellow fluorescence signal. This can be explained by the fact that usually in the stationary phase, predominantly the amount of storage lipids increase.

Foam occurred already after 60 h. The effect was amplified by the higher amount of cell debris, which is visible in Fig. 36. Rapeseed oil was added as antifoam reagent, since it was recognized in prior experiments in the STR that the usually used PPG 2000 stopped the growth of the algae (appendix, Fig. 81). However, rapeseed oil was not sufficient to prevent foam formation, which resulted in an overspilling of the bioreactor after approximately 100 h; overspilling always raises the risk of contamination drastically. In order to circumvent these problems in the following cultivation, a third stirrer was installed in the head space of the bioreactor to destroy the foam mechanically. Additionally, an empty flask was installed, connecting the bioreactor and the off gas filter, in order to minimize the contamination risk.

However, also in the second cultivation described in chapter 4.6.1, foam formation was a problem. The additional stirrer in the headspace together with a regular addition of rapeseed oil was not sufficient enough to prevent the foam formation adequately. The bioreactor was overspilling already after 60 h to the empty flask connected to the off gas.

This construction was successful to protect against contamination, but it could not prevent the loss of biomass and product. Similar problems were also observed in a STR cultivation at the 100 L scale. Even with the larger head space, an additional stirrer and 1 % rape seed oil could not prevent overspilling of the bioreactor.

4.3.4.2 Comparison of the STR cultivation with the CELL-tainer

The main disadvantage of the cultivations carried out in the STR was the prior discussed foam formation. In the CELL-tainer foam formation is avoided, since the system is aerated via the headspace and shear forces are lower. Thus, cell lyses rates might be reduced.

The cultivation shown in Fig. 37 was carried out in the CELL-tainer with 1 L cultivation broth under the same conditions as in the STR (shown in Fig. 35).

The maximum DCW was reached in both cultivations after 165 h and was slightly higher in the CELL-tainer (51.7 g L^-1) than in the STR (47.8 g L^-1). Nevertheless, the volumetric DHA content was the same (STR: 1.8 g L^-1 (3.9 %); CELL-tainer: 1.8 g L^-1 (3.4 %)), as well as the maximum production rate (STR: 22.5 mg L^-1h^-1; CELL-tainer: 22.1 mg L^-1h^-1) (Fig. 37 B and C).

Set point of the pH control was adjusted due to an increased offset between the offline and online measurement (Fig. 37 D).

Results: Comparison of different types of single-use bioreactors (SUBs)

Fig. 37: Fed-batch cultivation with glucose in CELL-tainer with 1 L cultivation broth. Medium:

25 g L^-1 glucose, 10 g L^-1 yeast extract, SOW, 0.75 g L¹ Na2HPO4, 0.2 mg L^-1 thiamine hydrochloride, 0.006 mg L^-1 biotin, feed: 500 g L^-1 glucose + 13 g L^-1 ammonium sulfate.

Lines shown in A and B were fitted with MATLAB smoothing spline and used for the calculation of µ, r_P and q_P shown in C.

-The YX/S of the growth and the stationary phase were higher in the CELL-tainer cultivation than in the STR. In the STR the amount of destroyed cells, which stuck to the bioreactor wall, was high. This amount was not considered in the DCW and therefore, it was missing in the YX/S, which might explain the lower value. Additionally, the STR spilled over, which also decreased the yield. YP/S was only higher in the growth phase in the CELL-tainer, in the stationary phase the value is similar.

Fig. 38: Y_x/s and Y_P/S for the cultivation in the STR (black bars) and in the CELL-tainer (white bars) with glucose, calculated for the growth (between zero and 88 h) and the stationary phase (with cross stripes).

Results: Comparison of different types of single-use bioreactors (SUBs)

Fig. 39: Results of the flow cytometric measurements during the cultivation with glucose as main carbon source in 1 L CELL-tainer. 1: Density plot of the measurement of the unstained cells (a) and the BOX stained cells (b). Percentages of the subcultures are marked in the graph. 2: Medians of the flow cytometric signals for a) unstained cells, b) percentage of PI stained cells, cells stained with c) Box and with d) Nile red.

The comparison of the flow cytometric measurements shown in Fig. 36 and Fig. 39 reveals a comparable pattern of the populations. However, the amount of debris visible in the lower part of the upper graphs and the quantity of cells with a totally collapsed cell membrane potential (on the right bottom of the BOX stained cells) was higher in the STR cultivations than in CELL-tainer trials. This indicates a higher amount of destroyed cells in the STR. The median for the signal of the BOX-staining and also the amount of PI-stained cells were lower in the end of the cultivation in the CELL-tainer than in the STR.

In addition to the flow cytometry measurements, the protein content in the supernatant was measured with the Bradford assay. The aim was to elucidate, if more cells were destroyed in the STR, which should be visible in a higher protein content in the supernatant.

Results: Optimization of the growth phase

Fig. 40: Protein content in the supernatant in STR and CELL-tainer cultivation with glucose as main carbon source and in the STR cultivation with glucose and acetate.

However, the protein amount in the CELL-tainer was slightly higher than in the STR. Since more debris was visible with the flow cytometry, it can be concluded that the debris was centrifuged partly to the cell pellet. Interestingly, the protein content was smaller in the stationary phase, when acetate is added. Cells seem to resist higher shear forces when no starvation for a carbon substrate occurs during the production phase.

4.4 Optimization of the growth phase

In the following, the progress of the medium development is described. The main focus lay on the improvement of the medium for the growth phase. Therefore, different carbon sources were tested, the reef salt was replaced by a specific mixture of inorganic salts and the amount of yeast extract, necessary for the cultivation, was successfully reduced. The process development was performed in different scales, starting with the DWP, to the shake flask scale and the implementation of the process in different bioreactor systems, prior to the later methodology plattform development (chapter 4.2).