Cultivation in shake flasks

Results: Development of a methodology platform for consistent process development

which is faster than manual sampling, but it still takes some time. During this procedure, the cultures were less shaken and probably oxygen limitations occurred, which might result in a decrease of DHA production without PFD.

The removal of CO2 was indicated by the increase of CO2 in the PFD phase and a lower pCO2

in the medium phase than in the culture without gas carrier (Fig. 24 C). A lower pCO2 can improve the growth as well.

However, when C. cohnii was cultivated with a higher amount of yeast extract (5 g L^-1 instead of 1 g L^-1), the cell densities increased, but oxygen limitation appeared already after 66 h (Fig.

25 A and B). Due to the low oxygen concentration in the system, the specific DHA concentration decreased compared to the experiments shown in Fig. 24.

Fig. 25: Cultivation in DWP with and without oxygenated PFD with modified medium to achieve higher cell densities. A: Growth curve, B: specific DHA concentration and C: pO2 concentration in PFD phase and culture broth. DWP experiments: 25 g L^-1 glucose, 1 g L^-1 yeast extract, 0.75 g L^-1 Na₂HPO₄, SOW, biotin, thiamine. Published first in Hillig et al. (2014), reprinted with kind permission of Springer+Business Media.

To avoid any oxygen limitations during the experiments, a medium adapted to the lower oxygen transfer rates and PFD to protect the cells during sampling was applied. In chapter 4.5.2 the screening system is used to investigate the influence of different additives on the DHA production.

Results: Development of a methodology platform for consistent process development

Fig. 26: Comparison of the growth, DHA production and the yields between UYF and TubeSpin bioreactor.

Medium: 25 g L^-1 glucose, 10 g L^-1 yeast extract, 0.75 g L^-1 Na2HPO4 H2O, SOW, thiamine, biotin, addition of ammonia sulfate and glucose within the production phase in dependence of the specific consumption. After 96 h 1 % v/v rapeseed oil was added. Temperature was decreased after 161 h from 25 to 15 °C. Results obtained after 270 h, whole experimental results in the appendix (Fig. 73). YX/S and YP/S is related to the added glucose concentration and does not include the amount of yeast extract and additive, which was similar in all experiments.

UYF, baffled on the bottom, guarantee kLa values of up to 400 h^-1 (Glazyrina et al. 2011).

Since the oxygen supply is a crucial factor in the cultivation of C. cohnii cells (Hu et al. 2010), the applicability of the UYF was tested. The DCW and the DHA concentration were higher in the UYF than in the TubeSpins (Fig. 26 A and B), but also a higher number of lipid droplets were visible during the microscopic examination in the culture broth in the UYF (Fig. 27). This might be an indication that shear forces caused by the baffles on the bottom of the UYF are provoking higher cell lysis rates in C. cohnii cultures. This higher number of destroyed cells led to a lower specific DHA content in relation to the DCW as well as to the cell number (Fig.

26 C and D). In general, yields were low in the UYF (Fig. 26 E and F). This indicates that maybe the amounts of larger cells, which were already in the stationary phase, were destroyed in the UYF or that the cells were destroyed before they reach the stationary phase leading to a decrease in the specific DHA concentration.

Fig. 27: Microscopic pictures of a C. cohnii cell suspension grown in TubeSpin bioreactor (A) and UYF (B).

Arrows indicating lipid droplets in the medium (Magnification 1:630).

Published first in Hillig et al. (2014), reprinted with kind permission of Springer+Business Media.

Because of the high number of lipid droplets in the culture, the influence of the shear forces was investigated by testing different shaking speeds.

Results: Development of a methodology platform for consistent process development

4.2.2.2 Influence of shaking speeds in UYF

For the experiments of this study, 100 mL medium was applied in 500 mL UYF. The influence of hydrodynamic forces on the morphology of the cells was investigated using different shaking speeds.

Fig. 28: Influence of different shaking speeds in UYF. A: cell number, specific and volumetric DHA concentration. B, C: flow cytometric measurements of the cell size (FSC) and the cell granularity (SSC) (B.1:

180 rpm, B.2: 230 rpm, B.3: 315 rpm) and the red fluorescence signal of nile red stained cells (C.1: 180 rpm, C.2:

230 rpm, C.3: 315 rpm). All measurements were carried out one day after reaching the stationary phase.

Cultivations were carried out in 500 mL UYF, sealed with AirOtop-membranes with 100 mL cultivation volume.

Published first in Hillig et al. (2013), Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

Fig. 28 A shows that the cell number and the volumetric DHA content decrease with higher shaking speed. When the shaking speed was set to 315 rpm, the cell growth ceased. The volumetric and the specific DHA content was lower at 230 rpm than at 180 rpm, which indicates that the cells containing higher amounts of DHA (“older” cells) were destroyed or the cells had generally accumulated less DHA. The granularity of the cells is slightly lower at 230 rpm, the intensity of the Nile red staining, which is correlated to the DHA content (de la Jara et al. 2003), also decreases in parallel to the shaking speed (Fig. 28 B and C). The diversity between cells with a high and a low specific DHA content, measured with the flow

Results: Development of a methodology platform for consistent process development

cytometry, is higher at 230 rpm (Fig. 28 C.2), pointing to higher stress conditions at this shaking speed.

4.2.2.3 Influence of shaking speed in the TubeSpin bioreactor 600

As an alternative to the UYF the TubeSpin Bioreactor 600 was applied. This system is characterized by lower shear forces, since it lacks of any baffles inside. A DoE was carried out in the TubeSpin Bioreactor 600 to investigate the influence of shaking speed and filling volume on growth and DHA production under the same nutrition conditions.

Fig. 29: Contour plots for the response surface model for the influence of shaking speed and filling volume on growth and DHA production of C. cohnii cells. A: cells mL^-1 (R² = 0.96), B: volumetric DHA content [g L^-1] (R² = 0.96) and C: specific DHA content [mg 10⁶cells mL^-1] (R² = 0.90). All models are significant (α < 0.05) and without significant lack of fit (α > 0.05). Data obtained after reaching the stationary phase. Published first in Hillig et al. (2013), Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

Three quadratic models were created from the response surface design, which was carried out in the TubeSpin bioreactor 600. In order to improve the models the factors, which had no significant influence on the response, were deleted stepwise (first: factor with the smallest influence). All three models are statistically significant at a significance level of 0.05 (A: p = 0.013, B: p = 0.000, C: p = 0.032). Additionally, the lack of fit test was not significant in all three cases (A: p = 0.217, B: p = 0.090, C: p = 0.459), which shows that there is no significant lack of fit of the three models to the experimental data.

The results in Fig. 29 demonstrate that the cell number and the volumetric DHA content was the highest with the highest shaking speed and the lowest filling volume applied. The filling volume had a minor influence on the cell number at a high shaking speed, but the sensitivity to the DHA content is obvious. This is probably due to a higher oxygen transfer caused by the lower filling volume, which still influences the production phase. For the specific DHA content two optima occurred. One at a high shaking speed and a low filling volume and another at a low shaking speed and high filling volume (Fig. 29 C). Maybe “older” cells, which are already in the stationary phase and exhibit a higher DHA content, were not destroyed with these setting, since the shear forces have been lower.

The experiments with 180 and 230 rpm have been repeated and completed with measurements at 315 rpm, in order to compare the physiology of the cells using the flow

Results: Development of a methodology platform for consistent process development

cytometry. In these experiments a higher amount of glucose was added to the culture during growth.

Fig. 30: Influence of shaking speed in TubeSpin Bioreactors 600. A. Cell number (A.1), volumetric (A.2) and specific (A.3) DHA content. B, C: flow cytometric measurement of the cell size (FSC), cell granularity (SSC) (B.1:

180 rpm, B.2: 230 rpm, B.3: 315 rpm) and Nile red staining intensity (C.1: 180 rpm, C.2: 230 rpm, C.3: 315 rpm);

data from the TubeSpin Bioreactor and SB200-X after reaching the stationary phase, flow cytometry measurement 24 h before growth terminated. Cultivations were performed with 100 mL filling volume in 600 mL TubeSpin Bioreactor (except SB200-X: 100 L medium). Published first in Hillig et al. (2013), Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

The comparison among 180, 230 and 315 rpm reveals that the cell number was relatively unaffected by the shaking speed, whereas the DHA content increased up to a shaking speed of 230 rpm (Fig. 30 A). The comparison of the cell physiology with the flow cytometry demonstrates a higher granularity of the cells, when the shaking speed was set to 230 rpm.

The Nile red intensity was also the highest for 230 rpm, which correlates with the higher specific DHA content (Fig. 30 B and C). Fig. 30 A indicates that the DHA content and the cell number in the TubeSpin bioreactor 600 are comparable to the amounts reached in the SUB SB200-X from Kühner shaker, which underlines the scalability of this system. The complete cultivation in the SB200-X is presented in chapter 4.3.3.

Results: Development of a methodology platform for consistent process development

The best results were obtained with 230 rpm and 100 mL medium. Therefore, these parameters were chosen for experiments, comparing a traditional Erlenmeyer flask design with the TubeSpin design.

4.2.2.4 Comparison between the Standard Erlenmeyer flask and the TubeSpin Bioreactor Experiments were carried out in parallel in the TubeSpins and a standard 500 mL Erlenmeyer flask with 100 mL filling volume.

Fig. 31: Comparison between Erlenmeyer flask and TubeSpin bioreactor 600. A.1: Growth curves for Erlenmeyer flask (triangle symbol) and TubeSpin Bioreactor 600 (circle symbol).

A.2: specific DHA content (black:

TubeSpin, gray: Erlenmeyer flask). B, C:

flow cytometric measurement of the cell size (FSC), cell granularity (SSC) (B.1:

TubeSpin Bioreactor 600, B.2:

Erlenmeyer flask), intensity of the red fluorescence signal of the Nile red stained cells (C.1: TubeSpin Bioreactor 600, C.2: Erlenmeyer flask). Samples for flow cytometry and DHA measurements originate from the stationary phase.

Published first in Hillig et al. (2013), Copyright Wiley-VCH Verlag GmbH & Co.

KGaA. Reproduced with permission.

Fig. 31 shows the superiority of the TubeSpin bioreactor 600 to the Erlenmeyer flask. The cell number in the Erlenmeyer flask reached only 60 % of the cell number in the TubeSpin Bioreactor 600; the specific amount of DHA was 40 % (Fig. 31 A). The growth of the cells was exponential in the TubeSpin bioreactor until the nitrogen was depleted after 150 h to induce the production phase. The growth in the Erlenmeyer flask was linear until this time point.

The flow cytometry measurements revealed that the diversity in cell size and granularity was higher in the Erlenmeyer flask (Fig. 31 B), which might hint to stress during the cultivation, probably caused by oxygen limitations. The granularity and also the Nile red intensity were higher in the TubeSpin Bioreactor 600 than in the Erlenmeyer flask (Fig. 31 B and C).

Surprisingly, the PI staining showed no negative influence of the flask geometry as well as the oxygen limitation on the cell membrane integrity in all experiments. The amount of stained cells was lower than 2 % in all flasks.

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