4 Results
4.5 Optimization of the production phase
4.5.1 Application of different strategies in the CELL-tainer
The growth performance was the best in the CELL-tainer compared to the other tested systems (chapter 5.2). Therefore, this SUB was used for the optimization experiments.
4.5.1.1 Comparison of different cultivations in CELL-tainer bioreactor
In the following an overview is given about several cultivations carried out in the CELL-tainer with the conditions described above. The growth phase will be discussed shortly, but the main focus will be put on the production phase. In chapter 4.4.5.1, a typical cultivation in the CELL-tainer was described. This cultivation is labeled as B in the following part. The described
Results: Optimization of the production phase
described cultivation B, therefore biotin, thiamine (after 120 h), and phosphate (after 146 h) was given at a later time point of the cultivation, causing a longer growth phase. The cultivation B and D were started with a new bag containing 8 L of the medium. The other four cultivations were carried out as a repeated fed-batch, which means that about 1 L of the last cultivation remained in the bag, which was refilled with 8 L fresh medium and the cultivation was started again. In all cultivations (except F) 300 mL of preculture were added.
Ammonia sulfate was provided partly via the glucose feed. The concentration was increased in order to enhance the maximum DCW, but unfortunately this was not successful.
Additional ammonia sulfate was added in the growth phase to avoid ammonia limitation.
Yeast extract was added to push the growth, when the growth rate decreased. The differences in nitrogen addition together with the maximum reached DCWs are shown in Table 10.
Table 10: Comparison of different cultivations in CELL-tainer bioreactor
A B C D E F
Cultivation regime Repeated fed-batch
Fed-batch
Repeated fed-batch
Fed-batch
Repeated fed-batch
Repeated fed-batch
(NH4)2SO4 in feed [g L-1] 10 12 12 13 13 13
Additional (NH4)2SO4 [g L-1] 5.5 0.9 0 1.0 0.73 0.35
(NH4)2SO4 addition in total [g L-1]
12.1 6.7 1.6 5.0 4.3 4.0
Additional yeast extract [g L-1]
5.4 (261 h) 4.2 (477 h)
2 (168 h) 8.2 (149-217 h)
0 0 4 (168 h)
Nitrogen in total [g L-1]* 3.76 2.15 1.45 1.67 1.52 1.70
Maximum DCW [g L-1] 47.4±0.2 45.9±0.4 22.1±0.6 47.8±1.1 45.8±0.6 35.2±2.2
*Total nitrogen was calculated from the yeast extract contained in the cultivation medium and the added yeast extract and ammonium sulfate in course of the cultivation
Cultivation C was carried out as repeated fed-batch. When this cultivation was started, a bacterial contamination was visible in the beginning. Therefore, ampicillin was added to the culture in order to stop bacterial growth. The comparison with the other cultivations shows clearly that this had a negative influence on cell growth. It was not possible to push the growth with additional yeast extract. Nevertheless, it was possible to observe the influence of sodium propionate on DHA production with this cultivation. The lag phase was shortened in E and F with the repeated fed-batch approach. In cultivation F the maximum DCW might be lower, because of the missing preculture.
4.5.1.2 Comparison of the influence of different additives on the DHA production in the CELL-tainer
Fig. 55 provides an overview about the DHA production in these different CELL-tainer cultivations. Since the increase in the bioreactor volume was larger when sodium salts were applied, the total produced DCW and DHA amount were compared. The nitrogen concentration in the culture broth has an influence on the DHA production (de la Jara et al.
2003; Ratledge 2004; Mendoza et al. 2008). Therefore, the ammonia content in each cultivation is shown in Fig. 55 on the lower part of the graphs (added amounts are provided
Results: Optimization of the production phase
in Table 10) together with the temperature regime, which was applied in addition to the supplementation of different additives.
Fig. 55: Influence of different additives on DHA-production in different CELL-tainer cultivations: vertical line marks the time point were the particular additive addition started. Additives: A-C and F: see Table 11, D: 150 mL sunflower oil (within 24 h), E: 150 mL rapeseed oil (within 24 h), growth conditions described in chapter 4.5.1.1.
A more detailed overview of the different additions of the additives and the observed DHA concentrations is provided in Table 11 for the cultivations with propionate and acetate.
Results: Optimization of the production phase
Table 11: Overview about the amount and duration of additive addition and observed DHA concentrations A: propionic acid B sodium
propionate
C: sodium propionate
F: sodium acetate
Period of addition [h] 603 238 - 288 289 - 459 216 - 362
Conc. of additive [mM] 124 287 397 717
Specific conc. of additive [µM 106cells-1]
6.7±0.2 5.2±0.1 29.8±0.2 19.0±0.1
DHA total [g]1 31.7 12.3 19.2 24.9 5.9 9.4 10.9 21.3
Specific DHA concentration [% DCW]1
4.3±0.1 2.2±0.1
3.7±0.2 4.6±0.1
2.5±0.1 4.4±0.1
2.5±0.2 4.6±0.5 Specific DHA conc.
[mg 106cells-1]1
0.107±0.003 0.054±0.001
0.033±0.001 0.040±0.001
0.039±0.001 0.090±0.003
0.026±0.001 0.043±0.002
rP whole process [mg L-1 h-1]2 5.363 8.30 1.89 6.24
rP stationary phase [mg L-1 h-1]4 5.845 3.59 1.56 4.77
qP whole process [mg g-1 h-1] 0.0723 0.122 0.088 0.121
qP stationary phase [mg g-1 h-1] 0.1215 0.074 0.072 0.132
1 at the time of the additive addition and in the end of the cultivation, 2related to the start volume, 3calculation at the time point of propionic acid addition, 4increase in DHA from the time point of additive addition to the end, related to the volume at the start point of the addition, 5calculated in the stationary phase before propionic acid was added.
The addition of propionic acid did not result in the desired success (A). Propionic acid was added slowly to the reactor to maintain the pH-value at the desired level via the pH control, but probably due to local pH gradients, cells had been destroyed. This was visible in the cell number and DCW. Also the specific and the volumetric DHA concentration decreased. This is because the DHA measurement considers only the DHA, which is incorporated in the cells, the supernatant is discarded prior to the analysis. When the cells were destroyed, the DHA, which was produced so far in the cells will be not measured anymore and would also hamper the downstream process. It should be noted, that the volumetric and specific DHA content were exceptionally high, before the addition of propionic acid was started. The addition of propionic acid was relatively late in comparison to the other cultivations. The prolonged growth phase performed with lower ammonia addition than in the other cultivations might increase the DHA production. On the other side the longer cultivation time would increase the costs, too.
In cultivation B and C, only sodium propionate was added (B: 287 mM, C: 397 mM). The addition of sodium propionate in B increased the DHA content slightly (30 %). The added sodium propionate was already consumed after 329 h. This can be recognized from the HCL consumption, which increased drastically after 238 h, when sodium propionate was added for the first time and remained constant until 329 h (data not shown). Eventually, the temperature was decreased with 1K h-1 to 15 °C. This had a positive influence on the DHA production. The specific and volumetric amount increased.
Specific DHA content increased drastically in C (per DCW: 76 %, per cell number: 131 %), probably caused by the larger specific amount of sodium propionate, which was added in the stationary phase (B: 5.2 µM 106cells-1, C: 29.9 µM 106cells-1). Furthermore, the glucose concentration was 5 g L-1 when the addition of sodium propionate started. 24 h later, all glucose was consumed and sodium propionate remained as the sole carbon source. This
Results: Optimization of the production phase
might support the specific DHA production and caused the large increase 48 h after the first sodium propionate addition. The specific DHA concentration probably had been higher, when the specific DHA amount would have been as high as it was in cultivation B at the beginning of the stationary phase.
As seen in from Fig. 55 D and E, the addition of sunflower or rapeseed oil did not increase the DHA content. However, the ammonia concentration was still high in the stationary phase in these cultivations, which might negatively influence the DHA production (de la Jara et al.
2003; Ratledge 2004; Mendoza et al. 2008). This unwanted high ammonia concentration was a result of a spontaneous transition from the growth in the stationary phase, which had not been recognized early enough. The feed should be stopped immediately, when the growth phase terminates, as long as the carbon feed is coupled with the ammonia feed. In order to achieve these conditions, the addition of the additives should start already in the very early stationary phase to avoid the accumulation of ammonia, as it happened in cultivation D and E. Otherwise, the ammonia concentration was also high in the stationary phase in cultivation C, but nevertheless, the DHA content increased. The temperature shift had no influence on DHA production in D and E, which can be caused either by the high ammonia concentration in the reactor or that the stationary phase already took too long, so that the metabolism was not active anymore.
Cultivation F demonstrated the positive effect of sodium acetate on DHA production. In the growth phase, the specific DHA content was nearly constant and increased drastically when the addition of sodium acetate starts. 738 mM sodium acetate was added between the 220 h and 360 h, which correspond to 19.6 µM 106cells-1. Glucose was consumed in parallel and depleted after 334 h. The rise in hydrochloric acid consumption lasted until the end of the cultivation indicating that acetic acid was metabolized over the whole second part of the cultivation. Sodium acetate was added in parallel to the temperature shift to avoid limitations of the carbon source. Therefore, the influence of the sodium acetate and the temperature shift cannot be evaluated separately. With this strategy, a specific DHA concentration was obtained, which was the highest among the five cultivations, coupled with the highest increase in the production phase (Fig. 55, Table 11).
When the different production rates are compared (Table 11), it becomes obvious that the overall volumetric production rate was the highest with sodium propionate in cultivation B.
Nevertheless, the specific DHA production rate was the same in cultivation B and F. Since the growth of the algae in the cultivations was different, it is more important to compare the specific DHA production rates after the addition of a certain additive. This rate was highest for the addition of acetate, when it was added in a high concentration (cultivation F).
However, the comparison of the influence of various additives in different cultivations was difficult. Therefore, it was important to establish a screening system, which allows experiments in parallel. The results with these screening systems will be discussed in the next chapter.
Results: Optimization of the production phase