In Situ Extraction from Microalgal Cultures using ROKAnol NL-5

At first, the cloud point extraction with ROKAnol NL-5 was performed in a batch mode. The aim was to examine the general applicability of the separation technique. Two different feedstocks from the BIQ Algae house were implemented:

(1) an authentic culture with a CD equal to 1.0 ± 0.1 g L^-1; (2) a cell slurry from the flotation unit, regarded to as flotate (see chapter 4.4). That feedstock had a higher CD of 3.90 ± 0.01 g L^-1. The batch cloud point extraction was performed at 45 °C as described in chapter 5.10.

The following general observations were made. Firstly, a stable phase separation took place during the experiments with both feedstocks. As expected, the micellar extract was the upper phase.

Secondly, an intense pigment accumulation was observed in both extract phases.

The color of the extract from the culture was green-brownish, while the coacervate of the flotate extraction was dark brownish. The coloring was more intense during the ROKAnol NL5 extraction than in the Triton X-114 experiments. A possible explanation for that can be the more pronounced toxicity of the surfactant in combination with the higher extraction temperature (45 > 40 °C). Therefore, a partial lysis of the cells could occur and thus release more intracellular pigments [44,122,129].

Lastly, biomass flocculation was exhibited in both experiments, whereby more massive flocks occurred during the flotate extraction. That was expected considering the extreme stress conditions for the cells (45 °C in combination with long-term surfactant toxicity) [136]. However, in case of ROKAnol NL5, the biomass sedimentation towards the bottom of the vessel, while the extract accumulated at the top. Hence, the solids could not penetrate the micellar phase.

Due to analytical limitations, no surfactant concentrations could be determined for the coexisting phases. However, a UV-Vis spectrophotometric measurement was performed for each phase and was compared to a ROKAnol NL-5 aqueous solution.

The corresponding spectra are presented in Figure 6.39.

Figure 6.39: UV-Vis spectra of the micellar phase from the batch extraction of microalgae culture and flotate using ROKAnol NL5. The dashed line represents a mixture of ROKAnol NL5 and dist.

water.

As depicted in Figure 6.39, UV-Vis spectra of the extracts could be obtained without being influenced by the surfactant. The absorbance of the 20 wt% Rokanol NL5 solution was neglectable. Overall, higher absorbance was observed for the extract from the flotate in comparison to the culture extract. That corresponded to the observed darker coloring of the micellar phase during the flotate extraction. Similar maxima in the spectra were obtained for both samples. The increase in the absorbance at 273 nm could be due to aromatic compounds. Also, the absorbance peaks at 337, 438, 614 and 664 were typical for plant pigments [75,111].

However, only a row estimation of the extracted compounds could be made based on the spectra. Nevertheless, the stable separation in the ROKAnol NL5/algae suspension was promising for a possible continuous extraction as well. Therefore, a first feasibility test of the continuous extraction from authentic Acutodesmus obliquus culture using ROKAnol NL5 was conducted in pilot scale. The process parameters were roughly chosen based on the observation in technical scale (see chapter 5.10).

Overall, a formation of a micellar phase was observed at the top of the column. In addition, after approx. 2 hours a recirculation of the extract was initiated as well.

However, at the end of the experiment a weight loss of 58 % was stated for the solvent. Hence, a flooding could be assumed. Therefore, a more precise parameter transfer (as for Triton X-114) in combination with a reliable analytics for the

ROKAnol NL5 concentration were needed to maintain a steady state in the pilot scale equipment.

Nevertheless, a color accumulation in the extract phase was observed during the continuous extraction as well. The spectra of samples, obtained during the experiment are presented in Figure 6.40.

Figure 6.40: UV-Vis spectra of the extract stream from the continuous extraction of microalgae culture using ROKAnol NL5 after 2.5, 3.5, 4.5, and 5.5 hours after the beginning of the experiment.

The spectra obtained for the extract during the continuous experiment with ROKAnol NL5 possess an analogous pattern to the results from the batch extraction. In addition, at the areas of maximal absorbance, the intensity of the signal is increasing with the experiment duration. Hence, an accumulation of the algae products in the micellar phase could be concluded.

Overall, the experiments in pilot scale with the surfactant ROKAnol NL5 proved the applicability of the surfactant for the separation of algae products from the authentic algae culture. The spectrophotometric analysis indicated for an accumulation of pigments in the micellar phase. Therefore, an HPLC pigment analysis of the extracts was conducted in collaboration with the “Cell biology and phycology” department of the University of Hamburg. The corresponding pigment concentration in the different extracts is presented in Table 6.4.

0 1 2 3 4 5

200 300 400 500 600 700

absorbance

wavelenght [nm]

extract, 2.5 h extract, 3.5 h extract, 4.5 h extract, 5.5 h

Table 6.4: Pigment concentration [mg·L^-1] in micellar extracts obtained from the cloud point extraction with ROKAnol NL5 in pilot scale

extract from: neoxanthin violaxanthin lutein

microalgae culture - - 0.092

flotate 0.088 0.089 0.493

continuous experiment 0.055 0.047 0.340

The pigment lutein was accumulated in all extracts. The highest accumulation of lutein was registered for the batch extraction from flotate, followed by the continuous experiment and the batch separation with algae culture. On the other hand, neoxanthin and violaxanthin were obtained only during the continuous experiment and from the flotate feedstock. The batch extraction from flotate led to higher concentration of neoxanthin and violaxanthin than the continuous approach. The more pronounced accumulation of lutein than of the other two pigments was in good agreement with the results by Wiltshire et al [65]. During their studies, the authors wholly permeabilized Acutodesmus obliquus dried biomass and thus achieved the highest recovery of lutein in the extract phase, composed of acetone and water. The corresponding violaxanthin and neoxanthin yield were lower. In this thesis, the highest pigment accumulation was achieved during the flotate extraction. That can be correlated to the higher cell density of the feedstock. However, a more systematic study was needed to characterize the influence of the process conditions and the feed composition on the extraction efficiency.

Overall, the results in chapter 6.5, presented the first known implementation of the surfactant-based in situ continuous extraction from green microalgae in pilot scale. By utilizing the technical scale study with a subsequent up-scale, the in situ product removal from A. obl. using the Triton X-114 cloud point system was realized in a stirred contactor with a higher capacity. Hence, the batch application, reported by Glembin et al. [9,132], was not only extended to a continuous application but was performed in a pilot plant. That process development was ultimately evident for the potential of the cloud point extraction for the large-scale product recovery from authentic biological feedstocks.

Based on the observations in this thesis, a general scheme for the design of an in situ cloud point extraction from a genuine feed solution is proposed in the next chapter.

6.6 IMPORTANT POINTS FOR THE DESIGN OF THE IN SITU CLOUD POINT EXTRACTION