3.1. Beads Analysis
Before immobilizing the microalgae, we first sought to determine the concentrations of polymers and gelling agents for generating macroscopically homogeneous beads. For this, alginate and CaCl2
concentrations were chosen arbitrarily and the ball manufacturing tests were performed. The results obtained are summarized in Table 1
, which were the average from three replicate experiments.
It has been found that both constituents (alginate and CaCl2) generated beads regardless of their concentrations, except for the 3% alginate solution (m/v). At this concentration of alginate solution, the formation of beads was almost impossible because the dissolution of alginate in water was very difficult even with the heating.
The 2.5% alginate solution generated beads of larger diameters than the other concentrations. However, the high viscosity of this solution interfered with its flow and high pressures were applied to make the beads. In addition, the latter had a non-regular shape.
In view of these results, the 2.5% and 3% alginate concentrations were considered unsuitable for the elaboration of the beads and were therefore not used in the following experiments.
Increasing the alginate concentration and decreasing the CaCl2
concentration increases the size of the beads (Figure 1
These observations are consistent with the findings of other articles. In 1985, Boyaval et al. [43
] obtained beads with a diameter of 3.5 mm using a concentration of 2.5% alginate and 0.5% CaCl2
. This present study reports a slightly higher value of 3.8 ± 0.2 mm for the same concentrations of alginate and CaCl2
. Other authors obtained lower average diameters of 2.5 mm [44
] and 2.6 mm [45
The differences observed with the literature could be attributed to the types of alginates and extrusion devices used.
3.2. Effects of Alginate and CaCl2 Concentrations on R. subcapitata Growth
shows the evolution of the cellular concentrations of R. subcapitata
in the alginate beads along the batch culture, according to the different concentrations of alginate and CaCl2
studied, over a period of 10 days. The experiments were performed in duplicate. The initial concentrations of microalgae cells, which varied between 2.6 × 105
± 0.26 and 3 × 105
± 0.3 cells/bead, were recorded inside the beads for all formulations. After a lag phase of three days, the OD685nm
measured using the spectrophotometer revealed a significant increase in the microalgae concentrations inside the beads made from all the formulations.
The beads made from the 1.5% and 2% alginate solutions (Figure 2
b,c) exhibited remarkably higher concentrations of microalgae compared to the 1% alginate beads (Figure 2
a) regardless of the CaCl2
concentrations used. The maximum concentrations of the cells reached within the beads of each formulation are presented in Table 2
The beads formed from the 1% alginate solution (Figure 2
a) were soft and easily dissolved, showing inadequate results after day 4, 5, and 7 for 0.2%, 0.5%, and 1% CaCl2
, respectively. The beads began to shrink and resulted in the release of cells into the culture medium. A high number of free cells were recorded on the 10th day (Table 3
These observations have shown that a low concentration of alginate causes the instability of the beads due to the thin layer of the formed biofilm. Furthermore, the Ca2+ ions play an important role in the hardening of the beads and in the maintenance of their stability for a long time. Low CaCl2 concentrations reduce the stability of the beads, which causes them to easily release the cells into the culture medium. Thus, a 1% concentration of alginate is certainly not suitable for the culture of microalgae in the beads for a long time.
The formulation based on 1.5% alginate (Figure 2
b) allowed for good growth of the microalgae. Nevertheless, a shrinkage of the beads formed from 0.2% and 0.5% CaCl2
solutions was observed after the 8th and 9th day, respectively. The 0.1% concentration of CaCl2
allowed us to maintain the stability of the beads during the 10 days of culture. In addition, the free cells detected on the 10th day were reduced compared to the beads from 1% alginate (Table 3
The beads synthesized with the 2% alginate solution (Figure 2
c) demonstrated a continuous growth of microalgae even after the exponential growth phase, without remarkable shrinkage of the beads. Furthermore, the free cell concentrations recorded on the 10th day of culture were considerably minimized (Table 3
). There was a maximum reduction of 40% compared to those synthesized using 1.5% alginate, while the maximum reduction was 88% when compared to those produced using 1% alginate for the same concentration of CaCl2
In a second step, the growth rates (Table 4
and Figure 3
) and the generation times (Table 4
) obtained from all the formulations tested were compared in order to fulfil the objective of this present study, which was the optimization of the growth rate of the alga to obtain a large quantity of cells to produce biodiesel. The results obtained showed that the beads made with the combination of 1% alginate and 1% CaCl2
had the lowest growth rate and therefore, the slowest growth (μ = 0.17 ± 0.01 cells/bead/day, G = 4.05 ± 0.4 days) of all the combinations studied.
On the other hand, the growth rate values recorded with the beads from the 1.5% and 2% alginate solutions and the studied concentrations of CaCl2 indicated that the highest growth rate was obtained with the formulation of 1.5% alginate and 0.2% CaCl2 (μ = 0.27 ± 0.2 cells/bead/day, G = 2.55 ± 0.25 days). These concentrations of alginate and CaCl2 appear to improve the mass transfer of nutrients, light and CO2 in the beads due to the relatively thin layer of alginate biofilm formed and the soft texture of the beads. However, they were not sufficient to ensure the stability of the beads for a long time; it is the same for the concentrations of alginate 1.5% and CaCl2 0.5%.
The combination of 1.5% alginate and 1% CaCl2
as well as all the combinations with 2% alginate were the best because they maintained the stability of the beads during the 10 days of culture. However, the lowest number of free cells detected in the medium at the end of culture was recorded with the combination of 2% alginate and 1% CaCl2
. As a result, these alginates and CaCl2
concentrations were determined as the optimal conditions for the immobilization of microalgae and will be used in the following work. An observation of the beads made under these conditions and containing R. subcapitata
cells in the exponential phase of growth, was carried out using a stereomicroscope (SteREO Discovery.V8) (Figure 4
), equipped with a low-magnification objective (G × 1.0) and image capture software (Zen 2.3 lite, blue edition).
The growth value of R. subcapitata
obtained in this study under the optimal conditions of immobilization (2% alginate and 1% CaCl2
) was higher than the values reported in other articles. Li-Juan Zhang et al. [46
] obtained a growth of 18.5 × 104
± 0.3 cells/mL under the optimal immobilization conditions, which were determined to be 4% alginate and CaCl2
, after three days of culture. If the growth value of the study presented here is converted into the same unit, it gives a growth of 34.3 × 104
± 0.34 cells/mL after three days of culture.
The differences in the growth values observed in the study of Li-Juan Zhang et al. [46
] could be attributed to the alginate and CaCl2
concentrations used. The researchers used concentrations higher than what is proposed in this paper because their objective was to obtain dissolution-resistant beads in contaminated freshwater sediments.
3.3. Comparison of Free and Immobilized R. subcapitata Growth in Alginate Beads
A comparison was established with the cells immobilized on the beads using 2% alginate and 1% CaCl2
). The results obtained showed that the cell division termination is almost concomitant in both culture conditions. Nevertheless, the final concentration of cells is greater in the free culture (7.1 × 106
± 0.71 cells/mL against 1.52 × 106
± 0.15 cells/mL for the immobilized culture). The immobilized cells expressed a growth rate of 0.203 ± 0.02 cells/mL/day during the exponential phase of growth, while the free cells had a growth rate of 0.305 ± 0.03 cells/mL/day. This is a decrease in the order of 34.42%. These results first confirmed that immobilization in natural polysaccharides often causes material transfer limitations, which has been described in the literature [32
]. Moreover, in the free culture, the suspension of algae in fermentation is homogeneous at any point of the reactor. In contrast, in the immobilized culture, the distribution of the alginate beads in the reactor and the distribution of the cells in the beads are heterogenous.
However, it should be noted that the use of immobilization technology in microalgae culture has made it easier to separate the cells from the water. The alginate beads are relatively large compared to free cells and can be easily collected through a simple filtration method such as sieving without a significant amount of energy consumed. In addition, although R. subcapitata
was incubated under standard microalgal culture conditions, its growth exhibited better results than was found in the literature [46
]. This research therefore suggests that optimizing the conditions of culture could provide even more promising results.