Several characteristics of PLA make this material particularly suitable to be used in specific fields such as medicine and cell, tissue, and organ engineering. PLA is a biodegradable and biocompatible material of relatively low cost and allows customized designs. For these reasons and trying to optimize a previously tested bioprocess based on the microalga Chlorella vulgaris, we designed a 3D-printable PLA device and manufactured it in three different colors, to test their performances as confinement scaffolds for this microorganism in an optimal (MS) and a more realistic (Cildáñez stream water) environment. Despite the higher efficiency in terms of biomass production for immobilized algae not confined in the developed PLA devices, free alginate beads have reduced practical usefulness owing to the relatively short shelf life of this organic matrix in natural environments, where predators cannot be controlled. In this work, we designed and tested a 3D-printed polylactic acid device with a considerable mechanical resistance, where the alginate matrix entrapping C. vulgaris cells can be internally retained.
Synthetic culture media and water of Cildáñez stream (Cil.W) used to feed the system allowed microalga growth after 5 days in the confined or unconfined system. The highest growth rate (µ) and the lowest duplication time (dT) was achieved when algae were immobilized as alginate beads. One possible reason that would explain these differences regarding the algal growth rate is the shape of the alginate-immobilization matrix. In the case of the unconfined beads, the spherical shape is expected to allow greater gas diffusion than the cylindrical structures of the PLA devices, and therefore, better oxygenation. Besides, the higher nutrient availability due to the direct contact between the alga-trapping matrix and the substrate may account for this result. In this sense, it must be mentioned that even when the PLA device has semipermeable walls, diffused oxygen and nutrients may not achieve sufficient homogenization inside these devices to allow maximum algae growth.
It is well known that high nitrogen and phosphorous concentrations in water bodies promote the development of primary producers, and their excess ultimately results in the eutrophication of aquatic systems. In light of this scenario, it becomes essential to reduce the concentration of these solutes. The bioremediation performance of the system proposed shows a successful reduction in the physicochemical parameters measured. In line with this finding, the denitrification rate calculated was significantly higher in the CfS, representing a 2.5-fold increase as compared to the UcS. This finding is of particular relevance in the context of recent research, which highlights the usefulness of solid carbon sources to sustain the growth of heterotrophic denitrifying communities able to ultimately reduce nitrate ion to nitrogen gas (Schipper et al. 2010). Solid carbon substances have the additional advantage that they can also act as biofilm carriers (Chu and Wang, 2013). Several biodegradable polymers such as poly-3-hydroxybutyric acid (PHB), polycaprolactone (PCL), polybutylene succinate, and polylactic acid have been developed and tested for solid-phase denitrification of eutrophicated water bodies (Honda and Osawa, 2002; Gibbs et al., 2004; Walters et al., 2009). The PLA device here designed and used in our experiments may have favored the recruitment of denitrifying populations thriving in this water body due to its rough surface, allowing optimal nitrate reductase activity, as previously suggested (Wu et al. 2015).
Phosphorus consumption by microalgae is generally related to lipid metabolism and biomass accumulation. We verified a Pearson's inverse correlation coefficient (ƿxy = − 0.75) between the remaining phosphorus levels in the substrate and the growth of algae, in line with previous reports (Liang et al., 2013). The results suggest that the increase in biomass could be accompanied by an increase in lipids, which is in agreement with an economy circuit in order to reuse, for example, in future biodiesel production.
Lead content became undetectable or reduced in UcS and CfS treatments, respectively. We ascribe this result to adsorption, a surface phenomenon that definitely favored the UcS: about 804 cm2 of total exposed area (100 beads; app. 8.04 cm2 each) versus approximately 91.85 cm2 of total exposed area in the CfS (25 devices; app. 3.7 cm2 each).
Although these devices are nearly opaque, they allowed enough light and access to nutrients for algae growth to be possible. Therefore, the acquisition of biomass led to significant reductions in the content of various solutes that contaminated this water because they served as nutrients to support the growth of microalgae. Furthermore, microbial communities (including coliforms) were significantly reduced, probably as a consequence of the established competition between the introduced microalgae and the native microbial community. In this way, the C. vulgaris population could carry out an efficient remediation process, probably with the help of other native populations.
On the other hand, the presence of coliforms in a given natural watercourse reveals fecal contamination and therefore entails health risk. Of note is that also coliforms significantly decreased under both systems tested. However, given their high initial numbers, only the unconfined bioprocess could effectively reduce the number of these microorganisms to such an extent that acceptable levels according to standard guidelines could be achieved. Nevertheless, significant reductions were also observed with the confined system. We consider that competition for nutrients between the introduced alga, and these heterotrophic populations were the main driver of this phenomenon. Nevertheless, specific antagonistic effects cannot be ruled out.
Immobilized algae inside the PLA devices could bioremediate this heavily polluted stream with reductions of over 90% for key anions, including nitrate and nitrite, as well as phosphorus and potentially pathogenic bacteria such as coliforms. The use of the biodegradable PLA device could have favored the development of an active denitrifying community, which probably contributed to this N-load reduction. The efficiency of the bioremediation process proposed was corroborated through cytotoxicity tests using the well-known bioindicator plant A. cepa. These results are in line with the reduced GIs already reported (Groppa et al. 2019). Likewise, seeds imbibition with UW was associated with lower MIs, while the use of DW and TW resulted in higher values. According to Leme and Marin-Morales (2009), low MIs are typically linked to the adverse effects of xenobiotics on plant growth and development. The watering with TW led to a higher root length compared to UW and even also DW. This result may be ascribed to the presence of some root promoting substance or biological community in the treated water.
Chlorella vulgaris is a fast-growing microalga with minimal culture requirements and considerable resistance to stress conditions. This makes it a valuable tool to be used for optimized bioremediation processes.