Biotechnology of Stepwise Acclimatization of Azolla Caroliniana Willd. to Fishpond Wastewater and Its Bioconversion Under Field Conditions

In this study, a simple and reproducible biotechnological scheme was developed for the stepwise acclimatization of Azolla caroliniana Willd. to wastewater discharged from fishponds, its controlled clonal propagation under field conditions, and the preparation of the resulting biomass for subsequent nutritional bioconversion. Initially, 0.1×, 0.4× and 1.0× concentrations of a nitrogen-free modified Hoagland medium were tested. Each experiment used 4.0 g of fresh biomass; growth was evaluated in terms of final biomass, covered area and relative growth rate (RGR). Across three independent temporal replicates, the 0.4× medium gave the highest performance: 47.04 ± 0.28 g biomass, 320.0 ± 2.82 cm² area and an RGR of 0.246 ± 0.001 day⁻¹. Thereafter, three parallel clonal lines were sequentially transferred to 25, 50, 75 and 100% fishpond wastewater. As the wastewater proportion increased, day-10 biomass rose consistently from 48.20 ± 0.63 g to 53.88 ± 0.24 g, and RGR from 0.24890 ± 0.00130 to 0.26005 ± 0.00045 day⁻¹. The lines acclimatized to 100% wastewater were transferred to floating field units with a surface area of 0.49 m². In the 10-day field validation, biomass reached 68.12 ± 0.28 g, covered area 1055.67 ± 4.25 cm² and RGR 0.28349 ± 0.00040 day⁻¹; net fresh biomass productivity amounted to 13.09 ± 0.06 g FW·m⁻²·day⁻¹. Drying 50.0 g samples of field biomass at 45°C for 6 h yielded 3.57 ± 0.13 g of dried mass, corresponding to a yield of 7.13 ± 0.25%. The results demonstrate the feasibility of clonal propagation and biomass production of Azolla caroliniana Willd. in fishpond wastewater without the addition of external mineral nitrogen. This approach integrates the conversion of wastewater biogenic elements into plant biomass, the reuse of water resources and the production of an alternative feed raw material for local aquaculture within a single technological chain.