Unveiling the role of ferrous ion in driving microalgae granulation from salt–tolerant strains for mariculture wastewater treatment

Development of microalgal–bacterial granular sludge (MBGS) from saline–adapted microalgae is a promising approach for efficient mariculture wastewater treatment, whereas the elusive mechanisms governing granulation have impeded its widespread adoption. In this study, spherical and regular MBGS were successfully developed from mixed culture of pure Spirulina platensis and Chlorella sp. GY–H4 at 10 mg/L Fe 2+ concentration. The addition of Fe 2+ was proven to induce the formation of Fe–precipitates which served as nucleation sites for microbial attachment and granulation initiation. Additionally, Fe 2+ increased the prevalence of exopolysaccharide–producing cyanobacteria, i.e. Synechocystis and Leptolyngbya, facilitating microbial cell adhesion. Furthermore, it stimulated the secretion of extracellular proteins (particularly tryptophan and aromatic proteins), which acted as structural backbone for the development of spherical granule form microalgal flocs. Lastly, it fostered the accumulation of exogenous heterotrophic functional genera, resulting in the efficient removal of DOC (98 %), PO 4 3− –P (98 %) and NH 4 + –N (87 %). Nevertheless, inadequate Fe 2+ hindered microalgal floc transformation into granules, excessive Fe 2+ expanded the anaerobic zone within the granules, almost halved protein content in the TB–EPS, and inhibited the functional genes expression, ultimately leading to an irregular granular morphology and diminished nutrient removal. This research provides valuable insights into the mechanisms by which Fe 2+ promotes the granulation of salt–tolerant microalgae, offering guidance for the establishment and stable operation of MBGS systems in mariculture wastewater treatment.