Short-term preservation and reactivation of fungal-bacterial granules for wastewater treatment under low-temperature conditions

Abstract

Reliable preservation and reactivation of functional fungal-bacterial granules (FBGs) is a key barrier to fungal-based wastewater treatment, particularly in cold regions where biomass start-up and recovery are slow and unstable. Preservation has been studied for aerobic granular sludge (AGS) and fungal pellets, but a systematic framework for FBGs is lacking. Here, the short-term preservation and reactivation of FBGs after three days of storage was systematically evaluated by comparing deep freezing (−80 °C), moderate freezing (−15 °C), and room-temperature drying, followed by structural and functional reactivation under room-temperature and low-temperature (15 °C) wastewater treatment conditions. Freezing preserved granule integrity and biomass quality, whereas drying reduced the organic fraction and impaired settleability, particularly under low-temperature conditions. At room temperature, all preserved FBGs achieved high chemical oxygen demand (COD) removal (93%–97%) and effective NH₄⁺-N removal (74%–79%), with −15 °C preservation providing the most stable reactivation and settleability. Microsensor profiling indicated that −15 °C preservation supported deeper oxygen penetration at room temperature, whereas −80 °C FBGs maintained higher near-surface oxygen at 15 °C. At 15 °C, COD removal remained high (84%–87%), whereas NH₄⁺-N removal was moderate (35%–37%), suggesting limited ammonium-transformation capacity. One-way ANOVA indicated statistically insignificant differences (p > 0.05) among preservation strategies for COD, NH₄⁺-N, or NO₃⁻-N removal after acclimation. Preserved FBGs retained bacterial and fungal taxa associated with biofilm formation, stress tolerance, or granule stability, whereas control FBGs disintegrated under cold stress. Overall, FBGs withstood short-term preservation and were reactivated without compromising structural integrity or organic-matter removal under the conditions tested.

Graphical abstract

Keywords

Biomass preservation and reactivation / Cold-region wastewater treatment / Low-temperature wastewater treatment / Granule restorability / Microbial community structure / Oxygen microsensing

Highlight

	● We systematically compared preservation strategies for fungal-bacterial granules.• FBGs remain structurally robust after freezing and drying.
	● Preservation alters oxygen transport without impairing granule restorability.
	● COD removal is resilient across temperatures and preservation methods.
	● Low-temperature nitrogen removal is limited by redox and microbial factors.

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