Recovery of structure and activity of disintegrated aerobic granular sludge after long-term storage: Effect of exogenous N-acyl-homoserine lactones

Highlights

• AGS after 33 months storage was used as inocula for start-up of AGS bioreactors.

• Successful recovery of structure and activity of stored AGS was demonstrated.

• Appropriate AHLs accelerated granule recovery and improved granule characteristics.

• Short-term exogenous AHLs regulation could sustain positive effects on AGS.

• Enrichment of functional genera contributed to rapid recovery of stored AGS.

Abstract

Long-term storage of aerobic granular sludge (AGS) may lead to granule inactivation and disintegration. Granule recovery in both structure and activity is important for scale-up and stability of AGS, but information about the structure recovery of stored AGS is limited. In addition, whether short-term exogenous N-acyl-homoserine lactones (AHLs) regulations could accelerate the granule recovery and sustain positive effects on AGS is unknown. Herein, the recovery of 33-month stored AGS was performed in three reactors for 38 days (phase I) at different exogenous AHLs concentrations (0, 50 and 500 nM of AHL-mixtures in R0, R1 and R2, respectively) and for an extended 45 days without exogenous AHLs (phase II). Results demonstrated successful recovery of disintegrated AGS in all reactors, although it was relatively time-consuming in R0. The treatment performance was similar among the reactors and steady-state removal of COD (90%) and NH₄⁺-N (94%) could be recovered within 7 and 21 days, respectively. However, exogenous AHLs regulation (especially in R1) obviously accelerated bioactivity recovery of heterotrophs and nitrifiers and improved granule characteristics, including biomass, density, hydrophobicity and extracellular polymeric substance (EPS). During phase II, sustainable positive effects remained in R1, but granule characteristics deteriorated in R2. The abundance of functional genera Thauera, Nitrosomonas and Candidatus_Nitrotoga, contributed to the rapid recovery and helped maintain the structure and activity of AGS. The predictive functional profiling of bacterial communities also demonstrated sustainably higher activities of metabolism, growth and signal sensing under exogenous AHLs regulation at an appropriate content.

Introduction

Aerobic granular sludge (AGS) is one of the most promising biological wastewater treatment technology, owing to its high biomass retention, excellent settle ability, simultaneous removal of organic matter and nutrients, and less area and equipment requirement (Sengar et al., 2018). It has been implemented in full-scale treatment of municipal and industrial wastewater (Li et al., 2014a; Pronk et al., 2015). However, a long start-up time of AGS has restricted its large-scale application (Lin et al., 2020). Some authors reported start-up periods up to 10 months for establishing reasonable granulation and reaching stable performance in pilot- and full-scale reactors (Ni et al., 2009; Nancharaiah et al., 2019). For similar technology, anaerobic granular sludge has been applied worldwide and it is stable in the granular form and maintains bioactivity for long-term storage (from months to years) under starvation conditions (Wu et al., 1995). Hence, today most full-scale reactors are fed with granular seeding sludge to reduce the start-up period.

Development of AGS is also expected to be shortened by starting with AGS from other plants (Pijuan et al., 2011; Pronk et al., 2015). Unlike anaerobic granular sludge, the source of feeding fresh AGS is still limited, because the applications of AGS have not prevailed. Stored AGS, if can be recovered sufficiently and quickly, presents a promising alternative option for fast start-up of AGS bioreactors. The bioactivity of AGS can be recovered in a short time after a storage period of weeks or months (Wang et al., 2008; Lee et al., 2010; Ogura et al., 2020). However, the start-up performance of the bioreactors inoculated with AGS after a long-term storage (say years) requires further investigation. Moreover, AGS may lose structural integrity due to anaerobic core hydrolysis or cell lysis after stored over a long period of time (Adav et al., 2009; Lee et al., 2009; Gao et al., 2011). To the best of our knowledge, the structural recovery of disintegrated AGS after long-term storage has not been reported before. The question of whether the disintegrated AGS can recover both in structure and activity is important for scale-up and stability of AGS bioreactors.

Development of AGS is regulated by quorum sensing (QS), which refers to cell-cell communication through secretion and sensing specific chemical signal molecules named autoinducers (Chen et al., 2018; Huang et al., 2019). As a major class of autoinducers, N-acyl-homoserine lactones (AHLs) are strongly and positively correlated with the aerobic granulation process, as they promote secretion of extracellular polymeric substances (EPS) and aggregation of bacteria (Lv et al., 2014; Tan et al., 2014; Chen et al., 2019; Li et al., 2020b). While inactivation or lack of AHLs may result in reduction of the bacterial attachment potential, damage to the EPS matrix and disintegration of the granular structure (Li and Zhu, 2014; Li et al., 2014b; Yuan et al., 2017; Zhang et al., 2019b). Owing to the positive effects of AHLs, exogenous dose of AHLs has been applied to enhance the formation of AGS. For example, exogenous AHLs during the early start-up stage enhanced biomass growth rate, EPS production and nitrifying sludge granulation (Li et al., 2015; Wu et al., 2017). Moreover, exogenous AHLs can accelerate the recovery of damaged biofilm (Batchelor et al., 1997; Gamage et al., 2011) and improve microbial activity and pollutants removal in wastewater treatment systems (De Clippeleir et al., 2011; Lv et al., 2018; Gao et al., 2019; Huang et al., 2020; Yan et al., 2020). Accordingly, adding AHLs might be a feasible strategy for accelerating the start-up in terms of reformation and reactivation of disintegrated AGS after long term storage, but it has received no attention. Besides, AHLs cannot be persistently added to the bioreactors due to economic reasons. Whether short-term addition of AHLs has sustainable positive effects on the characteristics and performances of AGS is still unknown.

To address these questions, this work investigated the feasibility of long-term stored AGS as seed for reactor start-up and the effects of exogenous AHLs on structure and bioactivity recovery. The recovery of 33-month stored AGS was performed in three sequencing batch reactors (SBRs) for 38 days (phase I) at different exogenous AHLs concentrations (0, 50 and 500 nM in R0, R1 and R2, respectively) and for an extended 45 days without exogenous AHLs (phase II). To the best of our knowledge, this is the first report in the literature exploring the structure recovery of disintegrated AGS and adopting exogenous AHLs to accelerate the recovery of long-term stored AGS.