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The evidence of high-attachment bacteria in aerobic granule formation revealed by mCherry-labeled Stenotrophomonas AGS-1.

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Title: The evidence of high-attachment bacteria in aerobic granule formation revealed by mCherry-labeled Stenotrophomonas AGS-1.
Authors: Liu, Jia1,2 (AUTHOR), Liu, Fan1,2 (AUTHOR), Gao, Yiyun1,2 (AUTHOR), Zhu, Jianrong1,2 (AUTHOR) zjrtshua@bnu.edu.cn
Source: Journal of Environmental Sciences (Elsevier). Jun2026, Vol. 164, p805-814. 10p.
Subject Terms: *Microbiology, *Wastewater treatment, Bacterial adhesion, Stenotrophomonas maltophilia, Fluorescent proteins, Biofilms, Microbial aggregation
Abstract: • Synthetic mc-AGS-1 with RFP-mCherry revealed AGS formation. • High-attachment mc-AGS-1 strongly interacted with other bacteria during AGS cultivation. • Low-attachment eGFP- E.coli dispersed homogeneously in liquid and was easily lost. • mc-AGS-1 localized on the AGS biofilm surface and migrated to the core layers. • High-attachment AGS-1 colonization promote the AGS formation. The mechanism by which biofilm formation driven by high-attachment bacteria promotes aerobic granular sludge (AGS) formation remains underexplored. Here, a red fluorescent protein (RFP) mCherry system was used to label high-attachment bacterium Stenotrophomonas AGS-1, to visualize its distribution and contribution during AGS development. The mCherry-labeled strain (mc-AGS-1) retained wild-type traits and exhibited robust fluorescence. Fluorescence tracking images revealed the persistent presence of mc-AGS-1 in both flocculent and granules, closely interacted with other bacteria throughout AGS cultivation. In contrast, the low-attachment eGFP-labeled Escherichia coli exhibited a homogeneous dispersion in liquid and was easily washed out during reactor operation. In terms of spatial distribution, mc - AGS-1 both localized on the surface layers of the AGS and then migrated to the core. Augmentation with mc-AGS-1 accelerated granulation, increasing the granule size by 43 %± 2.7 % (> 200 µm) after 10 days. A second augmentation resulted in 92 % ± 2.7 % granulation efficiency by day 40, with an average granule size of 526.3 µm. High-throughput sequencing revealed that the abundance of Stenotrophomonas increased from 0 % to 24 % post-augmentation and stabilized at 0.6 %-0.8 %, whereas the abundances of related aerobic granule-forming and pollutant-removing bacteria increased, demonstrating the inductive role of AGS-1 in promoting AGS formation. This study demonstrated that the RFP-mCherry labeling system can serve as a powerful tool for tracking the distribution patterns and microbial interactions of AGS-1 in AGS systems, highlighting its theoretical and practical application value in elucidating biofilm-mediated granulation mechanisms and advancing the development of optimized AGS technology. [Display omitted] [ABSTRACT FROM AUTHOR]
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Abstract:• Synthetic mc-AGS-1 with RFP-mCherry revealed AGS formation. • High-attachment mc-AGS-1 strongly interacted with other bacteria during AGS cultivation. • Low-attachment eGFP- E.coli dispersed homogeneously in liquid and was easily lost. • mc-AGS-1 localized on the AGS biofilm surface and migrated to the core layers. • High-attachment AGS-1 colonization promote the AGS formation. The mechanism by which biofilm formation driven by high-attachment bacteria promotes aerobic granular sludge (AGS) formation remains underexplored. Here, a red fluorescent protein (RFP) mCherry system was used to label high-attachment bacterium Stenotrophomonas AGS-1, to visualize its distribution and contribution during AGS development. The mCherry-labeled strain (mc-AGS-1) retained wild-type traits and exhibited robust fluorescence. Fluorescence tracking images revealed the persistent presence of mc-AGS-1 in both flocculent and granules, closely interacted with other bacteria throughout AGS cultivation. In contrast, the low-attachment eGFP-labeled Escherichia coli exhibited a homogeneous dispersion in liquid and was easily washed out during reactor operation. In terms of spatial distribution, mc - AGS-1 both localized on the surface layers of the AGS and then migrated to the core. Augmentation with mc-AGS-1 accelerated granulation, increasing the granule size by 43 %± 2.7 % (> 200 µm) after 10 days. A second augmentation resulted in 92 % ± 2.7 % granulation efficiency by day 40, with an average granule size of 526.3 µm. High-throughput sequencing revealed that the abundance of Stenotrophomonas increased from 0 % to 24 % post-augmentation and stabilized at 0.6 %-0.8 %, whereas the abundances of related aerobic granule-forming and pollutant-removing bacteria increased, demonstrating the inductive role of AGS-1 in promoting AGS formation. This study demonstrated that the RFP-mCherry labeling system can serve as a powerful tool for tracking the distribution patterns and microbial interactions of AGS-1 in AGS systems, highlighting its theoretical and practical application value in elucidating biofilm-mediated granulation mechanisms and advancing the development of optimized AGS technology. [Display omitted] [ABSTRACT FROM AUTHOR]
ISSN:10010742
DOI:10.1016/j.jes.2025.07.052