Enhanced Bacterium-Phage Symbiosis in Attached Microbial Aggregates on a Membrane Surface Facing Elevated Hydraulic Stress.

Phages are increasingly recognized for their importance in microbial aggregates, including their influence on microbial ecosystem services and biotechnology applications. However, the adaptive strategies and ecological functions of phages in different aggregates remain largely unexplored. Herein, we used membrane bioreactors to investigate bacterium-phage interactions and related microbial functions within suspended and attached microbial aggregates (SMA vs AMA). SMA and AMA represent distinct microbial habitats where bacterial communities display distinct patterns in terms of dominant species, keystone species, and bacterial networks. However, bacteria and phages in both aggregates exhibited high lysogenicity, with 60% lysogenic phages in the virome and 70% lysogenic metagenome-assembled genomes of bacteria. Moreover, substantial phages exhibited broad host ranges (34% in SMA and 42% in AMA) and closely interacted with habitat generalist species (43% in SMA and 49% in AMA) as adaptive strategies in stressful operation environments. Following a mutualistic pattern, phage-carried auxiliary metabolic genes (pAMGs; 238 types in total) presumably contributed to the bacterial survival and aggregate stability. The SMA-pAMGs were mainly associated with energy metabolism, while the AMA-pAMGs were mainly associated with antioxidant biosynthesis and the synthesis of extracellular polymeric substances, representing habitat-dependent patterns. Overall, this study advanced our understanding of phage adaptive strategies in microbial aggregate habitats and emphasized the importance of bacterium-phage symbiosis in the stability of microbial aggregates.

Phage Predation Promotes Filamentous Bacterium Piscinibacter Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates.

Although bacteria-phage interactions have broad environmental applications and ecological implications, the influence of phage predation on bacterial aggregation and structural stability remains largely unexplored. Herein, we demonstrate that inefficient lytic phage predation can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates, improving the structural and hydraulic stability of the dual-species aggregates. Specifically, phage predation at 103-104 PFU/mL (i.e., multiplication of infection at 0.01-0.1) promoted initial Piscinibacter colonization by 10-15 folds and resulted in 29-31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates without phage predation. Transcriptomic analysis revealed upregulated genes related to quorum sensing (by 15-92 folds) and polysaccharide secretion (by 10-90 folds) within the treated aggregates, which was consistent with 120-172% higher content of polysaccharides for the treated dual-species aggregates. Confocal laser scanning microscopic images further confirmed the increase of filamentous bacteria and polysaccharides (both with wider distribution) within the dual-species aggregates. Accordlingly, the aggregates' structural strength (via atomic force microscopes) and shear resistance (via hydraulic stress tests) increased by 77 and 42%, respectively, relative to the control group. In the long-term experiments, the enhanced hydraulic stability of the treated aggregates could facilitate dwelling bacteria propagation in flow-through conditions. Overall, our study demonstrates that phage predation can promote bacterial aggregation and enhance aggregate structural stability, revealing the beneficial role of lytic phage predation on bacterial symbiosis and environmental adaptivity.

Hitchhiking Behavior in Bacteriophages Facilitates Phage Infection and Enhances Carrier Bacteria Colonization.

Interactions between bacteriophages (phages) and biofilms remain poorly understood despite the broad implications for microbial ecology, water quality, and microbiome engineering. Here, we demonstrate that lytic coliphage PHH01 can hitchhike on carrier bacteria Bacillus cereus to facilitate its infection of host bacteria, Escherichia coli, in biofilms. Specifically, PHH01 could adsorb onto the flagella of B. cereus, and thus phage motility was increased, resulting in 4.36-fold more effective infection of E. coli in biofilm relative to free PHH01 alone. Moreover, phage infection mitigated interspecies competition and enhanced B. cereus colonization; the fraction of B. cereus in the final biofilm increased from 9% without phages to 43% with phages. The mutualistic relationship between the coliphage and carrier bacteria was substantiated by migration tests on an E. coli lawn: the conjugation of PHH01 and B. cereus enhanced B. cereus colonization by 6.54-fold compared to B. cereus alone (6.15 vs 0.94 cm2 in 24 h) and PHH01 migration by 5.15-fold compared to PHH01 alone (10.3 vs 2.0 mm in 24 h). Metagenomic and electron microscopic analysis revealed that the phages of diverse taxonomies and different morphologies could be adsorbed by the flagella of B. cereus, suggesting hitchhiking on flagellated bacteria might be a widespread strategy in aquatic phage populations. Overall, our study highlights that hitchhiking behavior in phages can facilitate phage infection of biofilm bacteria, promote carrier bacteria colonization, and thus significantly influence biofilm composition, which holds promise for mediating biofilm functions and moderating associated risks.

Deciphering defense system modulating bacteria-mobile genetic elements symbiosis in microbial aggregates under elevated hydraulic stress.

Bacterial defense systems are under strong evolutionary pressures to defend against mobile genetic elements (MGEs), yet their distribution in microbial aggregates in engineered systems remains largely unexplored. Herein, we investigated the bacterial defensome and MGEs within activated sludge flocs (AS) and membrane-attached biofilm (MF) in a full-scale membrane bioreactor. Similar distribution pattern of bacterial defense systems (63 types) was observed in prokaryotic genome in AS and MF, including RM system (∼40 %), Cas system (∼18 %) and TA-Abi system (∼28 %), exhibiting a dependency on the genome size and bacterial taxonomy in microbial aggregates under elevated hydraulic stress (MF). In contrast to plasmid and provirus, which carried defense systems (22 types) similar to their associated hosts, virome (61 %) carried novel defense systems (40 types) absent in their associated hosts. With 54 % of which involved in MGEs geneflow network, 69 % of high quality bacterial genome bins were associated with horizontal gene transfer (HGT), facilitating the exchange of mobile core functional genes. This potentially conferred competitive advantages to hosts through habitat-specific payload genes related to biotic defense, antibiotic resistance, and nitrogen metabolism. The longer growth cycle and varied defense gene density suggested the potential defense redundancy and trade-off of metabolic expense and immunity in bacterial host-MGE symbionts. Furthermore, enhanced cooperative network modules of cross-feeding and defense were observed in the MF, potentially helped the symbiotic microbial communities in coping with hostile conditions under elevated hydraulic stress. These findings shed light on the dynamics of bacterial defense systems in host-MGE coevolution and provide new perspectives of microbial aggregates manipulation for ecological and engineering application.

Uncover the secret of granule calcification and deactivation in up-flow anaerobic sludge bed (UASB) reactor with long-term exposure to high calcium.

Calcification of anaerobic granular sludge causing the decrease of microbial activity has been focused for several decades, but the mechanism of calcification and deactivation of calcified granule are still lacking. In this study, the calcification process of anaerobic granular sludge was analyzed in the UASB reactor with long-term exposure to a high calcium level of 2 gCa2+L-1. Greyish-white calcified granules with larger size and density first appeared at the bottom of sludge bed. Characterization of granular surface demonstrated that calcified granules had smaller specific surface area and pore volume with the disappearing channels in granules visualized over calcification. The coverage of calcite on granular surface with a thickness of 500-600 µm was observed by a microscope, which is responsible for the blockage of channels for substrate diffusion and transport. The evaluation of mass transfer showed that the 'effectiveness factor' η of calcified granules was higher than 1.23, proving the existence of mass transfer resistance. Furthermore, the interactions between calcium and extracellular polymeric substances (EPS) revealed the possible mechanism of calcite location at the outer layer of anaerobic granular sludge: The bulk calcite directly binds the EPS such as humic acids via charging functional groups, and bulk CO32- could react easily with Ca2+ bound to EPS according to thermodynamic analysis.

Enhancement of PPCPs removal by shaped microbial community of aerobic granular sludge under condition of low C/N ratio influent.

The frequent occurrence of pharmaceuticals and personal care products (PPCPs) in domestic wastewater has caused great concern. In this study, the removal of two typical pharmaceuticals (Roxithromycin, ROX; Sulfamethoxazole, SMZ) in aerobic granular sludge (AGS) reactors was investigated under condition of different C/N (carbon to nitrogen) ratios. Results showed that higher removal efficiencies of ROX and SMZ (95.2 % and 92.9 %) were achieved in the AGS reactor fed with low C/N influent. Batch experiments further revealed that the removal of ROX was influenced by the adsorption ability of the AGS while SMZ removal was mainly enhanced by biodegradation process. Analysis of extracellular polymeric substances (EPS) showed that the humic acid-like substances were enriched under low C/N condition, which was in accordance with dynamic change of microbial community. The microbes, like Thauera spp. and Xanthomonadaceae, were highly enriched in the reactor with high nitrogen loading rate and functioned as refractory organics degrader. Overall, the AGS process could achieve enhanced pharmaceuticals removal performance by the regulation of microbial community under low C/N influent, which provides insights into a feasible solution for simultaneous removal of nitrogen and trace organic pollutants in AGS reactor.

Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing.

Aerobic granular sludge (AGS) reactors with different sludge retention times (SRTs) were established for enhanced functional microorganism enrichment and granular formation. Results showed that higher total nitrogen (TN) removal efficiency and compact granules were achieved in the 6-day-SRT reactor. Also, Xanthomonadaceae, Rhodobacteraceae and Hyphomonadaceae with AHL-producing and EPS-secreting functions also enriched under 6-day SRT. For investigating the enhanced mechanism of sludge granulation, typical quorum sensing signals of acylated-homoserine-lactones (AHLs) and extracellular polymeric substances (EPS) were analyzed. Tryptophan-and-protein-like substances were major EPS components in granules formed at 6-day SRT. Meanwhile, most detected AHLs, i.e. C8-HSL and 3OHC8-HSL, were correlated positively with contents of tryptophan-and-protein-like substances. According to AHLs add-back test, AHLs especially those with 8-carbon sidechains, played important roles in aerobic sludge granulation via secreting special extracellular proteins by functional microbes enrichment.

Optimization of F/M ratio for stability of aerobic granular process via quantitative sludge discharge.

Food to microorganisms (F/M) ratio is one of the most important factors affecting microbial growth and pollutant removal in biological wastewater treatment system. For stability of aerobic granular process, optimal range of F/M ratio and a maneuverable F/M ratio control method via quantitative sludge discharge were investigated in this study. Results showed that stable aerobic granules were achieved with good settleability, high pollutant removal efficiency and microbial diversity when F/M ratio was controlled at 0.4-0.5 gCOD/gSS d. In addition, a maneuverable F/M ratio control method via quantitative sludge discharge was developed to verify the feasibility for optimization of F/M ratio. By this method, aerobic granules cultivated with the F/M ratio of 0.4 ±â€¯0.02 gCOD/gSS d had better pollutant removal performance and stable structure with higher protein-like components in EPS. This study further revealed the importance of F/M ratio in the stability of aerobic granular sludge process.

Stability of aerobic granular sludge under condition of low influent C/N ratio: Correlation of sludge property and functional microorganism.

Aerobic granular sludge process treating domestic wastewater with low C/N ratio is necessary to be studied for rapid urbanization in China and other countries. In this study, two parallel reactors with different influent C/N ratio (15 in R1, 5 in R2) were established. Compared to the disintegrated granule in R1 with high influent C/N ratio, granules with large size (650 µm) and compact structure (integrity coefficient <0.1) were stable in R2 along with influent C/N ratio decreased to 5. High-through sequencing illustrated the functional microbes like Thauera and Paracoccus enriched under low influent C/N ratio, and principal component analysis further showed these microbes were positive correlation with tryptophan and protein-like substances in extracellular polymeric substances (EPS) and granular strength. It was indicated that under low influent C/N ratio, several resistant microbes like Thauera (19.5%) enriched and then secreted tryptophan and protein-like substances, and stable granules with multi-functional microbes could be formed finally.