Deciphering the adaptation mechanism of anammox consortia under sulfamethoxazole stress: A model coupling resistance accumulation and interspecies-cooperation.

Sulfamethoxazole (SMX) is frequently detected in wastewater where anammox applications are promising. While it has been demonstrated that anammox consortia can adapt to SMX stress, the underlying community adaptation strategy has not yet been fully addressed. Therefore, in this study, we initially ascertained anammox consortia's ability to co-metabolize SMX in batch tests. Then, a 200-day domestication process of anammox consortia under SMX stress was carried out with community variations and transcriptional activities monitored by metagenomic and metatranscriptomic sequencing techniques. Despite the initial drop to 41.88 %, the nitrogen removal efficiency of the anammox consortia rebounded to 84.64 % post-domestication under 5 mg/L SMX. Meanwhile, a 4.85-fold accumulation of antibiotic resistance genes (ARGs) under SMX stress was observed as compared to the control group. Interestingly, the anammox consortia may unlock the SMX-inhibited folate synthesis pathway through a novel interspecies cooperation triangle among Nitrospira (NAA), Desulfobacillus denitrificans (DSS1), and the core anammox population Candidatus Brocadia sinica (AMX1), in which the modified dihydropteroate synthase (encoded by sul1) of NAA reconnected the symbiotic cooperation between AMX1 and DSS1. Overall, this study provides a new model for the adaptation strategies of anammox consortia to SMX stress.

Systematic evaluation of the impact of standard storage conditions on plasmid conjugation behavior in wastewater samples.

Filter mating experiment is widely used to study the conjugation behavior of plasmids and associated antibiotic resistance in environmental settings, however, the influence and biases brought by sample storage conditions (temperature and duration) were not yet systematically elaborated. This study systematically investigated the influence of standard storage conditions (4 °C, -20 °C, -80 °C) on plasmid conjugation behavior in influent (Inf) and activated sludge (AS) samples from sewage treatment plants (STP). The findings revealed a significant reduction in conjugation efficiency under all the tested storage conditions except for 1-week storage at 4 °C. Notably, storing at -80 °C maintained conjugation activities in activated sludge more effectively compared to -20 °C. However, the preservation performance was less effective for influent samples, which consist mainly of anaerobe-dominant communities. Systematic loss of IncH-type plasmids was observed in influent samples stored at 4 °C and -20 °C. Correspondingly, the plasmid-carrying resistome genotypes detected in the influent samples showed a clear downward trend with the increase in storage duration when stored at 4 °C and -20 °C. A relatively uniform composition in terms of incompatibility type and resistome profile was observed across activated sludge samples, regardless of the varied storage conditions. This study highlights the critical impact of storage conditions on plasmid conjugation behavior and resistome composition, offering valuable insights for optimal sample handling in resistome research.

Electric-Inducive Microbial Interactions in a Thermophilic Anaerobic Digester Revealed by High-Throughput Sequencing of Micron-Scale Single Flocs.

Although conductive materials have been shown to improve efficiency in anaerobic digestion (AD) by modifying microbial interactions, the interacting network under thermophilic conditions has not been examined. To identify the true taxon-taxon associations within thermophilic anaerobic digestion (TAD) microbiome and reveal the influence of carbon cloth (CC) addition, we sampled micron-scale single flocs (40-70 µm) randomly isolated from lab-scale thermophilic digesters. Results revealed that CC addition not only significantly boosted methane yield by 25.3% but also increased the spatial heterogeneity of the community in the sludge medium. After CC addition, an evident translocation of Pseudomonas from the medium to the biofilm was observed, showing their remarkable capacity for biofilm formation. Additionally, Clostridium and Thermotogaceae tightly aggregated and steadily co-occurred in the medium and biofilm of the TAD microbiome, which might be associated with their unique extracellular sugar metabolizing style. Finally, CC induced syntrophic interaction between Syntrophomonas and denitrifiers of Rhodocyclaceae. The upregulated respiration-associated electron transferring genes (Cyst-c, complex III) on the cellular membranes of these collaborating partners indicated a potential coupling of the denitrification pathway with syntrophic acetate oxidation via direct interspecies electron transfer (DIET). These findings provide an insight into how conductive materials promote thermophilic digestion performance and open the path for improved community monitoring of biotreatment systems.

Strategies and tools in illumina and nanopore-integrated metagenomic analysis of microbiome data.

Metagenomic strategy serves as the foundation for the ecological exploration of novel bioresources (e.g., industrial enzymes and bioactive molecules) and biohazards (e.g., pathogens and antibiotic resistance genes) in natural and engineered microbial systems across multiple disciplines. Recent advancements in sequencing technology have fostered rapid development in the field of microbiome research where an increasing number of studies have applied both illumina short reads (SRs) and nanopore long reads (LRs) sequencing in their metagenomic workflow. However, given the high complexity of an environmental microbiome data set and the bioinformatic challenges caused by the unique features of these sequencing technologies, integrating SRs and LRs is not as straightforward as one might assume. The fast renewal of existing tools and growing diversity of new algorithms make access to this field even more difficult. Therefore, here we systematically summarized the complete workflow from DNA extraction to data processing strategies for applying illumina and nanopore-integrated metagenomics in the investigation in environmental microbiomes. Overall, this review aims to provide a timely knowledge framework for researchers that are interested in or are struggling with the SRs and LRs integration in their metagenomic analysis. The discussions presented will facilitate improved ecological understanding of community functionalities and assembly of natural, engineered, and human microbiomes, benefiting researchers from multiple disciplines.

Reduction performance of microplastics and their behavior in a vermi-wetland during the recycling of excess sludge: A quantitative assessment for fluorescent polymethyl methacrylate.

Large amounts of microplastics (MPs) that have accumulated in excess sludge may increase the environmental risk for its subsequent treatment. This study aimed to investigate the performance and mechanism of the reduction of MPs in excess sludge in a vermi-wetland. For this, 1 µm, 100 µm, and 500 µm of fluorescent MPs stained with Nile red were added to raw sludge, and their decreased numbers were quantified during the treatment of sludge. The results showed that the removal rates of chemical oxygen demand and total solids from the excess sludge were 63.44%-90.98% and 37.61%-51.56% in the vermi-wetland, respectively. The numbers of 1 µm, 100 µm, and 500 µm MPs could be reduced by 86.62%-95.69%, 95.44%-99.52%, and 100% in the vermi-wetland, respectively. These results indicate that the vermi-wetland is more effective at eliminating MPs. Further insight into the vermi-wetland stratification was obtained, and more than 74.87% of the MPs were intercepted in the vermicompost layer. Moreover, all the particle sizes of MPs were found in the excrement of earthworms. However, only 1 µm MPs were detected in their digestive organs. This study suggests that the interception effect is primarily responsible for elimination of MPs in excess sludge, and the bioturbation of earthworms plays an important role in the mobilization of MPs in vermi-wetlands.

Performance and stratified microbial community of vermi-filter affected by Acorus calamus and Epipremnum aureum during recycling of concentrated excess sludge.

Vermi-filter combined with wetland plant (VFP) is an eco-friendly and sustainable approach for recycling of excess sludge by joint action of earthworms, wetland plants and microorganisms. However, the effects of wetland plants on sludge decomposition and involved microorganisms are remain unclear. This study investigated the effects of wetland plants on the treatment performance of VFP for concentrated excess sludge and microbial community distribution inside the VFP. The wetland plants' species of Acorus calamus and Epipremnum aureum were separately planted on the surface layer of vermi-reactors by earthworms Eisenia fetida. Results showed that the growth rate of E. fetida in VFPs significantly (P < 0.001) increased by 75%, compared to VF. In addition, the removal rates of total solids and chemical oxygen demand in VFPs could reach 61%-79% and 36%-68%, respectively, displaying a better performance of sludge reduction than in the VF. The surface layer of VFPs was a hotspot for degradation of organic matter, where bacteria played an important role in bio-decomposition rather than eukaryotes. Moreover, wetland plants could significantly (P < 0.001) enrich the eukaryotic population, rather than bacterial population. Compared to the VF, the wetland plants could promote the diversities of bacterial community in VFPs, showing specific functioned genus in different layers. In contrast, A. calamus could be a better candidate than E. aureum in the VFP. This study suggests that the inoculation of wetland plants can improve the performance for treating concentrated excess sludge by changing the biomass of earthworms and the structure of microbial community within the VFP.