Aerobic composting represents an efficacious strategy for the disposal of human feces, yet investigations into the effects of different bulking agents on this process remain limited. This study investigated the effects of composting human feces with four types of bulking agents—wheat straw, corn straw, millet straw, and sawdust—in a foam composting device, as well as the impacts of the process on the microbial community structure and metabolic functions adopting sequencing data analysis and metagenomic analysis. The results demonstrate that aerobic composting can safely treat human feces, resulting in a mature compost product. Comparative assessments of compost quality and microbial profiles with various bulking agents indicated superior performance of corn straw compost, surpassing those produced with wheat straw, millet straw, and sawdust in terms of humification level (HA/FA = 2.9), peak temperature reached (71.2°C), composting duration (20 days), and nutrient composition (TN 42.87 g/kg). Additionally, the diversity and dominance of certain microbial colonies (Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidota) were significantly higher in composts formulated with corn straw. The metagenomic data analysis reveals significant differences in the abundance of “carbon metabolism” and “microbial metabolism” among different groups, further indicating that the addition of different bulking agents affects the utilization of metabolic products, amino acids, and carbohydrates as carbon sources by microbes in human feces compost. Consequently, leveraging corn straw as a bulking agent, given its abundant availability, could potentially improve the efficiency and outcome of the human feces composting process.

Microbial community composition, function, and viability are important for biofilm-based sewage treatment technologies. Most studies of microbial communities mainly rely on the total deoxyribonucleic acid (DNA) extracted from the biofilm. However, nucleotide materials released from dead microorganisms may interfere with the analysis of viable microorganisms and their metabolic potential. In this study, we developed a protocol to assess viability as well as viable community composition and function in biofilm in a sewage treatment system using propidium monoazide (PMA) coupled with real-time quantitative polymerase chain reaction (qPCR) and metagenomic technology. The optimal removal of PMA from non-viable cells was achieved by a PMA concentration of 4 μM, incubation in darkness for 5 min, and exposure for 5 min. Simultaneously, the detection limit can reach a viable bacteria proportion of 1%, within the detection concentration range of 10²–10⁸ CFU/mL (colony forming unit/mL), showing its effectiveness in removing interference from dead cells. Under the optimal conditions, the result of PMA–metagenomic sequencing revealed that 6.72% to 8.18% of non-viable microorganisms were influenced and the composition and relative abundance of the dominant genera were changed. Overall, this study established a fast, sensitive, and highly specific biofilm viability detection method, which could provide technical support for accurately deciphering the structural composition and function of viable microbial communities in sewage treatment biofilms.

Urban sewage, aquaculture wastewater, and medical wastewater are significant reservoirs and transmission sources of Salmonella. Rapid detection of Salmonella is crucial for effectively reducing the risk of disease transmission and safeguarding public health. Differentiating viable Salmonella from inactivated cells presents significant challenges, affecting the accurate assessment of pathogen risks. Moreover, current detection methods face several limitations, including lengthy detection periods, high costs, and limited applicability, underscoring the need for rapid, sensitive, and visual detection diagnostic approaches. In this study, we combined propidium monoazide (PMA) with recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeats (CRISPR)/Cas12a systems to develop a rapid detection system for viable Salmonella targeting the fimY gene. DNA of viable Salmonella was amplified and visually detected within 60 min and dead cells were effectively excluded. We assessed the specificity and sensitivity of the PMA-RPA-CRISPR/Cas12a assay. The results showed that the assay had a high level of specificity, with no reactions observed with other pathogens. The application of PMA has no effect on the sensitivity of RPA-CRISPR/Cas12a technology and the visibility of the fluorescence reporting system. We successfully detected viable Salmonella in wastewater with a minimum detection limit of 10¹ CFU/mL. In summary, the PMA-RPA-CRISPR/Cas12a system developed in this study allows for the rapid and visual detection of viable Salmonella in wastewater at concentrations as low as 10¹ CFU/mL. By integrating PMA with the RPA-CRISPR/Cas12a technology, this system offers valuable technical support for the efficient, sensitive, and clear detection of viable Salmonella in wastewater.