ABSTRACTWastewater is considered a reservoir of antimicrobial resistance genes (ARGs), where the abundant antimicrobial-resistant bacteria and mobile genetic elements facilitate horizontal gene transfer. However, the prevalence and extent of these phenomena in different taxonomic groups that inhabit wastewater are still not fully understood. Here, we determined the presence of ARGs in metagenome-assembled genomes (MAGs) and evaluated the risks of MAG-carrying ARGs in potential human pathogens. The potential of these ARGs to be transmitted horizontally or vertically was also determined. A total of 5,916 MAGs (completeness >50%, contamination <10%) were recovered, covering 68 phyla and 279 genera. MAGs were dereplicated into 1,204 genome operational taxonomic units (gOTUs) as a proxy for species ( average nucleotide identity >0.95). The dominant ARG classes detected were bacitracin, multi-drug, macrolide-lincosamide-streptogramin (MLS), glycopeptide, and aminoglycoside, and 10.26% of them were located on plasmids. The main hosts of ARGs belonged to Escherichia, Klebsiella, Acinetobacter, Gresbergeria, Mycobacterium, and Thauera. Our data showed that 253 MAGs carried virulence factor genes (VFGs) divided into 44 gOTUs, of which 45 MAGs were carriers of ARGs, indicating that potential human pathogens carried ARGs. Alarmingly, the MAG assigned as Escherichia coli contained 159 VFGs, of which 95 were located on chromosomes and 10 on plasmids. In addition to shedding light on the prevalence of ARGs in individual genomes recovered from activated sludge and wastewater, our study demonstrates a workflow that can identify antimicrobial-resistant pathogens in complex microbial communities.IMPORTANCEAntimicrobial resistance (AMR) threatens the health of humans, animals, and natural ecosystems. In our study, an analysis of 165 metagenomes from wastewater revealed antibiotic-targeted alteration, efflux, and inactivation as the most prevalent AMR mechanisms. We identified several genera correlated with multiple ARGs, including Klebsiella, Escherichia, Acinetobacter, Nitrospira, Ottowia, Pseudomonas, and Thauera, which could have significant implications for AMR transmission. The abundance of bacA, mexL, and aph(3")-I in the genomes calls for their urgent management in wastewater. Our approach could be applied to different ecosystems to assess the risk of potential pathogens containing ARGs. Our findings highlight the importance of managing AMR in wastewater and can help design measures to reduce the transmission and evolution of AMR in these systems.

Biogas fermentation is a widely used technology of commercial and environmental importance, but the presence of nitrogenous compounds can strongly impact reactor productivity. Here, we investigated methane production in the presence of nitrite inhibition. The effects of lactate, glucose, acetate, formate and maize silage were evaluated. Methane production depended on substrate type, substrate concentration, and the buffer system. Nitrite inhibited methane production, but amendment with anammox bacteria mitigated this inhibition, resulting in 81.1 % recovery of methanogenic activity. Furthermore, anammox bacteria were efficient in removing nitrogenous compounds. A microbial community analysis showed significant shifts depending on the applied conditions, suggesting that a syntrophic relationship of Anaerolinaceae with Methanosaeta, Methanoculleus or Methanospirillum played a crucial role in methanogenesis via the acetoclastic and hydrogenotrophic pathways. These findings can help with stabilizing reactors for methane production and emission reduction.

Abstract Background The need for addition of external electron donors such as ethanol or lactate impairs the economic viability of chain elongation (CE) processes for the production of medium-chain carboxylates (MCC). However, using feedstocks with inherent electron donors such as silages of waste biomass can improve the economics. Moreover, the use of an appropriate inoculum is critical to the overall efficiency of the CE process, as the production of a desired MCC can significantly be influenced by the presence or absence of specific microorganisms and their metabolic interactions. Beyond, it is necessary to generate data that can be used for reactor design, simulation and optimization of a given CE process. Such data can be obtained using appropriate mathematical models to predict the dynamics of the CE process. Results In batch experiments using silages of sugar beet leaves, cassava leaves, and Elodea/wheat straw as substrates, caproate was the only MCC produced with maximum yields of 1.97, 3.48, and 0.88 g/kgVS, respectively. The MCC concentrations were accurately predicted with the modified Gompertz model. In a semi-continuous fermentation with ensiled sugar beet leaves as substrate and digestate from a biogas reactor as the sole inoculum, a prolonged lag phase of 7 days was observed for the production of MCC (C6–C8). The lag phase was significantly shortened by at least 4 days when an enriched inoculum was added to the system. With the enriched inoculum, an MCC yield of 93.67 g/kgVS and a productivity of 2.05 gMCC/L/d were achieved. Without the enriched inoculum, MCC yield and productivity were 43.30 g/kgVS and 0.95 gMCC/L/d, respectively. The higher MCC production was accompanied by higher relative abundances of Lachnospiraceae and Eubacteriaceae. Conclusions Ensiled waste biomass is a suitable substrate for MCC production using CE. For an enhanced production of MCC from ensiled sugar beet leaves, the use of an enriched inoculum is recommended for a fast process start and high production performance.

Huge phages have genomes larger than 200 kilobases, which are particularly interesting for their genetic inventory and evolution. We screened 165 wastewater metagenomes for the presence of viral sequences. After identifying over 600 potential huge phage genomes, we reduced the dataset using manual curation by excluding viral contigs that did not contain viral protein-coding genes or consisted of concatemers of several small phage genomes. This dataset showed seven fully annotated huge phage genomes. The phages grouped into distinct phylogenetic clades, likely forming new genera and families. A phylogenomic analysis between our huge phages and phages with smaller genomes, i.e., less than 200 kb, supported the hypothesis that huge phages have undergone convergent evolution. The genomes contained typical phage protein-coding genes, sequential gene cassettes for metabolic pathways, and complete inventories of tRNA genes covering all standard and rare amino acids. Our study showed a pipeline for huge phage analyses that may lead to new enzymes for therapeutic or biotechnological applications.

The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting.

Creating structurally and functionally stable microbiomes would be greatly beneficial to biotechnology and human health but so far has proven challenging. Here, we propose a looped mass transfer design that keeps microbiomes constant over long periods of time. The effluent of five parallel reactors that began with the same inoculum, was mixed in a reactor that represented a regional pool. Part of this pool was transferred back to the five reactors. Community dynamics were monitored and visualized by quantitative microbial flow cytometry and selected taxonomic sequencing of whole communities and sorted subcommunities. The rescue effect, known from metacommunity theory, was the main stabilizing mechanism that led to the survival of subcommunities with zero netgrowth, especially at high mass transfer rates. The looped mass transfer approach promises to overcome notorious stochastic structural fluctuations in bioreactors and has the potential to design and stabilize communities that can perform desired functions.