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Background Composting is an important technique for environment-friendly degradation of organic material, and is a microbe-driven process. Previous metagenomic studies of composting have presented a general description of the taxonomic and functional diversity of its microbial populations, but they have lacked more specific information on the key organisms that are active during the process. Results Here we present and analyze 60 mostly high-quality metagenome-assembled genomes (MAGs) recovered from time-series samples of two thermophilic composting cells, of which 47 are potentially new bacterial species; 24 of those did not have any hits in two public MAG datasets at the 95% average nucleotide identity level. Analyses of gene content and expressed functions based on metatranscriptome data for one of the cells grouped the MAGs in three clusters along the 99-day composting process. By applying metabolic modeling methods, we were able to predict metabolic dependencies between MAGs. These models indicate the importance of coadjuvant bacteria that do not carry out lignocellulose degradation but may contribute to the management of reactive oxygen species and with enzymes that increase bioenergetic efficiency in composting, such as hydrogenases and N2O reductase. Strong metabolic dependencies predicted between MAGs revealed key interactions relying on exchange of H+, NH3, O2 and CO2, as well as glucose, glutamate, succinate, fumarate and others, highlighting the importance of functional stratification and syntrophic interactions during biomass conversion. Our model includes 22 out of 49 MAGs recovered from one composting cell data. Based on this model we highlight that Rhodothermus marinus, Thermobispora bispora and a novel Gammaproteobacterium are dominant players in chemolithotrophic metabolism and cross-feeding interactions. Conclusions The results obtained expand our knowledge of the taxonomic and functional diversity of composting bacteria and provide a model of their dynamic metabolic interactions.

Background: Thermophilic composting is a semi-engineered process carried out by diverse microbial communities. Composting is an environment friendly way of degrading biomass; its study may help uncover important biomass-degrading organisms and key enzymes. DNA sequence-based previous studies have presented a general description of the microbial-molecular features of composting, but they have lacked more specific information on the key organisms that are active during the process and their genomes. Methods: We present an analysis of metagenome-assembled genomes (MAGs) obtained from time-series samples of a thermophilic composting process in the São Paulo Zoological Park (Brazil). Our results are based on a careful analysis of MAG gene content and on metabolic modeling of their interactions. Results: We recovered 60 MAGs from sequencing datasets from two separate composting cells. Phylogenetic analysis shows that 47 of these MAGs represent novel taxa at the genus or higher levels. We have analyzed the gene repertoire of these MAGs in terms of lignocellulose degradation, secondary metabolite production, antibiotic resistance genes, denitrification genes, sulfur metabolism, hydrogen metabolism, and oxygen metabolism. For one of the composting cells we also had metatranscriptome data, which allowed a deeper analysis of 49 MAGs. This analysis showed the presence of three distinct clusters of MAGs with varying activity during the 99-day composting process. The interaction model pointed to Sphaerobacter thermophilus and Thermobispora bispora as key players in the process, as well as other bacteria that are novel. Our results also show the importance of coadjuvant bacteria and of microbial functions related to efficient bioenergetic processes during biomass conversion, such as N2O reduction and hydrogenases. A novel acidobacteria MAG encodes N2O reductase hallmark genes (nosZD). Strong metabolic dependencies predicted between MAGs revealed that cross-feeding in composting can be determined by complementary functions found in the genomes of producers and consumers, supporting the Black Queen hypothesis for co-evolutionary interactions. Conclusions: This study reveals for the first time the key bacterial players in thermophilic composting and provides a model of their dynamic metabolic interactions. These findings pave the way for more rational composting procedures and provide information that could help the development of novel biomass-degrading technologies.

Thanks in large part to newer, better, and cheaper DNA sequencing technologies, an enormous number of metagenomic sequence datasets have been and continue to be generated, covering a huge variety of environmental niches, including several different human body sites. Comparing these metagenomes and identifying their commonalities and differences is a challenging task, due not only to the large amounts of data, but also because there are several methodological considerations that need to be taken into account to ensure an appropriate and sound comparison between datasets. In this chapter, we describe current techniques aimed at comparing metagenomes generated by 16S ribosomal RNA and shotgun DNA sequencing, emphasizing methodological issues that arise in these comparative studies. We provide a detailed case study to illustrate some of these techniques using data from the Human Microbiome Project comparing the microbial communities from ten buccal mucosa samples with ten tongue dorsum samples in terms of alpha diversity, beta diversity, and their taxonomic and functional profiles.

Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the Sao Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new Thermobacillus species; the first Bacillus thermozeamaize genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as Geobacillus thermoglucosidasius and Caldibacillus debilis. The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new Thermobacillus species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built, using compost metagenome and metatranscriptome datasets generated in a previous study. We obtained strong evidence that five of the six recovered genomes are indeed present and active in that composting process. We have thus discovered three (perhaps four) new thermophillic bacterial species that add to the increasing repertoire of known lignocellulose degraders, whose biotechnological potential can now be investigated in further studies.