The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition.

Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomic- and metatranscriptomic-based analyses of a large composting operation in the São Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50 °C to 75 °C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the process; and that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomass-degrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.

The size of bacterial genomes is often associated with organismal metabolic capabilities determining ecological breadth and lifestyle. The recently proposed Candidate Phyla Radiation (CPR)/Patescibacteria encompasses mostly unculturable bacterial taxa with relatively small genome sizes with potential for co‐metabolism interdependencies. As yet, little is known about the ecology and evolution of CPR, particularly with respect to how they might interact with other taxa. Here, we reconstructed two novel genomes (namely, Candidatus Saccharibacter sossegus and Candidatus Chaer renensis) of taxa belonging to the class Saccharimonadia within the CPR/Patescibacteria using metagenomes obtained from acid mine drainage (AMD). By testing the hypothesis of genome streamlining or symbiotic lifestyle, our results revealed clear signatures of gene losses in these genomes, such as those associated with de novo biosynthesis of essential amino acids, nucleotides, fatty acids and cofactors. In addition, co‐occurrence analysis provided evidence supporting potential symbioses of these organisms with Hydrotalea sp. in the AMD system. Together, our findings provide a better understanding of the ecology and evolution of CPR/Patescibacteria and highlight the importance of genome reconstruction for studying metabolic interdependencies between unculturable Saccharimonadia representatives.

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 São 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.

Biodiversity underlies ecosystem functioning. While aboveground biodiversity is often well studied, the belowground microbiome, in particular protists, remains largely unknown. Indeed, holistic insights into soil microbiome structures in natural soils, especially in hyperdiverse biomes such as the Brazilian Cerrado, remain unexplored. Here, we study the soil microbiome across four major vegetation zones of the Cerrado, ranging from grass-dominated to tree-dominated vegetation with a focus on protists. We show that protist taxon richness increases towards the tree-dominated climax vegetation. Early successional habitats consisting of primary grass vegetation host most potential plant pathogens and least animal parasites. Using network analyses combining protist with prokaryotic and fungal sequences, we show that microbiome complexity increases towards climax vegetation. Together, this suggests that protists are key microbiome components and that vegetation succession towards climax vegetation is stimulated by higher loads of animal and plant pathogens. At the same time, an increase in microbiome complexity towards climax vegetation might enhance system stability.