Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics

Abstract

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.

Figure 1. Relative abundance of bacteria in ZC4 samples at the order level.

Taxonomic assignments were obtained from 16S rRNA amplicon (A) and shotgun (S) sequencing datasets using RDP classifier and MG-RAST (M5NR) database, respectively. Only orders with relative abundance ≥2% are shown. Unassigned 16S (10–30%) and DNA shotgun reads (10.1–14.6%) were excluded. Samples are referred to by the letter D followed by the collection day. Asterisk indicates one day after the turning procedure.

Figure 2. ZC4 phylogenetic diversity variation.

Profile of the number of OTUs estimated by 16S rRNA amplicon sequencing and phylogenetic diversity index (alpha rarefaction) during ZC4 composting. Intervals on the horizontal axis do not represent chronological time. Samples are referred to by the letter D followed by the collection day.

Figure 3. Maximum likelihood phylogenetic tree based on nucleotide sequences of 113 ortholog genes.

Node numbers represent bootstrap support. Scale is number of substitutions per site. Thermaerobacter marianensis (clostridiales) was chosen as outgroup; all other species are members of the Bacillaceae family.

Figure 4. Functional profile of the metatranscriptomes based on COG categories.

(a) Hierarchical clustering of metatranscriptome CDSs. The tree was generated using an IMG/M tool, selecting the COG pathways method. Samples are referred to by the letter D followed by the collection day. (b) Number of COGs (y axis) of each COG functional category (x axis) that had differential representation, based on relative abundance of CDSs. A COG functional category was considered differentially represented if the relative abundance value for a given sample was at least 1.5 times the interquartile range for the third quartile (i.e., only overrepresented outliers were noted) considering relative abundance values for that category in all samples. Each histogram corresponds to the same-color group of samples to its left.

Figure 5. Analysis of CDSs involved in lignocellulosic degradation during ZC4 composting process.

Relative abundance (%) of the CDSs annotated as hemicellulases, cellulases, ligninases and pectinases in the metatranscriptome contigs throughout the composting process, according to the IMG-M pipeline. Samples are referred to by the letter D followed by the collection day. Intervals on the horizontal axis do not represent chronological time.

Figure 6. Taxonomic profile of the ZC4 metatranscriptome.

Columns labeled by A show taxonomic profiles considering all CDSs annotated with COGs. Columns labeled by B show taxonomic profiles considering CDSs annotated with COGs related to plant biomass (lignocellulose and pectin) degradation. The CDSs were classified with program myTaxa. Samples are referred to by the letter D followed by the collection day. The asterisk indicates one day after the turning procedure.

Figure 7. Molecular overview of a thermophilic composting operation.

The top panel shows the three stages identified in this work: start (D01, D03 and D64), middle (D07, D15 and D30), and end (D78 and D99). The box for each stage shows the microbiota composition at the order level. The relative abundance of microorganisms is roughly proportional to the font size, and only orders with relative abundance ≥2.5% are shown. The middle panel depicts the degradation process, with vertical correspondence to the stages shown in the top panel. The bottom panel shows relative abundance of COG functions associated to lignocellulolytic enzymes, also with vertical correspondence to the stages shown in the top panel. The results in this figure were based on ZC4 metatranscriptomes.