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The enzymatic arsenal of several soil microorganisms renders them particularly suitable for the degradation of lignocellulose, a process of distinct ecological significance with promising biotechnological implications. In this study, we investigated the spatiotemporal diversity and distribution of bacteria and fungi with 16S and Internally Trascribed Spacer (ITS) ribosomal RNA next-generation-sequencing (NGS), focusing on forest mainland Abies cephalonica and insular Quercus ilex habitats of Greece. We analyzed samples during winter and summer periods, from different soil depths, and we applied optimized and combined targeted meta-omics approaches aiming at the peroxidase-catalase family enzymes to gain insights into the lignocellulose degradation process at the soil microbial community level. The microbial communities recorded showed distinct patterns of response to season, soil depth and vegetation type. Overall, in both forests Proteobacteria, Actinobacteria, Acidobacteria were the most abundant bacteria phyla, while the other phyla and the super-kingdom of Archaea were detected in very low numbers. Members of the orders Agaricales, Russulales, Sebacinales, Gomphales, Geastrales, Hysterangiales, Thelephorales, and Trechisporales (Basidiomycota), and Pezizales, Sordariales, Eurotiales, Pleosporales, Helotiales, and Diaporthales (Ascomycota) were the most abundant for Fungi. By using optimized “universal” PCR primers that targeted the peroxidase-catalase enzyme family, we identified several known and novel sequences from various Basidiomycota, even from taxa appearing at low abundance. The majority of the sequences recovered were manganese peroxidases from several genera of Agaricales, Hysterangiales, Gomphales, Geastrales, Russulales, Hymenochaetales, and Trechisporales, while lignin -and versatile-peroxidases were limited to two to eight species, respectively. Comparisons of the obtained sequences with publicly available data allowed a detailed structural analysis of polymorphisms and functionally relevant amino-acid residues at phylogenetic level. The targeted metagenomics applied here revealed an important role in lignocellulose degradation of hitherto understudied orders of Basidiomycota, such as the Hysterangiales and Gomphales, while it also suggested the auxiliary activity of particular members of Proteobacteria, Actinobacteria, Acidobacteria, Verrucomicrobia, and Gemmatimonadetes. The application of NGS-based metagenomics approaches allows a better understanding of the complex process of lignocellulolysis at the microbial community level as well as the identification of candidate taxa and genes for targeted functional investigations and genetic modifications.

Additional file 3: Table S2. Primers, amplicons, and calibrator plasmids used for plasmid copy number determination.

Additional file 1: Figure S1. Completion of plasmid sequences by primer walking with a list of the primers used for each plasmid (A). PCR amplification of ZM4 chromosome region containing a 2.4-kb fragment near ZMO0133 locus that is absent in previously reported ZM4 genome sequence (B). A schematic is shown detailing the location of primers used to PCR, and PCR products on agarose gel are also shown. Figure S2. Customized rRNA depletion kit was developed with Life Technologies for Z. mobilis mRNA enrichment, and RNA-Seq result of the percentage of rRNA, tRNA, and mRNA in Z. mobilis total RNA was calculated (A). qRT-PCR measurement of rRNA content before and after rRNA depletion of total RNA using the customized kit (B). rRNA reduction is reported as the fold change in the target rRNA in total RNA relative to depleted RNA. Measurements were collected in WT (Z. mobilis strain 33C derived from Z. mobilis 8b) and MT (a mutant strain of 33C) grown in either rich media with 5% glucose (RMG) or rich media with 5% xylose (RMX) and collected in two biological replicates. Error is reported as standard deviation. Residual rRNA contamination and rRNA depletion efficiency of samples described in (B) was detected by RNA-Seq (C). Error is reported as standard deviation. An example of pairwise replicate correlation of RNA-Seq pseudo read counts (i.e. log2 transformed following addition of La Place constant of 1) for two biological replicates after rRNA depletion (D). Figure S3. Heatmap of RNA-Seq data from 6% and 9% ACSH, anaerobic (AN) and aerobic (AE) conditions. Coloring by condition (left color bar) corresponds to the one used for the Fig. 4. Blue, NREL, fermentor with biomass hydrolysates; black, NREL, flasks with rich RMG medium; light grey, GLBRC, 6% ACSH; Orange, GLBRC, 9% ACSH; light green, Univ. Athens, anaerobic; dark green, Univ. Athens (UA), anaerobic; dark green, UA, aerobic. Top index bar shows expression clusters (see Additional file 6: Table S5 for gene-cluster assignments). Right annotation bar shows generalized factor that is applicable to experimental designs across all the 3 research centers: “Early” and “Late” are growth stages.

Zymomonas mobilis subsp. mobilis is one of the most rigorous ethanol-producing organisms known to date, considered by many to be the prokaryotic alternative to yeast. The two most applied Z. mobilis subsp. mobilis strains, ZM4 and CP4, derive from Recife, Brazil, and have been isolated from sugarcane fermentations. Of these, ZM4 was the first Z. mobilis representative strain to be sequenced and analyzed. Here, we report the finishing of the genome sequence of strain CP4, which is highly similar but not identical to that of ZM4.