Bacterial and fungal contributions to delignification and lignocellulose degradation in forest soils with metagenomic and quantitative stable isotope probing

Delignification, or lignin-modification, facilitates the decomposition of lignocellulose in woody plant biomass. The extant diversity of lignin-degrading bacteria and fungi is underestimated by culture-dependent methods, limiting our understanding of the functional and ecological traits of decomposers populations. Here, we describe the use of stable isotope probing (SIP) coupled with amplicon and shotgun metagenomics to identify and characterize the functional attributes of lignin-, cellulose-and hemicellulose-degrading fungi and bacteria in coniferous forest soils from across North America. We tested the extent to which catabolic genes partitioned among different decomposer taxa; the relative roles of bacteria and fungi, and whether taxa or catabolic genes correlated with variation in lignocellulolytic activity, measured as the total assimilation of 13C-label into DNA and phospholipid fatty acids. We found high overall bacterial degradation of our model lignin substrate, particularly by gram-negative bacteria (Comamonadaceae and Caulobacteraceae), while fungi were more prominent in cellulose-degradation. Very few taxa incorporated 13C-label from more than one lignocellulosic polymer, suggesting specialization among decomposers. Collectively, members of Caulobacteraceae could degrade all three lignocellulosic polymers, providing new evidence for their importance in lignocellulose degradation. Variation in lignin-degrading activity was better explained by microbial community properties, such as catabolic gene content and community structure, than cellulose-degrading activity. SIP significantly improved shotgun metagenome assembly resulting in the recovery of several high-quality draft metagenome-assembled genomes and over 7,500 contigs containing unique clusters of carbohydrate-active genes. These results improve understanding of which organisms, conditions and corresponding functional genes contribute to lignocellulose decomposition.


Introduction
The incomplete decomposition of woody biomass in coniferous forests is an important resolve the catabolic and ecological traits of decomposers, we must utilize culture-independent 38 methods, like stable isotope probing (SIP), that better reflect in situ conditions. 39 The nature of microbial lignin-degradation is poorly described beyond the canonical 40 breakdown of lignin in woody biomass by specialized, aerobic, litter-inhabiting wood-rot fungi.  Table S3. 167     used to compare control and labeled microcosms to identify OTUs and taxonomic groups that 205 incorporated 13 C from the labeled substrate. An OTU or taxon had to be selected by at least one 206 of these methods and be, on average, 3-fold more abundant in 13 C libraries in at least one 207 geographic region. Random forest classification, implemented in boruta (Kursa and Rudnicki, 208 2010), was used to identify taxa or CAZymes predictive of total 13 C incorporation into DNA or 209 PLFA. Correlation between features selected by boruta and total 13 C were subsequently 210 performed using 'rcorr' from the R package Hmisc. The relative importance of soil layer, 211 geographic region, total carbon, CAZy composition and community structure in linear models 212 for each substrate was assessed using the R package relaimpo (Grömping, 2006) using the 213 primary and secondary axes of NMDS as measures of community structure and CAZy 214 composition.

Results
We utilized SIP microcosm-based experiments to investigate the taxonomic identity and  Table S4). The enrichment of degradative 227 populations was also apparent in shotgun metagenomes, as evidence by the increased proportion   Figure S4). Notably, several OTUs belonging to unclassified clades of 242 Caulobacteraceae were highly enriched in 13 C-DNA pools from lignin ( Figure S5) and were 243 significantly more abundant in mineral than organic soils (21.5 vs. 6.0 counts per thousand reads, 244 respectively; t-test; p=0.02). Differences in predisposition for cellulose-versus lignin-catabolism 245 in Asticcacaulis versus Caulobacter, respectively, was evident in the catabolic gene content of 246 metagenome assemblies (Figure 4bc). 247 We anticipated that lignocellulolytic species would commonly degrade more than one of  Fungicide-treatment greatly reduced assimilation of 13 C from lignin into fungal PLFAs, 301 but also into gram-positive bacteria and, to a lesser extent, gram-negative bacteria (Figure 7a).

302
While overall bacterial activity was decreased by fungicide-treatment, the assimilation of 13 C by   (Table S8).

Discussion
We failed to find support for our initial hypothesis that individual species would degrade 345 multiple components of lignocellulose. Instead, we identified a diverse array of taxa assimilating           Table S5. considered when comparing between them. Also, δ-13 C enrichment is weighted against total 12 C, 714 and thus reflects the total lignocellulolytic activity relative to total biomass which is 3-to 20-715 fold higher in organic layer soils (details in Table S1).

721
Taxa comprising fewer than 0.05% of total reads are not displayed.  • Quantify 13 C in Fungi/Bacteria

Shotgun Sequencing
• Identify functional genes • Identify functional taxa (fungi) • Bacteria-speci c activity       Figure S1. Reaction scheme for the synthesis of 13 C ring-labeled coniferyl alcohol which was subsequently polymerized into lignin with an average of fourteen coniferyl alcohols.   Figure S2. Evidence for the improved assembly of metagenomes derived from 13 C-enriched DNA in soil microcosms amended with 13 C-labeled (blue) or unlabeled (pink) cellulose or lignin. Statistically supported differences between paired labeled and unlabeled treatments (t-test; p < 0.01) are designated with an asterix (*). Statistical testing could not be performed for cellulose with single 12 C-libraries. When composited, 12 C-libraries cellulose libraries had significantly lower assembly than 13 C-libraries in organic (Wilcoxon; p < 0.003) and mineral layer soils (Wilcoxon; p < 0.001).   Figure S3. The relative abundance of prominent lignocellulolytic members of Burkholderiales based on differential abundance between 12 C-and 13 C-libraries. Plots are organized in a hierarchical structure displaying family and genus for all active taxa within each group. Error bars correspond to one standard error of the mean. Significant (Tukey HSD; p adj < 0.05) pairwise differences are grouped by lettering.   Figure S4. The relative abundance of all OTUs classified to the genera of Caulobacteraceae. Differences in hemicellulolytic, cellulolytic and lignolytic can be observed among genera and unclassified Caulobacteraceae sequences based on differential abundance between 12 C-and 13 C-libraries. Error bars correspond to one standard error of the mean. Significant (Tukey HSD; p adj < 0.05) pairwise differences are grouped by lettering.    Figure S6. Abundances of OTUs enriched in both lignin and cellulose 13 C-pyrotag libraries within the same region. Plots are titled with the lowest supported taxonomic classification and include representative read names. Where two OTUs were combined, both independently exhibited the trend in multiple substrate use. The unclassified Caulobacteraceae OTU belongs to the clade 'LH3_1' presented in Supplementary Figure 3b. 10