Acidotolerant Bacteria and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil

Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi.


List of supplementary Figures
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nMDS analyses of bacterial and fungal communities in the 'heavy' and 'middle' fractions of both SIP experiments.

Figure S2.
Gene numbers of mmoX genes of treatments with different pH in the pH shift SIP experiment.

Figure S7.
Diversity and richness estimators of mxaF sequences from pyrosequencing amplicon pools at similarity level of 90%.

Figure S8.
Diversity and richness estimators of ITS gene sequences from pyrosequencing amplicon pools at similaritiy level of 97% (species level).

Figure S9.
Composition of the various mxaF phylotypes after different substrate or pH treatments.  Tables   Table S1. Relative abundances of labeled bacterial taxa (OTU) based on 16S rRNA gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]and [ 13 C 1 ]-methanol treatment of Substrate SIP experiment. Table S2.

List of supplementary
Taxonomic affiliation of bacterial phylotypes (OTUs with family-level cutoff 90.0% based on 16S rRNA gene sequences) in numerical order. Table S3.
Relative abundances of labeled fungal taxa (OTU) based on ITS gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]-and [ 13 C 1 ]-methanol treatments of Substrate SIP experiment. Table S5.
Taxonomic affiliation of fungal phylotypes (ITS gene sequences clustered at species-level 97% similarity cut-off) in numerical order. Table S6.
Relative abundances of labeled bacterial taxa (OTU) based on 16S rRNA gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]and [ 13 C 1 ]-methanol treatment at pH 4 of pH SIP experiment. Table S7.
Relative abundances of labeled bacterial taxa (OTU) based on 16S rRNA gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]and [ 13 C 1 ]-methanol treatment at pH 7 of pH SIP experiment. Table S8.
Relative abundances of labeled taxa (OTU) based on mxaF gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]-and [ 13 C 1 ]-methanol treatments at pH 4 of pH SIP experiment. Table S9.
Relative abundances of labeled taxa (OTU) based on mxaF gene sequences in all fractions (H, heavy; M, middle; L, light) of [ 12 C]-and [ 13 C 1 ]-methanol treatments at pH 7 of pH SIP experiment. Table S10. Relative Table S13. Similarity analyses of bacterial communities (family-level with 90.1% cutoff of 16S rRNA gene sequence) of both SIP experiments based on ANOSIM (Analysis of Similarity) and NPMANOVA (non-parametric multivariate analysis of variance). Table S14. Similarity analyses of fungal communities (family-level with 97.0% cut-off of ITS gene sequence) of both SIP experiments based on ANOSIM (Analysis of Similarity) and NPMANOVA (non-parametric multivariate analysis of variance). Table S15. Similarity analyses of mxaF-possessing methylotrophic communities (90% cut-off) of both SIP experiments based on ANOSIM (Analysis of Similarity) and NPMANOVA (non-parametric multivariate analysis of variance).   1% for Bacteria (16S rRNA gene sequences, family-level; reduced dataset, for detailed information see Supplemental Materials and Methods) and 97% for Fungi (ITS gene sequences species-level). Stress values are given in brackets. All analyses are based on Bray-Curtis similarity index. Symbols according to SIP experiment: , substrate SIP; , pH 4; , pH 7. ' 12 C' indicates [ 12 C]-substrates and ' 13 C' indicates [ 13 C u ]-substrates. Symbols according to supplemented [ 13 C u ]-substrate: , methanol; , acetate +; , glucose +; , xylose +; , vanillic acid +; , CO 2 +; , CO 2 (cross indicates additional supplementation of [ 12 C]-methanol in substrate SIP experiment).               Table S2) and confirmed by positioning in phylogenetic tree (data not shown) b Sequence identity with BLASTn < 90 % as well as ambiguous position in phylogenetic tree (for further information see Table S2)  Percentage of labeled taxa to total fraction [%] . 24 51 a Taxonomic affiliation was done with BLASTn (November 2015) and is based on the next cultivated hit for each OTU (for further information see Table S8) b Sequence identity of next cultured hit < 90 %, phylogenetic affiliation up to order level d Sequence identity of next cultured hit ≥ 95 %, phylogenetic affiliation up to genus level e Query of next cultured hit was only 72 % with BLASTn analysis Percentage of labeled taxa to total fraction [%] . 43 48 a Taxonomic affiliation was done with BLASTn (November 2015) and is based on the next cultivated hit for each OTU (for further information see Table S8) c Sequence identity of next cultured hit < 95 %, phylogenetic affiliation up to family level d Sequence identity of next cultured hit ≥ 95 %, phylogenetic affiliation up to genus level  Table S2) and confirmed by positioning in phylogenetic tree (data not shown) b Sequence identity with BLASTn < 90 % as well as ambiguous position in phylogenetic tree (for further information see S2) Percentage of labeled taxa to total fraction [%] . 75 50 a Taxonomic affiliation was done with BLASTn (December 2015; for further information see Table S2) and confirmed by positioning in phylogenetic tree (data not shown) b Sequence identity with BLASTn < 90 % as well as ambiguous position in phylogenetic tree (for further information see Table S2)   2015) and was done with a bayesian classifier implied with MOTUHR based on the best hit of consensus taxonomy after 100 bootstrapped assignments (for further reference sequences based on 'massBLASTer' of UNITE see Table S5) b Taxa in brackets dominated fungal order or family c Treatment with methanol (MeOH), acetate (Ace), glucose (Glu), xylose (Xyl), vanillic acid (Van) and carbon dioxide (CO 2 ); cross (+) indicates additional methanol supplementation d Treatment with methanol at different pH conditions (pH 4 and pH 7) a Treatment with methanol (MeOH), acetate (Ace), glucose (Glu), xylose (Xyl), vanillic acid (Van) and carbon dioxide (CO 2 ); cross (+) indicates additional methanol supplementation b Treatment with methanol at different pH conditions (pH 4 and pH 7) c Comparison between t 0 of Substrate SIP experiment and pH-SIP experiment (Sub vs pH) and between both t0 of pH-SIP (pH4 vs pH7)

Table S14. Similarity analyses of fungal communities (family-level with 97.0% cut-off of ITS gene sequence) of both SIP experiments based on ANOSIM (Analysis of Similarity) and NPMANOVA (non-parametric multivariate analysis of variance).
Values of total analyses in bold, pairwaise analyses in cursive. a Treatment with methanol (MeOH), acetate (Ace), glucose (Glu), xylose (Xyl), vanillic acid (Van) and carbon dioxide (CO 2 ); cross (+) indicates additional methanol supplementation b Treatment with methanol at different pH conditions (pH 4 and pH 7) c Comparison between t 0 of Substrate SIP experiment and pH-SIP experiment (Sub vs pH) and between both t0 of pH-SIP (pH4 vs pH7) a Treatment with methanol (MeOH), acetate (Ace), glucose (Glu), xylose (Xyl), vanillic acid (Van) and carbon dioxide (CO 2 ); cross (+) indicates additional methanol supplementation b Treatment with methanol at different pH conditions (pH 4 and pH 7) c Comparison between t 0 of Substrate SIP experiment and pH-SIP experiment (Sub vs pH) and between both t0 of pH-SIP (pH4 vs pH7)  Table S2) and confirmed by positioning in phylogenetic tree (data not shown) b Sequence identity with BLASTn <90% as well as ambiguous position in phylogenetic tree (for further information see Table S2) c Treatment with methanol (MeOH), acetate (Ace), glucose (Glu), xylose (Xyl), vanillic acid (Van) and carbon dioxide (CO 2 ); cross (+) indicates additional methanol supplementation d Treatment with methanol at different pH conditions (pH 4 and pH 7)