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

Abstract

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.

Using recirculating flumes and a response surface model to investigate the role of hyporheic exchange and bacterial diversity on micropollutant half-lives

“…Total bacterial community was quantied based on the 16S rRNA gene using quantitative real-time PCR according to published protocols. Results from quantitative real-time PCR were used as an estimate for bacteria biomass 40,41 to evaluate the effectivity of the pre-incubation period. Illumina Miseq amplicon sequencing targeting the 16S rRNA gene using the bacteria specic primer pair 341F and 806R was performed by LGC Genomics GmbH (Berlin, Germany) followed by post processing of the raw data as previously published.…”

Impact of plants on the diversity and activity of methylotrophs in soil

“…However, there are significant issues with the use of the 16S rRNA gene to infer function, especially with methylotrophs. For example, only a few members of the genera Bacillus and Flavobacterium are methylotrophs [11,26]. DNA-based diversity studies of methylotrophs therefore require use of a functional marker gene.…”

“…DNA-based diversity studies of methylotrophs therefore require use of a functional marker gene. Previous studies in the soil environment, using PCR primers targeting the large subunit encoding gene of the canonical methanol dehydrogenase, mxaF [8], have revealed a relatively low diversity [18,26,27], highlighting the necessity to incorporate the recently discovered novel MDH genes and enzymes, notably xoxF and mdh2, into analyses of methylotrophs in soil. PCR primers targeting different clades of xoxF are now available [28] but have not been extensively used in soil environments.…”

A robust, cost‐effective method for DNA, RNA and protein co‐extraction from soil, other complex microbiomes and pure cultures

“…As the novelty of our method lies in the recovery of proteins from the phenol phase, typically discarded in the protocol of Griffiths et al (), we further demonstrate that our biomolecule co‐extraction is suitable for metaproteomics analysis of other soil types with varying clay contents. Indeed, the DNA and RNA co‐extraction protocol from Griffiths et al () has been cited to date by over 1,250 papers including many reporting on downstream high‐throughput sequencing, including 16SrRNA and ITS amplicon analysis (Haas et al, ; Morawe et al, ; Sayer et al, ; Whitman et al, ), metagenomics (Malik, Thomson, Whiteley, Bailey, & Griffiths, ; Soares et al, ; Wilhelm, Hanson, Chandra, & Madsen, ) and metatranscriptomics (Alessi et al, ; de Menezes, Clipson, & Doyle, ; Hesse et al, ). Here, 16S rRNA profiling from DNA and cDNA was performed on three samples corresponding to three biological replicates from one soil type (with a clay content of 11.1%) while metaproteomics was conducted for nine samples corresponding to three biological replicates from three soil types (with clay content of 11.1%, 19.4% and 35.1%).…”