# Methanol consumption drives the bacterial chloromethane sink in a forest soil

Pauline Chaignaud, Mareen Morawe, Ludovic Besaury, Eileen Kröber, Stéphane Vuilleumier, Françoise Bringel, Steffen Kolb

► To cite this version:

Pauline Chaignaud, Mareen Morawe, Ludovic Besaury, Eileen Kröber, Stéphane Vuilleumier, et al. Methanol consumption drives the bacterial chloromethane sink in a forest soil. The International Society of Microbiological Ecology Journal, 2018, 12 (11), pp.2681-2693. 10.1038/s41396-018-0228-4. hal-01959653

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# Methanol consumption drives the bacterial chloromethane sink in a forest soil

Pauline Chaignaud<fnref n="1" />,<fnref n="2" /> · Mareen Morawe<fnref n="2" /> · Ludovic Besaury<fnref n="1" />,<fnref n="4" /> · Eileen Kröber<fnref n="3" /> · Stéphane Vuilleumier ID<fnref n="1" /> · Françoise Bringel ID<fnref n="1" /> · Steffen Kolb ID<fnref n="3" />

Received: 16 February 2018 / Revised: 1 June 2018 / Accepted: 15 June 2018 / Published online: 10 July 2018
© The Author(s) 2018. This article is published with open access

## Abstract

Halogenated volatile organic compounds (VOCs) emitted by terrestrial ecosystems, such as chloromethane (CH₃Cl), have pronounced effects on troposphere and stratosphere chemistry and climate. The magnitude of the global CH₃Cl sink is uncertain since it involves a largely uncharacterized microbial sink. CH₃Cl represents a growth substrate for some specialized methylotrophs, while methanol (CH₃OH), formed in much larger amounts in terrestrial environments, may be more widely used by such microorganisms. Direct measurements of CH₃Cl degradation rates in two field campaigns and in microcosms allowed the identification of top soil horizons (i.e., organic plus mineral A horizon) as the major biotic sink in a deciduous forest. Metabolically active members of *Alphaproteobacteria* and *Actinobacteria* were identified by taxonomic and functional gene biomarkers following stable isotope labeling (SIP) of microcosms with CH₃Cl and CH₃OH, added alone or together as the [¹³C]-isotopologue. Well-studied reference CH₃Cl degraders, such as *Methylobacterium extorquens* CM4, were not involved in the sink activity of the studied soil. Nonetheless, only sequences of the *cmuA* chloromethane dehalogenase gene highly similar to those of known strains were detected, suggesting the relevance of horizontal gene transfer for CH₃Cl degradation in forest soil. Further, CH₃Cl consumption rate increased in the presence of CH₃OH. Members of *Alphaproteobacteria* and *Actinobacteria* were also ¹³C-labeled upon [¹³C]-CH₃OH amendment. These findings suggest that key bacterial CH₃Cl degraders in forest soil benefit from CH₃OH as an alternative substrate. For soil CH₃Cl-utilizing methylotrophs, utilization of several one-carbon compounds may represent a competitive advantage over heterotrophs that cannot utilize one-carbon compounds.

## Introduction

Chloromethane (CH₃Cl) is the most abundant halogenated volatile organic compound (VOC) in the atmosphere and contributes substantially to destruction of stratospheric ozone [1, 2]. Global emissions reach about 2.0 ± 0.6 Tg CH₃Cl per year [3, 4]. Current knowledge also suggests that terrestrial CH₃Cl emissions are mainly associated with biological activities of the aboveground part of plants and with white rot fungi in soil [5–7]. CH₃Cl is produced upon burning of plant biomass, from methoxy groups of plant structural components such as lignin and pectin [8, 9]. CH₃Cl is also enzymatically produced by S-adenosylmethionine-dependent methylation of chloride [10]. Current estimates for the global sink are larger than the global source [3], to which forest soil could contribute as much as 1.0 Tg per year [3, 11]. Global budgets are still uncertain, as the nature of the biotic sink activity as well as its spatial and temporal variability are not known at the regional and global scale [12].

**Electronic supplementary material** The online version of this article (https://doi.org/10.1038/s41396-018-0228-4) contains supplementary material, which is available to authorized users.

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Françoise Bringel
francoise.bringel@unistra.fr

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Steffen Kolb
Kolb@zalf.de

<footnote n="1">¹ Department of Microbiology, Genomics and the Environment,
Université de Strasbourg, CNRS, GMGM UMR 7156,
Strasbourg, France</footnote>

<footnote n="2">² Department of Ecological Microbiology, University of Bayreuth,
Bayreuth, Germany</footnote>

<footnote n="3">³ Microbial Biogeochemistry, RA Landscape Functioning, ZALF
Leibniz Centre for Landscape Research, Müncheberg, Germany</footnote>

<footnote n="4">⁴ Present address: UMR FARE 614 Fractionnement des
AgroRessources et Environnement, Chaire AFERE, INRA,
Université de Reims Champagne-Ardenne, Reims, France</footnote>
Net emissions of CH₃Cl from terrestrial ecosystems are mitigated by soil and phyllosphere bacteria that utilize this VOC as a growth substrate [7, 13]. Such bacteria are aerobic methylotrophs, metabolically specialized for growth with one-carbon compounds [7]. The concentration of CH₃Cl in the atmosphere is low (approx. 590 ppt). This suggests similar concentrations of CH₃Cl in soils, although no experimental data are available to date [3, 14]. Previous studies suggest that aerobic soil methylotrophs can utilize CH₃Cl at environmentally relevant nanomolar to picomolar concentrations [15–20]. Such low concentrations likely do not yield sufficient energy for substantial bacterial growth with CH₃Cl. However, many known alphaproteobacterial CH₃Cl degraders also grow with methanol (CH₃OH) [13, 21], and this is also true in situ for soil methylotrophs of a deciduous forest [22]. Abundance of methylotrophs in O and A soil horizons is high, and ranges from 10⁶ to 3 × 10⁸ cells g soil⁻¹, consistent with their frequent isolation from soils [23, 24].

Which organisms define the bacterial CH₃Cl sink in soils is largely unknown at present. The only biochemically characterized pathway for CH₃Cl utilization is the *cmu* pathway, characterized in detail for *Methylobacterium extorquens* CM4 [21]. It has been found in various CH₃Cl-degrading bacterial strains, including several strains from forest soil [7, 18, 25–27]. The chloromethane dehalogenase gene *cmuA* has been used to detect CH₃Cl-degraders in various environments [15, 16, 18, 28–30]. Nonetheless, there is experimental and genomic evidence that the *cmu* pathway is not the only pathway for CH₃Cl utilization [13].

We hypothesized that (a) top soil (i.e., organic layer [O~f~, O~h~] plus A horizon) is the preferable habitat for CH₃Cl degraders, and (b) that the as yet largely unknown CH₃Cl bacterial sink in soils benefits from additional energy and carbon sources, such as CH₃OH as a widespread methylo-trophic growth substrate in soil [23, 24]. These hypotheses were tested for a European deciduous forest dominated by beech (*Fagus sylvatica* L.), and in a multi-treatment DNA stable isotope probing (SIP) laboratory experiment invol-ving combinations of ¹³C-labeled and unlabeled CH₃OH and CH₃Cl amendment. Amplicon sequencing of 16S rRNA, *cmuA*, and *mxaF/xoxF* genes as relevant gene mar-kers was employed to identify microorganisms potentially involved in the CH₃Cl sink.

## Materials and methods

Sampling site. Samples were collected in the natural forest reserve area Steigerwald located in South Germany (49° 37′ N, 10° 17′ O; sandy loam; Dystric Cambisol; pH of 4.6; mean annual temperature 7.5 °C; mean annual precipitation 725 mm). Steigerwald forest has not been managed for at

least 25 years and represents a quasi-pristine deciduous forest typical of Central Europe, i.e., predominance of European beech (*Fagus sylvatica* L.) with minor stocks of oak (*Quercus robur* L.) [31].

## Field closed-chamber measurements of CH₃Cl consumption

Eight closed top chambers were installed on-site at 12 am on 19 June 2013 and 20 August 2013. Air temperatures were 19 °C and 23 °C, respectively. The self-constructed stainless steel chambers had a total volume of 25.1 L (40 cm diameter; 20 cm height) and were equipped with an injec-tion port. Chambers were spiked with 100 ppm CH₃Cl [32] and gas samples were collected over a period of 180 min with gas-tight syringes, and stored in exetainers (Labco Limited, England) for subsequent analysis by gas chroma-tography (GC).

## First order rate constants of CH₃Cl-degradation

Fresh beech leaves, dead leaf litter, soil horizons (O~f~, A~h~, B), and rotting wood were investigated. For beech phyllosphere analysis, fresh beech leaves were sampled by cutting branches from trees and a pool of cut leaves were immediately used for experiments. Samples were taken on 20 August 2013 (8 locations per compartment pooled) and on 12 July 2016 (3 locations pooled), and transferred to duplicated gas-tight 125 mL serum bottles that were subsequently flushed with synthetic air (Rießner Gase GmbH, Germany). CH₃Cl (Rießner Gase GmbH, Germany, purity 99.99%) was injected to final concentrations of 60 ppb or 200 ppm for samples taken on 20 August 2013 and 12 July 2016, respectively. Controls included a substrate-free control, a biological process control (amended with 20 mM KCN to inhibit biological activity), and an anoxic control (headspace was pure N₂). All treatments and controls were performed in triplicate. Gas samples were taken with gas-tight syringes and stored in 3 mL, pre-evacuated exetainers (Labco ltm, UK) for further analysis by gas chromatography.

## Soil microcosm set-up for DNA stable isotope probing (DNA SIP)

Five samples from the upper soil layer within a 20 m circle, were taken on 20 August 2013, pooled, homogenized and sieved (2 mm-mesh size). To stimulate CH₃Cl consumption, sieved soil was aliquoted in batches of 500 g into sealed flasks, CH₃Cl (Sigma Aldrich) added to the head space at a final concentration of 1%, and incubated in 1 L glass flasks at 20°C in the dark. Mixing ratios of CH₃Cl were followed by GC until added CH₃Cl was consumed. Then, for each
<table><caption>**Table 1** Microcosm setup and carbon substrate amendment</caption><thead><tr><th>Carbon source added</th><th>Total carbon added (mM)<sup>a</sup></th><th>Gas phase amendment<sup>b</sup></th><th>Liquid phase amendment<sup>c</sup></th></tr></thead><tbody><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl</td><td>18</td><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl</td><td>H<sub>2</sub>O</td></tr><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl and CH<sub>3</sub>OH</td><td>36</td><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl</td><td>CH<sub>3</sub>OH</td></tr><tr><td>CH<sub>3</sub>Cl and [<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>36</td><td>CH<sub>3</sub>Cl</td><td>[<sup>13</sup>C]-CH<sub>3</sub>OH</td></tr><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>18</td><td>—</td><td>[<sup>13</sup>C]-CH<sub>3</sub>OH</td></tr><tr><td>CH<sub>3</sub>Cl</td><td>18</td><td>CH<sub>3</sub>Cl</td><td>H<sub>2</sub>O</td></tr><tr><td>CH<sub>3</sub>Cl and CH<sub>3</sub>OH</td><td>36</td><td>CH<sub>3</sub>Cl</td><td>CH<sub>3</sub>OH</td></tr><tr><td>CH<sub>3</sub>OH</td><td>18</td><td>—</td><td>CH<sub>3</sub>OH</td></tr><tr><td>Control</td><td>—</td><td>—</td><td>H<sub>2</sub>O</td></tr></tbody></table>

microcosm of the DNA SIP experiment, 70 g activated soil was transferred to a 500 mL flask sealed with a Viton stopper. Eight different treatments were prepared, each performed in duplicates. Four treatments were amended with <sup>13</sup>C-labeled substrates and four with unlabeled <sup>12</sup>C-substrates. [<sup>13</sup>C]-CH<sub>3</sub>Cl (Sigma Aldrich), [<sup>13</sup>C]-CH<sub>3</sub>Cl together with unlabeled CH<sub>3</sub>OH (Campro Scientific), [<sup>13</sup>C]-CH<sub>3</sub>OH (Sigma Aldrich) together with unlabeled CH<sub>3</sub>Cl, and [<sup>13</sup>C]-CH<sub>3</sub>OH (Table 1). Control incubations were amended with equivalent amounts of unlabeled substrates, or left without amendment (Table 1). CH<sub>3</sub>Cl and/or CH<sub>3</sub>OH were amended as unlabeled or <sup>13</sup>C-labeled isotopologue in 5 pulses over a period of 23 days (Fig. S1). CH<sub>3</sub>Cl and CO<sub>2</sub> headspace mixing ratios were monitored by GC, and amended again when CH<sub>3</sub>Cl was no longer detectable. After each pulse, 5 soil aliquots (1 g each) per microcosm were retrieved and stored at -80 °C until further analysis.

## pH and gravimetric water content in soil samples

pH was measured on sieved soil, before activation and after each substrate pulse using an InLab R422 pH electrode (InLab Semi-Micro; Mettler-Toledo, Gießen, Germany). Gravimetric soil moisture content was determined by weighing the soil before and after weight constancy following drying at 60 °C.

## Quantification of CH<sub>3</sub>Cl, CO<sub>2</sub>, and <sup>13</sup>CO<sub>2</sub>

On 19 June 2013 and 20 August 2013, CH<sub>3</sub>Cl and CO<sub>2</sub> mixing ratios were determined by GC (HP 5890 Series II,

Agilent) using a Porapak Q 80/100 column (Supelco, USA) and a helium-methane mixture (95:5) as the carrier gas. On 16 July 2016, CH<sub>3</sub>Cl mixing ratios were determined by ISQ™ Quadrupole mass spectrometer (MS) coupled with TRACE™ Ultra gas chromatograph (GC) (Thermo Fisher Scientific, Massachusetts, USA). CH<sub>3</sub>Cl and CO<sub>2</sub> mixing ratios were calculated by regression analysis based on a 5-point calibration with standard mixing ratios of both gases. In the SIP experiment, GC MS analysis (Perkin-Elmer GC Clarus 600 system) was carried out as described previously [22]. Further Details of gas analysis methods are given in Supplemental Information.

## Nucleic acid extraction and RNA removal

Nucleic acids from experimental replicates of all treatments and controls were extracted from each 0.5 g of soil when CH<sub>3</sub>Cl had been consumed after the third substrate pulse [33]. RNA was removed according to standard procedures including a treatment with RNAse followed by isopropanol precipitation (Supplemental Information). Recovered DNA was resuspended in DNAse-free water, quantified, and eventually stored at -80 °C until further processing through isopycnic centrifugation [22].

## DNA fractionation by isopycnic centrifugation

Separation of the heavy (H) and light (L) DNA in a cesium chloride gradient was described in detail previously [22, 34]. In brief, a cesium chloride gradient solution mixed with each RNA-free DNA was loaded into an ultra-centrifugation tube, placed in a Vti 65.2 vertical rotor (Beckman Coulter, Germany) and centrifuged for 40 h at 177,000×g in a LE-70 ultracentrifuge (Beckman Coulter, Germany). DNA was harvested according to established procedures in 10 gradient fractions [22, 34]. The main four fractions of H and of L DNA were pooled and DNA was quantified [22] (Fig. S2a-b, Supplemental Information). DNA concentrations ranged between 0.3 and 3.0 ng µL<sup>-1</sup> for H fractions, and 18.5 and 50.3 ng µL<sup>-1</sup> for L fractions.

## PCR amplification, high-throughput sequencing, and data processing

PCR amplification of the 16S rRNA gene, and of CH<sub>3</sub>Cl dehalogenase CmuA and methanol dehydrogenase MxaF/ XoxF encoding genes are described in detail in Table 2, and in Table S2 (Supplemental Information). New primers were designed to detect a larger spectrum of genetic diversity using NGS sequencing both for *cmuA* (Table S2; Fig. S7) and *mxaF* / *xoxF* (Supplemental Information, Table S2, Fig S8). For sequencing analysis following PCR

<footnote><sup>a</sup>Added per pulse (5 pulses in total)</footnote>

<footnote><sup>b</sup>A volume of 6 mL CH<sub>3</sub>Cl was added for each CH<sub>3</sub>Cl pulse. At each pulse, a total gas phase volume of 56 mL (air with or without CH<sub>3</sub>Cl) was added to 500 mL flasks in order to maintain overpressure in the flasks</footnote>

<footnote><sup>c</sup>Either 1 mL milliQ water or 216 mM CH<sub>3</sub>OH stock solution was added per pulse to each microcosm</footnote>
<table><caption>**Table 2** Analyzed gene markers and amplicon characteristics</caption><thead><tr><th>Gene marker</th><th>Function</th><th>PCR Primer</th><th>Sequence (5'-3')<sup>a</sup></th><th>Amplicon size (bp)</th><th>SNP/ OTU<sup>b</sup></th><th>Total OTUs<sup>c</sup></th><th>Labeled OTUs<sup>d</sup></th><th>Reference</th></tr></thead><tbody><tr><td rowspan="2">16 S rRNA gene</td><td rowspan="2">Ribosomal small subunit RNA</td><td>341for</td><td>CCTACGGNGGCWGCAG</td><td rowspan="2">464</td><td rowspan="2">9</td><td rowspan="2">117</td><td rowspan="2">5</td><td>[60]</td></tr><tr><td>785/805rev</td><td>GACTACHVGGGTATCTAATCC</td><td>[61]</td></tr><tr><td rowspan="2">cmuA</td><td rowspan="2">chloromethane methyltransferase</td><td>cmuAf422</td><td>GARGTBGGITAYAAYGGHGG</td><td rowspan="2">422</td><td rowspan="2">38</td><td rowspan="2">8</td><td rowspan="2">5</td><td rowspan="2">This study<sup>f</sup></td></tr><tr><td>cmuAr422</td><td>TCRTTGCGCTCRTACATGTCICC</td></tr><tr><td rowspan="3">mxaF/ xoxF<sup>e</sup></td><td rowspan="3">methanol dehydrogenase</td><td>mdh1</td><td>GCGGIWSÇAICTGGGGYT</td><td rowspan="3">430</td><td rowspan="3">39</td><td rowspan="3">6</td><td rowspan="3">6</td><td rowspan="3">This study<sup>f</sup></td></tr><tr><td>mdh2</td><td>GCGGIWSÇAICTGGGGYT</td></tr><tr><td>mdhR</td><td>GAASGGYTCSYARTCCATGCA</td></tr></tbody></table>

<sup>a</sup>Degenerate base mixtures: B (C,G,T), H (A,C,T), K (G,T), N (A,C,G,T), R (A,G), S (G,C), Y (C,T), V (A, C, T), W (A,T). Inosine (I) was used instead of the N mixture [62] in some cases

<sup>b</sup>Maximal Single Nucleotide Polymorphism (SNP) positions possible within an OTU

<sup>c</sup>Corresponding to the sum of OTUs detected in the 8 microcosms of the SIP experiment. Sequences were affiliated to the same OTU at cutoff values of 98, 90, and 80% sequence identity at the nucleotide level for 16S rRNA gene, *cmuA*, and *mxaF/xoxF* amplicons, respectively

<sup>d</sup>See Material and Methods for the criteria applied to define OTUs as 'labeled'

<sup>e</sup>Primer pairs allow to amplify both *mxaF* and *xoxF* types of methanol dehydrogenase (mdh) sub units. Amplifications were performed with two different forward primers (mdh1, mdh2) in order to reduce primer degeneracy and improve PCR efficiency. Amplicons obtained with primers mdh1/mdhR and mdh2/mdhR were pooled before sequencing.

<sup>f</sup>See Supplemental Information of Materials and Methods for details

amplification, briefly, a barcode oligonucleotide identifying sample origin was ligated to each PCR product. Equimolar pools of all libraries were assembled, and the resulting combined library was sequenced using Illumina MiSeq technology (LGC Genomics GmbH, Germany). Reads were assembled into contigs and analyzed using Mothur v.1.33.2 with default parameters (http://www.mothur.org/wiki/MiSeq_SOP) [35]. 16S rRNA reads <420 or >460 bp were discarded. Reads were pre-clustered into groups of sequences with up to 2 nucleotide differences. Chimera sequences were removed with UCHIME [36]. Remaining sequences were assigned by naïve Bayesian taxonomic classification using the SILVA reference database. Sequences that could not be assigned to *Bacteria* and *Archaea* were excluded from further analysis. OTUs were defined at 98% sequence similarity. Raw reads of *cmuA* and *mxaF/xoxF* amplicons with read lengths within 20 nucleotides of the expected amplicon length were clustered by USEARCH [37]. Sequences occurring only once in all libraries were considered artefactual and removed, but singletons within individual amplicon libraries were kept. Reads were clustered iteratively at progressively lower cut-off values, and the cut-off value at which the number of retrieved OTUs stabilized was selected [38]. These OTU sequences were compared against a gene-specific database generated from GenBank using BLAST (http://blast.ncbi.nlm.nih.gov) for identification. Taxonomic assignments of consensus sequences of each OTU were used to identify <sup>13</sup>C-labeled gene OTUs.

## Identification of <sup>13</sup>C-labeled OTUs

Labeled OTUs were defined according to a previously reported protocol developed to minimize false positives in 'H DNA' fractions [22, 39]. The following criteria were applied to identify <sup>13</sup>C-labeled OTUs: (1) OTU abundance in the 'heavy' fraction of a microcosm treated with <sup>13</sup>C-labeled substrate higher than in the 'heavy' fraction of the corresponding microcosm treated with unlabeled substrate; (2) OTU abundance in the 'heavy' fraction higher than in the 'light' fraction; (3) OTU abundance in the 'heavy' fraction of the microcosm treated with <sup>13</sup>C-labeled substrate ≥0.5%; (4) OTU abundance difference between 'heavy' and 'light' fractions higher by at least 0.3% (a threshold that considers the variance of each OTU abundance). Moreover, a lower limit of 5% was set for the labeling proportion (LP), (i.e., the relative frequency of a labeled OTU in a specific heavy fraction) of a given OTU to be considered as labeled [22].

## Statistical and phylogenetic analyzes

Richness and Simpson diversity indices were determined using Mothur and PAST (http://folk.uio.no/ohammer/past). Relationships between sequence datasets in different DNA fractions and microcosms were investigated by two-dimensional NMDS (non-metric multidimensional scaling) within Mothur, and visualized with Kaleidagraph (Synergy Software, Reading, PA, USA). Details on phylogenetic tree construction are given in figure legends.
<figure><img src="image_6.png" /><figcaption>**Fig. 1** Dissipation of added CH₃Cl in forest soil. Closed top chambers applied onto soil at the Steigerwald forest sampling site were amended with 100 ppm CH₃Cl (color symbols, chambers 1–5) or incubated without supplementation (black symbols, triplicate controls) on a 19 June 2013 and b 20 August 2013</figcaption></figure>

## Nucleotide sequence accession numbers

Sequence datasets were deposited to the NCBI BioSample database under the study accession number SUB3319582.

## Results

Localization of active CH₃Cl consumption in different forest compartments. Two sampling campaigns were performed, with the second campaign aiming to verify and justify that top soil samples were indeed the most active in CH₃Cl degradation, since only this horizon was selected to be assessed by SIP. Immediate consumption of CH₃Cl was observed in two field site campaigns at the temperate deciduous forest Steigerwald (Fig. 1a, b). In contrast, no net CH₃Cl emissions were detected from the forest floor (<1 ppb in chambers headspace). This strongly suggested that the investigated forest top soil represents a major sink for atmospheric CH₃Cl at the forest ecosystem level.

Results from lab-scale microcosms suggest that net CH₃Cl consumption was primarily due to biological activity. Indeed, CH₃Cl consumption was not observed in microcosms that were treated with KCN (Fig. 2a–e). O₂ and Aₕ horizons were the most active layers (Fig. 2b, c). Because no CH₃Cl consumption was detected under anoxic conditions (data not shown), top soil aerobic microorganisms likely represent the active sink for CH₃Cl (Fig. 2b). Results obtained for the phyllosphere of European beech were variable, as we detected substantial consumption of CH₃Cl in 2013 only (Fig. 2e).

## Mineralization and assimilation of CH₃Cl by the top soil microbial community

As expected, a net increase in CO₂ formation occurred in all microcosms in response to substrate amendment (Fig. S1a-c). A non-significant trend was observed towards larger CO₂ release upon addition of CH₃OH compared to CH₃Cl

(Fig. S1c). Rates of CO₂ production were in the range of 0.2–0.3 mmol g<sub>dry soil</sub>⁻¹ day⁻¹ for microcosms to which CH₃Cl or CH₃OH was added. No differences in CO₂ release between microcosms to which labeled or unlabeled substrates were added were observed, confirming that different carbon isotopologues did not affect carbon metabolism (t-test, *p* > 0.10; Fig. S1). Uncertainties were large, but about 10 mM (20%) of the added 54 mM of [¹³C]-CH₃Cl were converted to [¹³C]-CO₂ (Fig. S1). Hence, up to 80% of amended [¹³C]-CH₃Cl was assimilated into biomass. This rather high rate may be an overestimate, given measurement uncertainties and undetected losses by precipitation of carbonate. An non-significant increase in [¹³C]-CO₂ formation from labeled [¹³C]-CH₃Cl was observed when unlabeled CH₃OH was also provided (t-test, *p* > 0.10; Fig. S1c). In the reverse case of microcosms amended with [¹³C]-CH₃OH together with unlabeled CH₃Cl, the increase in [¹³C]-CO₂ formation was slightly less. Moreover, CH₃OH was mineralized to a larger extent than CH₃Cl when added alone (t-test, *p* ≤ 0.05). Taken together, these observations suggest that microbial CH₃Cl consumption activity is enhanced by CH₃OH.

## Overall microbial community response to amended substrates

Based on three gene markers, amendment of substrates led to significant changes in microbial community composition, basing on statistical analyzes combining ¹³C-labeled and unlabeled phylotypes (Fig. S3a–c, Table S3, Tables 3 and 4). Combined with the observation that CO₂ formation increased in amended microcosms (Fig. S1), this suggested increased growth of specific microorganisms with amended substrates.

## Diversity of ¹³C-labeled family-level OTUs based on the 16S rRNA gene

A total of 117 family-level OTUs (90% similarity) were detected (Table S1). Of these and across all four substrate treatments (Fig. 3a), several genera within five families
**Fig. 2** Dissipation of added CH₃Cl in microcosms of forest compartments. First order rate constants [k] of CH₃Cl-amended microcosms of Steigerwald forest sampled on 20 August 2013 (circles) and on 12 July 2016 (diamonds). **a** Leaf litter (2013, *k* = 2.35 h⁻¹; 2016, *k* = 0.19 h⁻¹); **b** O₁ horizon (2013, *k* = 3.04 h⁻¹; 2016, *k* = 2.42 h⁻¹); **c** A₁h horizon (2013, *k* = 6.93 h⁻¹; 2016, *k* = 2.00 h⁻¹); **d** B horizon (2016, *k* = 2.72); **e** fresh beech leaves (2013, *k* = 2.76 h⁻¹; 2016, *k* = 0.06 h⁻¹). Control experiments without amendment (triangles) or including KCN on top of CH₃Cl to inhibit biological activity (squares) were also performed. B horizon was only measured in 2016

<figure><img src="image_7.png" /></figure>

were identified that satisfied the criteria set for defining ¹³C-labeled OTUs (Fig. 3a): *Beijerinckiaceae* within *Alphaproteobacteria*, three families of *Actinobacteria* (*Acidothermaceae*, *Pseudonocardiaceae*, and *Streptomyce-taceae*) and OTU108₁₆S within the TM7 phylum, recently renamed as *Candidatus* Saccharibacteria (Fig. S4) [40]. *Methylovirgula*, *Acidothermus*, and *Streptomyces* repre-sented over 95% of the *Beijerinckiaceae*, *Acidothermaceae*, and *Streptomycetaceae*, respectively (Fig. 3a). In the microcosm amended with [¹³C]-CH₃Cl, three of the five family-level OTUs (*Acidothermaceae*, *Beijerinckiaceae*, and *Streptomycetaceae*) represented about 80% of all labeled OTUs (Fig. 3a). In the microcosm amended with [¹³C]-CH₃OH, in contrast, mainly *Beijerinckiaceae* was labeled. The labeled *Cand*. Saccharibacteria-like OTU108₁₆S and the *Pseudonocardiaceae* sp.-like OTU85₁₆S were both found at

low abundance in all ¹³C-amended microcosms (Fig. 3a). All labeled OTUs differed from the closest type strains as well as from previously described CH₃Cl-degrading isolates (Fig. S4). Another interesting observation was that several other detected *Actinobacteria*, i.e. *Gryllotalpicola*, likely represent CH₃OH utilizers based on labeling patterns (Fig. 3a, Fig S4).

## Diversity of CH₃Cl utilizers based on *cmuA*

Chloromethane:cobalamin methyltransferase *cmuA*, the biomarker for CH₃Cl consumption by the *cmu* pathway, was amplified with newly designed primers (Table S2). Eight OTUs were detected among which five satisfied the criteria defined for ¹³C-labeled OTUs (Table S3). Consensus sequences of these 8 OTUs were compared to known
<figure><img src="image_8.png" /><figcaption>**Fig. 3** Relative abundance of [¹³C]-labeled phylotypes. **a** Bacterial 16S rRNA genes, **b** *cmuA*, and **c** *mxaF/xoxF*. Only microcosms exposed to [¹³C]-labeling are shown. Specific OTUs are indexed with corresponding gene markers (e.g., OTU108₁₆S). Phylogenetic assignments are based on phylogenetic reconstructions (Fig. S3 [16S rRNA gene], Fig. S4 [*mxaF/xoxF*], and Fig. 4 [*cmuA*]). OTUs with >0.5% relative abundance in the H fraction but <5% labeling proportion were not considered as labeled. Unlabeled OTUs with relative abundance <0.5% are not shown</figcaption></figure>

*cmuA* of genome-sequenced cultivated strains, and of uncultivated OTUs identified in a previous SIP study of soil amended with [¹³C]-CH₃Cl [15]. The eight OTUs belonged to three distinct gene clusters (Fig. 4). Labeled OTUs were closely related to sequences of known CH₃Cl-degraders including *Methylobacterium extorquens* CM4 (>99 % identity) or *Hyphomicrobium* sp. MC1, as well as with *cmuA* sequences retrieved from a soil environment [15]. In the CH₃Cl treatment, OTU2*cmuA* and OTU3*cmuA* were

among the most dominant ¹³C-labeled OTUs. This labeling pattern was conserved in all ¹³C-labeled substrate treatments of our study (Fig. 3b). However, when methanol was the ¹³C-labeled substrate, an additional OTU6*cmuA* was detected (Fig. 3b). OTU6*cmuA* was very similar to *cmuA* genotypes of reference CH₃Cl-degrading *Hyphomicrobium* strains (Fig. 4). All other ¹³C-labeled OTUs represented minor populations with closest similarity to *cmuA* from *Methylobacterium* (Figs. 3b, 4).
<figure><img src="image_9.png" /><figcaption>**Fig. 4** Phylogenetic affiliations of detected *cmuA* OTUs. A maximum likelihood phylogenetic tree was reconstructed from a 422 nt-long sequence alignment based on the Tamura-Nei model [59]. Bold, labeled *cmuA* OTUs. Scale bar, 0.05 substitutions per site. Bootstrapping was performed with 1000 replicates. Reference sequences</figcaption></figure>

from previous studies were included: characterized CH3Cl-utilizing isolates (green diamonds); uncharacterized genome-sequenced isolates containing *cmu* genes (green open diamonds); sequences detected by SIP in the marine environment (blue circles [30]) or in soil (brown circles [18])

## Diversity of methanol utilizers based on *mxaF/xoxF*

Two types of methanol dehydrogenase (mdh) are predominantly found in methanol utilizers, MxaFI and XoxF, both harboring a pyrroloquinoline quinone catalytic center

[41]. Previously used *mxaF/xoxF* primers were biased against *xoxF* [42]. Thus, we designed new *mdh* primers targeting both types of MDH (Table 2 and S2; Fig. S8). A total of 15 distinct ¹³C-labeled *mdh* OTUs were found, all but one *xoxF* genes (Figs. 3c and S5). These OTUs were
mostly affiliated to one of the canonical XoxF clades, i.e., XoxF5 [41]. The underrepresentation of other clades might had been effected by the design strategy of the primers (Fig. S8, Supplemental Information). Only OTU15<sub>mdh</sub> was closely similar to a *mxaF* gene of *Beijerinckiaceae* (Fig. 3c and Fig. S5). Predominant <sup>13</sup>C-labeled *mxaF/xoxF* OTUs associated with known *xoxF* genes of *Bradyrhizobium* and *Sinorhizobium* were found in all four <sup>13</sup>C-labeled substrate treatments. On the other hand, <sup>13</sup>C-labeled *mxaF/xoxF* OTUs closely related with *xoxF* genes from *Acetobacteraceae* (*Acidiphilium*, *Gamm proteobacteria*) were detected in the [<sup>13</sup>C]-CH<sub>3</sub>OH-amended treatment only (OTU14<sub>mdh</sub>) (Figs. 3c and S5). We hypothesize that OTU14<sub>mdh</sub> corresponds to a methanol utilizer, not a CH<sub>3</sub>Cl utilizer, also because only methanol-utilizing strains are known in *Acetobacteraceae* [43, 44]. In both [<sup>13</sup>C]-CH<sub>3</sub>Cl-amended treatments, OTU25<sub>mdh</sub> was dominant, whereas in both [<sup>13</sup>C]-CH<sub>3</sub>OH amended treatments, OTU24<sub>mdh</sub> prevailed. Both OTUs correspond to *Bradyrhizobium*-like *xoxF* genes.

## Key Bacteria of the CH<sub>3</sub>Cl sink in the investigated top soil

At first glance, bacterial taxa suggested to be associated with utilization of CH<sub>3</sub>Cl differ depending on which of the three investigated gene biomarkers in the [<sup>13</sup>C]-CH<sub>3</sub>Cl SIP experiment is considered (Fig. 3, Tables 3 and 4). However, consideration of the labeling proportion (LP) of <sup>13</sup>C-labeled OTUs (Fig. 3) allows to refine the analysis. A high LP suggests strong <sup>13</sup>C-labeling, hinting at predominant transformation of the <sup>13</sup>C labeled-substrate by *Alphaproteobacteria*. In support of this, the taxonomic 16S rRNA gene biomarker indeed suggests that this class, and in particular strains closely related to *Methylovirgula* within the *Beijerinckiaceae* family, includes a major part of the primary utilizers of amended [<sup>13</sup>C]-CH<sub>3</sub>Cl soils (Figs. 3a, S4, S6). Beyond that, the LP analysis approach also allowed to identify *Actinobacteria* of the genus *Kineospora* as potential novel key [<sup>13</sup>C]-CH<sub>3</sub>Cl degraders (Figs. 3a, S4, Table S3).

## Discussion

We show in this study that the oxic compartment of top soil was the main sink for CH<sub>3</sub>Cl in the investigated forest soil, and that this degradation process was primarily biotic, in agreement with an early exploratory study of CH<sub>3</sub>Cl dissipation in various environments [27]. We performed a detailed study of potential CH<sub>3</sub>Cl degraders in top soil, and focused on oxic conditions, since anoxic incubations did not show evidence for CH<sub>3</sub>Cl consumption, as expected given that strict anaerobes are not frequently abundant in oxic soils [45].

<table><caption>**Table 3** Number of filtered sequences obtained from heavy DNA fractions</caption><thead><tr><th>Treatment</th><th>16S rRNA gene</th><th>cmuA</th><th>mxaF/xoxF</th></tr></thead><tbody><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl</td><td>46110</td><td>4282</td><td>786</td></tr><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>Cl and CH<sub>3</sub>OH</td><td>178042</td><td>3778</td><td>1534</td></tr><tr><td>CH<sub>3</sub>Cl and [<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>76690</td><td>614</td><td>918</td></tr><tr><td>[<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>98332</td><td>1886</td><td>588</td></tr><tr><td>CH<sub>3</sub>Cl</td><td>72522</td><td>370</td><td>2616</td></tr><tr><td>CH<sub>3</sub>Cl and CH<sub>3</sub>OH</td><td>47236</td><td>922</td><td>862</td></tr><tr><td>CH<sub>3</sub>OH</td><td>90822</td><td>246</td><td>2946</td></tr><tr><td>Unamended control</td><td>100032</td><td>184</td><td>2710</td></tr></tbody></table>

See Table S1 for further information

<table><caption>**Table 4** Diversity indices for 16S rRNA gene OTUs obtained from heavy and light fractions of SIP experiment</caption><thead><tr><th>Microcosm</th><th>SIP fraction</th><th>Sobs<sup>a</sup></th><th>Shannon index<sup>a</sup></th><th>Simpson diversity<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="2">[<sup>13</sup>C]-CH<sub>3</sub>Cl</td><td>H</td><td>1762</td><td>5.95</td><td>44</td></tr><tr><td>L</td><td>2870</td><td>7.82</td><td>223</td></tr><tr><td rowspan="2">[<sup>13</sup>C]-CH<sub>3</sub>Cl and CH<sub>3</sub>OH</td><td>H</td><td>1629</td><td>6.11</td><td>36</td></tr><tr><td>L</td><td>3150</td><td>7.55</td><td>354</td></tr><tr><td rowspan="2">CH<sub>3</sub>Cl and [<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>H</td><td>1566</td><td>6.52</td><td>120</td></tr><tr><td>L</td><td>3062</td><td>7.86</td><td>283</td></tr><tr><td rowspan="2">[<sup>13</sup>C]-CH<sub>3</sub>OH</td><td>H</td><td>1215</td><td>4.58</td><td>7</td></tr><tr><td>L</td><td>3148</td><td>7.40</td><td>322</td></tr><tr><td rowspan="2">Unamended control</td><td>H</td><td>1804</td><td>6.45</td><td>102</td></tr><tr><td>L</td><td>2904</td><td>7.39</td><td>217</td></tr></tbody></table>

## Potential bacterial degraders of CH<sub>3</sub>Cl in forest top soil are phylogenetically distinct from known CH<sub>3</sub>Cl-utilizing isolates

*Alphaproteobacteria* (i.e., *Beijerinckiaceae*), and *Actinobacteria* (i.e., *Kineosporaceae*) likely represent key CH<sub>3</sub>Cl degraders in the investigated forest soil (Fig. 3a). The phylogenetic affiliation of these CH<sub>3</sub>Cl utilizers was only distantly related to known CH<sub>3</sub>Cl degraders and thus a novel finding. Moreover, metabolically active *Actino-bacteria* show only limited sequence identity with the only known CH<sub>3</sub>Cl-degrading isolate of this phylum, *Nocar-dioidia* sp. strain SAC-4 [27] (Fig. S4). In contrast, almost all soil CH<sub>3</sub>Cl-degrading isolates known so far are affiliated with only a few genera of *Alphaproteobacteria* [15, 18, 29, 30], and are phylogenetically distinct from the key genus associated with CH<sub>3</sub>Cl degradation in our study (*Methylovirgula*, family *Beijerinckiaceae*). In addition, members of the candidate division *Cand*. *Saccharibacteria*

<footnote><sup>a</sup>Calculated at the 98% sequence identity level. See Materials and Methods for definitions. Simpson diversity is considered a conservative measure of the effective number of phylotypes [63]</footnote>
(syn. TM7) were also ¹³C-labeled in our experiments. Physiological knowledge within this division is limited, and methylotrophy unreported to date [40, 46]. Since cross-labeling via ¹³CO₂ cannot be fully ruled out in our experimental setup due to the long incubation period, further efforts will be required to confirm that this phylum indeed includes methylotrophs able to degrade CH₃Cl.

## Low diversity of the *cmuA* biomarker and potential horizontal gene transfer of *cmu* genes to *Beijerinckiaceae*

Amplicons of *cmuA* retrieved from ¹³C-labeled DNA were most closely similar to the *cmuA* gene of *Methylobacterium extorquens* CM4 in both [¹³C]-CH₃Cl and [¹³C]-CH₃OH treatments, despite the fact that newly designed *cmuA* primers cover a broader diversity of known *cmuA* sequences than previous ones (Fig. S7). However, abundance of *Methylobacteriaceae* in 16S rRNA gene datasets was <1% (data not shown), and no OTU affiliated to this family was defined as ¹³C-labeled with the applied criteria. Notably, *Beijerinckiaceae* was the key family associated with CH₃Cl degradation based on the 16S rRNA gene biomarker. This may suggest that CH₃Cl-degraders from *Beijerinckiaceae* use another CH₃Cl degradation pathway than the *cmu* pathway, or that they possess a *cmuA* gene that escapes detection with the used primers. Alternatively, such degraders may have acquired a known *cmuA* gene by horizontal gene transfer. Indeed, the *cmu* pathway is plasmid-borne in *M. extorquens* CM4 [47], and further experimental [48] as well as sequence-based [13] evidence for horizontal transfer of the capacity for chloromethane degradation is also available.

## Evidence for novel CH₃Cl degraders based on *mxaF/*xoxF

Whereas *mxaF* genes had been detected in a previous study of the same soil with canonical primers [22] (Fig. S5), mainly *xoxF* sequences were retrieved here with newly designed primers that detect both *mxaF* and *xoxF* (Fig. S5, Supplemental Information). Of the detected 25 *mxaF*/*xoxF* OTUs, only three OTUs, most similar to *xoxF* genes found in *Bradyrhizobium* and *Sinorhizobium* genomes, dominated microcosms irrespectively of the performed treatment (Fig. 3c). Worthy of note, growth with CH₃Cl has not been reported to date for representatives of these genera [23, 49, 50]. In addition, *xoxF* genes similar to those of *Methylobacterium* were also ¹³C-labeled upon [¹³C]-CH₃Cl amendment. Although some cross-feeding of ¹³CO₂ cannot be entirely excluded since *Bradyrhizobium*, *Sinorhizobium*, and *Methylobacterium* can assimilate CO₂ [49, 51], this possibility was minimized by regular exchange of the gas

phase to remove formed CO₂. 16S rRNA gene OTU analysis indicated members of *Beijerinckiaceae* as main alpha-proteobacterial CH₃Cl degraders (Fig. 3a). Basing on phylogenetic analysis (Fig. S5), the major ¹³C-labeled *xoxF* OTUs appear quite distinct from known *xoxF* genes of *Beijerinckiaceae Sinorhizobium* and *Bradyrhizobium*. Hence, we hypothesize that OTU24<sub>mdh</sub> and OTU25<sub>mdh</sub> represent hitherto unknown *xoxF* genes in *Beijerinckiaceae*, or that used primers discriminated against the genotype of *Beijerinckiaceae*. A XoxF-type MDH might be advantageous as it directly leads to formate, avoiding the more toxic formaldehyde produced by MxaFI-type MDH [52].

## Consumption of CH₃OH by CH₃Cl degraders and implications for the CH₃Cl sink in soil

*Beijerinckiaceae* were newly identified here as novel potential CH₃Cl and also CH₃OH utilizers. The latter finding agrees well with a previous study on the microbial methanol sink in the same soil [22]. None of the methylotrophic *Beijerinckiaceae* characterized so far utilize methyl halides [53]. *Beijerinckiacea* also comprise typical methanotrophs [54], and one such isolate was shown to transform CH₃Cl under laboratory conditions, but not to grow with this compound [55, 56]. Basing on the functional gene marker *pmoA* (encoding the beta-subunit of particulate methane monooxygenase), representatives of the uncultivated upland soil cluster α taxon represented the most abundant group of methanotrophs in Steigerwald forest soil. This *pmoA* type, however, is phylogenetically distinct from *pmoA* of *Beijerinckiaceae* [32]. However, a recent study suggest that USCα is indeed a member of *Beijerinckiaceae* [57]. Thus, we presently cannot rule out that methanotrophs were not involved in the observed CH₃Cl sink activity in Steigerwald forest soil.

The presence of CH₃OH as an alternative methylotrophic growth substrate supports the notion of enhanced assimilation of carbon from CH₃Cl by *Beijerinckiaceae* during CH₃OH-driven growth. This idea is supported by the observed high labeling percentages in combined substrate amendments with CH₃OH and [¹³C]-CH₃Cl (Fig. 3). Similarly, the reverse combined amendment of unlabeled CH₃OH with [¹³C]-CH₃Cl led to increased mineralization of [¹³C]-CH₃Cl (Fig. S1). Taken together, these findings suggest that activity and growth of soil microorganisms that define the bacterial CH₃Cl sink in the investigated soil strongly depend on availability of CH₃OH.

Many methylotrophs can simultaneously utilize several one-carbon compounds [58]. This is likely a selective advantage in natural environments when availability of potential substrates is variable and often limiting. As shown in a previous study on the same soil, *Beijerinckiaceae* can be either methylotrophic or non-methylotrophic [22]. On
the basis of the data reported here, we suggest that key CH₃Cl degraders in soil may be capable of assimilating several one-carbon substrates to optimize their growth. Such a metabolic lifestyle is likely to be of advantage in order to compete with other aerobes in the complex top soil environment, and suggests that the microbial CH₃Cl sink is linked to the availability of other key carbon sources in soil such as CH₃OH.

## Conclusions

Our study provides a first deep coverage exploration of bacterial diversity functionally linked with the CH₃Cl sink in soil. It has revealed that CH₃Cl consumption in forest soil may be driven by alternative carbon sources such as CH₃OH. It also uncovered new taxa associated with CH₃Cl degradation, including genera of *Alphaproteobacteria* and *Actinobacteria* that had not yet been identified in the context of previous SIP experiments with CH₃Cl, and for which no isolates are yet available. The used CH₃Cl concentrations, which are much higher than those encountered in the troposphere, might have harmed some bacteria that cannot grow with 1% CH₃Cl. However, we focused in the study on those soil bacteria that can deal with these concentrations and degraded it. We are aware that the used SIP approach might have been biased by label transfer. However, the low limit of detection of DNA SIP combined with the very conservative approach chosen to identify potential chloromethane degraders, only strongly-labeled microorganisms were identified, which maximizes the likelihood that they were directly labeled from amended labeled chloromethane and not through crossfeeding. Taxa corresponding to cultivated model CH₃Cl degraders, such as *Methylobacterium extorquens* CM4, were not relevant for CH₃Cl degradation in the investigated forest top soil. Thus, cultivation of new isolates requires future efforts to improve coverage of existing diversity chloromethane degraders by isolates. Detection of *cmuA* genes closely similar to those of such strains (>99%) suggests that horizontal transfer of the ability to degrade CH₃Cl is an important aspect of the CH₃Cl sink in soil. Our study also suggests that methylo trophs in top soil may have a competitive advantage over non-methylotrophs by their ability to utilize diverse one-carbon substrates simultaneously. Testing this hypoth esis, and addressing alternative metabolic strategies of CH₃Cl degradation in soil, will be the topic of future investigations.

**Acknowledgements** PC was funded through a PhD grant from Région Alsace (France) and the Deutsche Forschungsgemeinschaft (Germany). LB was supported by the French Agence Nationale de la Recheche ANR (ANR-14-CE35-0005-01). MM was financed through the Deutsche Forschungsgemeinschaft DFG (Ko2912/5-1 and

Ko2912/10-1). The Deutsche Akademischer Austauschdienst (DAAD) program PROCOPE funded travel expenses. We thank Y. Louhichi-Jelail for technical assistance in performing PCR amplifications, and H.L. Drake and M.A. Horn for hosting the project at the Department of Ecological Microbiology of the University of Bayreuth (Germany).

## Compliance with ethical standards

**Conflict of interest** The authors declare that they have no conflict of interest.

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