The impact of substrates and pH on methylotrophs

The mean coverage of all combined amplicon libraries for mxaF gene sequences based on 90 % similarity cut-off was 99.08 ± 0.64 % (Figure 50A). Only methylotrophic microorganisms possessing mxaF genes encoding for the large subunit of the PQQ-methanol dehydrogenase were addressed. Thus, other methylotrophic microorganisms possessing further methylotrophic marker genes such as xoxF (also encoding a PQQ-methanol dehydrogenase) or other methanol-utilising enzymes were not included in this analysis.

The detected numbers of family-level based phylotypes were on average 65 ± 15 OTUs for the substrate SIP experiment incubations. For the pH shift SIP experiment a difference between pH 4 and pH 7 incubations was observed. The number of detected phylotypes was lower at pH 4 with an average of 54 ± 7 OTUs compared to a nearly 2-fold higher value for pH 7 incubation with an average of 96 ± 15 OTUs. (Figure 50B). The Chao 1 indices estimating mxaF gene richness followed the same pattern with average values of 94 ± 26 expected phylotypes for the substrate SIP experiment, 75 ± 29 expected phylotypes for pH 4 incubations and 115 ± 19 expected phylotypes for pH 7 incubations of the pH shift SIP experiment (Figure 50F). Thus, a higher influence of different pH conditions on the overall diversity of mxaF gene sequences in the initial acidic soil was assumed.

In addition, no domination of a few or only one phylotype was observed in all combined amplicon libraries of both SIP experiments (Figure 50C, D & E). Thus, a diverse mxaF-possessing methylotrophic community was assumed at the beginning as well as after the incubation with different substrates or at different pH conditions.

Figure 50 Diversity and richness estimators of mxaF gene sequences from pyrosequencing amplicon pools at similarity level 90%.

Figures indicating coverage (%) (A), numbers of OTUs (B), dominance D (C), Shannon index H (D), equitability J (E) and Chao1 index (F) of t0 samples (no treatment, combined data sets of replicates for substrate SIP experiment t0) and after treatment for both SIP experiments. A ¹² indicates [¹²C]-isotopologue, ¹³ indicates [¹³C]-isotopologue. A cross indicates additional supplementation of methanol in substrate treatments. Symbols: , combined data sets of

‘heavy’, ‘middle’ and ‘light’ fractions; ●, ‘heavy’ fraction; ●, ‘middle’ fraction; ○, ‘light’ fraction. This figure has been published in Morawe et al. 2017.

In general, the methylotrophic community was significantly affected by different substrates as well as different pH incubations as shown with ANOSIM (R = 0.33, p = 0.02) and NPMANOVA (F = 2.02, p = 0.0023) analyses (Table A 7). As assumed by the numbers of detected and expected phylotypes (Chao 1 index) before, ANOSIM analysis revealed a high influence of pH (R = 0.85, p = 0.02) and only a minor effect of different substrates (R = 0.18, p = 0.13) on the methylotrophic community. This result was also confirmed by NPMANOVA analysis. The pairwise NPMANOVA indicated no obvious differences in the methylotrophic community between t₀ and t_End of different substrate incubations with the lowest influence of vanillic acid treatments (F = 0.9) and the highest influence for xylose treatments (F = 2.01) compared with t₀ (Table A 7). The same trend was shown for the comparison between methanol treatments and substrate treatments. The pairwise NPMANOVA of samples from the pH shift SIP experiment revealed a remarkable influence of a more neutral pH in

comparison with its t0 (F = 7.19) as well as in comparison with the incubation at pH 4 (F = also in accordance with the analysis of the bacterial community (see 3.5.1).

Only small differences between [¹²C]-, [¹³C]- and combined dataset-derived communities were observed in an nMDS plot, which accords to the other nMDS plots for bacteria (based on 16S rRNA gene sequences, see 3.5.1 & Figure 48) and fungi (based on ITS gene sequences, see 3.5.3 & Figure 55). In addition, a clear clustering corresponding to the incubations with different substrates and under different pH conditions was obvious (Figure 51). Although ANOSIM and NPMANOVA analyses revealed no strong dissimilarities between the t₀ replicates a noticeable scattering effect in the nMDS plot was represented. This was also observed with the 16S rRNA gene sequence analyses (see 3.5.1 & Figure 48) and could be due to methodical reasons like PCR-based differences and the amount of sequences obtained in the independently conducted pyrosequencing for the different amplicon libraries.

Another explainable reason for the scattering of t₀ replicates could be that the nMDS was conducted with the complete dataset and an nMDS plot attempts to illustrate as accurately as possible the pairwise dissimilarity between different samples based on a distance matrix in a two-dimensional plot. Nonetheless, a stress value below 0.2 indicates for an acceptable representation of the original structure of the data [Clarke, 1993].

colours: sets of pH shift SIP experiment; , pH 4 treatment;

, pH 7 treatment.

Cross: additional [¹²C]-methanol supplementation convex polygons

dashed lines: all data from Substrate SIP; grey lines:

all data for t0; dotted lines: all data from pH SIP experiment

Figure 51 nMDS analyses of the mxaF-possessing bacterial community after different substrate or pH treatments.

The nMDS analysis for the bacterial methylotrophic community was based on mxaF gene sequences (similarity cut-off 90 %). The analysis is based on the Bray-Curtis similarity index with a stress value of 0.1868. This figure has been published in Morawe et al. 2017.

3.5.2.1. Comparison of the methanol treated samples and multi-carbon treated samples

Although the effect of different substrates on the responding methylotrophic community was assumed as low, the position of the substrate SIP experiment derived-samples in the nMDS plot hypothesised the grade of similarities between different substrate incubated-samples compared to each other and with its t₀ samples (Figure 51). Methanol- and glucose-treated samples revealed a closer positioning, assuming higher similarity between these incubations.

Xylose- and acetate-treated samples showed the highest dissimilarity as indicated by their positioning of the samples (Figure 51).

Interestingly, the phylogenetic analysis revealed a decreasing amount of Methylobacterium-related phylotypes and an increase of Hyphomicrobium-Methylobacterium-related phylotypes in all incubations of the substrate SIP experiment (Figure 52 & Table A 10). In the methanol-treated samples only approximately 31 % of all OTUs were related to Methylobacterium. The at t₀ dominating OTU_mxaF 35 was no longer detectable, and instead OTU_mxaF 40 was the dominating sequence (i.e., approximately 20 % abundance) followed by OTUmxaF 55 (i.e., approximately 10 % abundance). Acetate, glucose as well as treatments with CO₂ with and without additional methanol showed the same trend with a domination of increased OTU_mxaF 40 followed by OTU_mxaF 55, and either no detection of OTU_mxaF 35 or a detection of OTU_mxaF 35 at a low level. Thus, a growth stimulating benefit for these phylotype possessing taxa might be suggested. Only xylose and vanillic acid treatments revealed another distribution of Methylobacterium-related phylotypes. OTU_mxaF 35 was still dominant in vanillic acid treatment, but decreased compared to t0 and OTUmxaF 55 was highly increased and thus dominant in xylose treatments.

The distribution of Hyphomicrobium-related sequences still showed the presence of OTU_mxaF 185 in all samples of the substrate SIP experiment with nearly equal amounts in methanol treatments (i.e., approximately 15 % abundance), a lower abundance in vanillic acid, glucose, xylose and both CO₂ treatments (i.e., approximately an abundance of 7.5 %, 7 %, 6

%, 6 % and 3 %, respectively) and an increase in acetate treatment (i.e., approximately 24 % abundance). Thus, acetate seemed to have a stimulating effect on OTU_mxaF 185, whereat other multi-carbon substrates supported growth of other taxa. Further phylotypes that were present in all samples were OTU_mxaF 266 and OTU_mxaF 172. The at t₀ more abundant phylotype OTUmxaF 266 was highly increased in methanol treatments (i.e., approximately 25

% abundance) as well as in treatments with vanillic acid, glucose and CO₂ without methanol (i.e., approximately an abundance of 14 %, 9 % and 9 %, respectively) but decreased in treatments with acetate (i.e., abundance below 1 %) as well as xylose and CO₂ with additional methanol (i.e., approximately 2.5 % abundance). The low abundant phylotype OTUmxaF 172 was increased but still low abundant in treatments with methanol, vanillic acid and CO₂ without additional methanol. Only in xylose treated samples OTU_mxaF 172 was detected as a high abundant Hyphomicrobium-related sequence.

Figure 52 Composition of various mxaF genotypes after different substrate or pH treatments.

Relative abundances of combined (¹²C and ¹³C) data sets of all mxaF-affiliated genotypes (A) and in more detail Methylobacterium-affiliated (B) and Hyphomicrobium-affiliated genotypes (C, D). Phylogenetic affiliation is indicated by equal colours; ambiguous affiliation (i.e., sequence identity with BLASTn < 90 % as well as ambiguous position in phylogenetic tree) is indicated by shading. Additional [¹²C]-methanol supplementation in Substrate SIP experiment is indicated by a cross. Shown are genotypes with relative abundances ≥ 0.5 % in combined (¹²C and ¹³C) data sets.

This figure has been published in Morawe et al. 2017.

In all substrate treatments the at t0 marginal abundant phylotype OTUmxaF 210 increased, but only in treatments with methanol, vanillic acid and CO₂ with additional methanol the abundance was high (i.e., approximately 8 %, 11 % and 19 % abundance, respectively). A similar observation was made for the phylotype OTU_mxaF 309, which was marginal abundant at t₀ (i.e., below 0.5 %), but highly increased in treatments with glucose and CO₂ with additional methanol (i.e., approximately 21 % and 14 % abundance, respectively).

3.5.2.2. Low abundant mxaF phylotypes in the substrate SIP experiment

Besides the domination of Methylobacterium- and Hyphomicrobium-related phylotypes smaller amounts (i.e., abundances mainly below 5 %) of phylotypes related to Methylorhabdus, Methylocystaceae and Beijerinckiaceae were detected (Figure 52 & Table A 10). Methylorhabdus-related phylotypes were present in all samples of the substrate SIP experiment but the abundance was almost always below 0.5 % (exception for the methanol treatment). Only in acetate and xylose treatments a slight higher abundance was detected (i.e., approximately 0.4 % abundance, data not shown) compared to other treatments, indicating a putative utilisation of these two multi-carbon substrates besides methanol. This is in accordance with the known substrate spectrum of this facultatively methylotrophic genera [Doronina et al., 1996]. Although the generation time of Methylorhabdus on glucose is higher than on methanol [Doronina et al., 1996] the abundance of Methylorhabdus-related phylotypes in glucose treated samples was only minimalistic, suggesting no utilisation of the hexose under the given conditions. Thus, it can be assumed that the competition for glucose in the forest soil is high and the low abundant Methylorhabdus is not competitive enough.

Methylocystaceae-related phylotypes were only detectable at low abundances in the initial t0

sample and after treatments with glucose and acetate. The abundance in the glucose treatment was high compared to the t0 samples, indicating a growth supporting effect for this phylotype. For a long time methylotrophic members of the Methylocystaceae were known to be obligately methanotrophic organisms, and multi-carbon substrates such as glucose were not utilised as energy source. However, multi-carbon compounds can be used as supporting carbon source when organisms grow in the presence of methanol or methane [Hanson, 1992]. Another multi-carbon substrate known to be utilised by Methylocystaceae, especially Methylocystis species in acidic peat [Belova et al., 2011] or bog [Im et al., 2011a]

environments, is acetate. Although the Methylocystaceae-related phylotype was detected in the acetate treatment a growth supporting effect of acetate on this phylotype was not observed, since the abundance was negligible.

Beijerinckiaceae-related phylotypes were detected in all samples from the substrate SIP experiment. This family is known to show a preference for acidic soils and includes facultatively methylotrophic genera such as Methylocella, Methylorosula and Methylovirgula [Dedysh et al., 2005a; Vorob’ev et al., 2009; Berestovskaya et al., 2012; Marín & Arahal, 2013; Crombie & Murrell, 2014]. Interestingly, in the initial t₀ sample of the acidic soil the abundance of Beijerinckiaceae-related phylotypes is marginal. Incubations with different substrates revealed a stimulation of growth for Beijerinckiaceae-related taxa, but after the treatment with methanol only less than 3 % of all detected mxaF-correlated phylotypes are related to this family. The treatment with acetate revealed comparable amounts, whereas in samples treated with glucose and xylose the amount of Beijerinckiaceae-related phylotypes was higher, indicating a more stimulating effect for taxa comprising these phylotypes.

Abundances for Beijerinckiaceae-related phylotypes in treatments with vanillic acid were less

than 2 % indicating a minor stimulating effect compared to methanol and other substrates tested. The highest and also the lowest abundance for Beijerinckiaceae-related phylotypes were detected in CO₂ incubations with (i.e., approximately 11 % abundance) and without additional methanol (i.e., approximately 0.9 % abundance), suggesting a clear growth supporting effect of CO₂ in combination with methanol for these phylotype included taxa.

Phylogenetic affiliation and reliable classification of mxaF sequences was not always possible. Contradictory, miscellaneous and ambiguous results from BLASTn analysis and phylogenetic trees made it impossible to affiliate the different phylotypes. The polyphyletic origin of methylotrophic organisms, gene transferring events such as horizontal gene transfer as well as the choice of a suitable primer pair set [Moosvi et al., 2005; see 2.5.7.1] and the limited length of amplicons from pyrosequencing (i.e., approximately 460 bp) contributed to this problem. In all substrate treatments such unclassifiable phylotypes were detectable (Figure 52). Interestingly, their abundance was low at t₀ (i.e., abundances below 2 %) and in methanol-, glucose- and CO2-treated samples (i.e., abundances below 1 %), whereas an increase in abundances was detected in treatments with acetate, vanillic acid and xylose (i.e., abundances of approximately 5 %, 12 % and 16 %, respectively), suggesting a growth stimulating effect on taxa including these unclassifiable phylotypes. Thus, putatively unknown methylotrophic species, which are capable of utilising acetate, aromatic compounds and pentoses and revealing supported growth through multi-carbon substrates, could be hypothesised. Since methanol did not led to higher abundances of this phylotypes it could be questioned how competitive these taxa are in nature and if methanol is not the preferred carbon source. However, missing phylogenetic affiliation prevented further conclusions and clearer assumptions.

3.5.2.3. Comparison of the

mxaF-possessing methylotrophic community

incubated under different pH conditions

As indicated by ANOSIM and NPMANOVA analyses, the effect of different pH conditions on the mxaF-possessing methylotrophic community was stronger than the treatment with different substrates (Table A 7). Interestingly, even in the different t₀ samples remarkable differences were obvious with a domination of Methylobacterium-related phylotypes at pH 4 and a domination of Hyphomicrobium-related phylotypes at pH 7 (Figure 52).

Similar to the methanol incubation of the substrate SIP experiment the amount of Methylobacteriaceae-related phylotypes was decreased, and Hyphomicrobium-related phylotypes were increased after the treatment with methanol at pH 4 (i.e., no adjustment of pH). The t₀ samples with the in situ pH 4 were dominated by the phylotypes OTU_mxaF 35, but only in the methanol treatments of the pH shift SIP experiment OTU_mxaF 35 was still most abundant (i.e., 36 % abundance), which revealed a different composition of Methylobacterium-related phylotypes of both methanol incubations (i.e., incubations of the substrate SIP and pH shift SIP experiment). The same was observed for

Hyphomicrobium-related phylotypes. The in the substrate SIP experiment detected OTUmxaF 185 was in general less abundant in the pH shift SIP experiment, though an increase in the methanol treatment at pH 4 compared to t₀ was detected. Instead, the most abundant Hyphomicrobium-related phylotypes at t₀ as well as after the treatment with methanol at pH 4 was OTU_mxaF 236, which was only minor abundant in methanol incubations of the substrate SIP experiment. Besides the Methylobacterium- and Hyphomicrobium-related phylotypes, minor proportions of Methylorhabdus- and Beijerinckiaceae-related phylotypes (i.e., approximately 3 % and 3.5 % abundance, respectively) were detected. The Methylorhabdus-related phylotype OTU_mxaF 18 was indicated to be enriched, whereas the amount of Beijerinckiaceae-related phylotypes remained rather constant.

Under the elevated pH 7 conditions, samples of t₀ as well as methanol treatments revealed a contrary composition of the mxaF-possessing methylotrophic community compared to all incubations under in situ pH 4. The initial domination of the Hyphomicrobium-related phylotypes at pH 7 (i.e., approximately 69 % abundance) was highly decreased. The initial high abundant phylotypes OTU_mxaF 257 and OTU_mxaF 236 (i.e., approximately 23 % and 21 % abundance, respectively) remained still abundant, but at low amounts (i.e., approximately 6

% and 4 % abundance, respectively). Interestingly, the initial lower abundant phylotype OTU_mxaF 185 increased from approximately 1 % up to approximately 4 %. After the methanol treatment at pH 7 Methylobacterium-related phylotypes increased in abundance up to 74 %, especially OTU_mxaF 55 followed by OTU_mxaF 40 (i.e., up to approximately 40 % and 30 % abundance, respectively). The abundance of Beijerinckiaceae-related phylotypes however decreased from initial approximately 8 % to 1 %, indicating an inhibitory effect of this more neutral pH to this family with its preference for acidic soils [Marín & Arahal, 2013].

3.5.2.4. The effect of the pH on mxaF- and mmoX-possessing methylotrophs

Despite the dissimilarities of the mxaF-possessing methylotrophic communities in the pH 4 and pH 7 treatments also the abundance of these bacteria was assumed to be influenced as well as the abundance of methanotrophs. Methanotrophs that were often detected in acidic soils are affiliated to Beijerinckiaceae. For example, the ‘restricted’ facultatively methanotrophic genus Methylocella seems to prefer acidic environments with a pH below 5 [Rahman et al., 2011]. Methylocella possess solely the sMMO [Marín & Arahal, 2013], wherefore Methylocella-specific mmoX genes were further targeted to briefly assess the effect of an elevated pH on methanotrophs in an inherently acidic forest soil.

The initial quantification of the bacterial abundance (based on 16S rRNA gene numbers) revealed an increase in both pH 7 treatments (i.e., the unsupplemented controls and methanol treatments), demonstrating the general growth restricting conditions at the in situ pH 4 for Bacteria (Figure 53). The same was observed for mxaF gene numbers.

Contrary to that, the gene numbers of mmoX were dramatically decreased in all pH 7 treatments emphasising that the acidic in situ pH conditions were advantageous especially for methanotrophic organisms such as Methylocella or further Beijerinckiaceae.

Figure 53 Influence of different pH conditions on 16S rRNA, mxaF and mmoX gene numbers in soil slurry treatments.

Comparison of the gene copy numbers ng^-1 DNA at t0 (no treatment; white) and after treatment (grey, unsupplemented control; black, methanol supplementation). Gene copy numbers for t0 were determined by duplicated qPCR measurements. Gene copy numbers for different treatments are mean values of replicates (filled columns). Gene copy numbers for each replicate were determined by duplicated qPCR measurements (shaded columns); error bars indicate standard error; n.d., not detectable.

Im Dokument Aerobic Methylotrophic Microorganisms in an Acidic Deciduous Forest Soil: Substrate Range and Effect of pH (Seite 166-174)