Multi-carbon compound assimilation by methanol-utilising microorganisms in an

3.3.1. Conversion of methanol and multi-carbon substrates and the formation of [

C]-CO

as evidence for substrate dissimilation

In order to investigate the multi-carbon substrate range of methylotrophic microorganisms homogenous soil slurry incubations of the acidic forest soil were conducted under methylotrophic and mixed substrate conditions (see 2.3.3). The initial pH of the soil slurry was low and stayed constant at 3.7 ± 0.1 during the incubation period. The only exception was the treatment supplemented with acetate that showed a slight increase of the pH up to 4.5 at the end resulting in a mean pH of 4.0 ± 0.2.

The native microbial community in an acidic soil was tested for their capacity to utilise selected C1 and multi-carbon substrates. As typical C1 compounds methanol and methane were chosen. Methanol was supplemented daily as 1 mM pulse to mimic in situ conditions as well as avoiding accumulation and toxication. The utilisation of methanol was assumed by an increased formation of CO₂ evidently after five days of incubation (Figure 46A). Methane was supplemented consistently to methanol, in which the concentrations of methane were 100 times higher than atmospheric conditions, but still low compared to other studies to address methanotrophic microorganisms with high-affinity enzymes for methane utilisation in particular. However, no utilisation of methane was observed in any incubation of the substrate SIP experiment (Figure 46B).

Apart from the C1 compounds – methane and methanol – the capacity to utilise multi-carbon compounds by the methylotrophic microorganisms in soils was analysed. The multi-carbon compounds tested were acetate as a common intermediate in the soil matrix, a hexose sugar (glucose), a pentose sugar (xylose) and an aromatic compound (vanillic acid). All these compounds are assumed to be plant-derived in situ.

Preliminary conducted substrate consumption tests revealed that supplemented acetate and sugars were no longer detectable in soil slurry samples within a few hours resulting in degradation rates of 0.11 mM acetate h^-1, 0.2 mM glucose h^-1 and 0.125 mM xylose h^-1 in soil slurries (data not shown). Thus, in the substrate SIP experiment acetate and sugars were pulsed daily. The aromatic compound vanillic acid was hardly detectable in the soil slurry supernatant indicating a binding to soil particles. However, degradation of vanillic acid could be observed resulting in a degradation rate of 0.02 mM vanillic acid h^-1 in soil slurries (data not shown). Thus, vanillic acid was pulsed for a second time after 3 days. An adaption to the supplementation of vanillic acid could be observed resulting in an increased uptake rate with

Figure 46 CO2 formation and conversion of different multi-carbon substrates in soil slurry treatments.

12CO2 and ¹³CO2 concentrations (cumulative) of substrate SIP experiment treatments pulsed with methanol (A) and other multi-carbon substrates (F) as well as methanol treatments of the pH shift SIP experiment with pH 4 (C) and pH 7 (D). A cross indicates additional methanol supplementation. Substrate utilisation is supposed by the conversion of supplemented substrates (E), methane utilisation in the substrate SIP experiment treatments is negligible (B, E).

Methanol treatments serve as control treatments for supplemented multi-carbon substrate treatments. All values are mean values of replicates; error bars represent standard deviations. White symbols, unsupplemented control; grey symbols, methanol treatments; black symbols, multi-carbon substrate treatments; , [¹³C]-CO2;, [¹²C]-CO2;, substrates; , methane. This figure has been published in Morawe et al. 2017.

The utilisation of multi-carbon substrates was indicated by the observed disappearance of supplemented substrates and a correlating increase of CO₂ formation (Figure 46E & F).

Since methanol was also pulsed in the substrate treatments, the methanol incubation served as control for the different substrate treatments. Even after one day differences in CO₂ formation between substrate incubations and the methanol incubation were already detectable (Figure 46F). During the SIP incubation experiment methanol, acetate, glucose, xylose and vanillic acid were supplemented as ¹²C- or ¹³C-isotopologue. For vanillic acid only carbon atoms of the aromatic ring were ¹³C-isotopes, carbon atoms of the carboxyl and methyl group were ¹²C-isotopes. Thus, only the fate of aromatic ring-derived carbon atoms can be tracked as [¹³C]-CO₂ for vanillic acid. The amount of CO₂ detectable in ¹²C- and ¹³ C-isotopologue substrate incubations was similarly, assuming no preference of [¹²C]-substrates utilisation (Figure 46F).

The utilisation of ¹³C-isotopologues was obviously proven by [¹³C]-CO₂ formation. On average the carbon recovery per 1 mM [¹³C1]-methanol pulse as [¹³C]-CO2 was approximately 20 % in the methanol supplemented incubations, and no increase of [¹³C]-CO₂ formation per 1 mM pulse was observed during the incubation period. In contrast, for all multi-carbon substrate treatments an increase in [¹³C]-CO₂ formation per 1 mM substrate pulse was observed. Acetate supplementation affected an approximately 5-fold increase per 1 mM of acetate assuming a carbon recovery of 4.30 % up to 22.26 % (on average 11.72 %).

Glucose supplementation affected an approximately 4-fold increase per 1 mM of glucose assuming carbon recovery of 4.32 % up to 17.48 % (on average 10.82 %). Xylose supplementation affected an approximately 3.7-fold increase per 1 mM of xylose assuming a carbon recovery of 5.96 % up to 22.19 % (on average 12.80 %). Vanillic acid supplementation affected an approximately 2.5-fold increase per 1 mM of vanillic acid assuming a carbon recovery of 6.35 % up to 15.67 % (on average 11.37 %).

3.3.2. Influence of the soil pH on methanol utilisation and the [

C]-CO

formation

Soil samples for pH shift SIP experiment were taken at another time point than for the substrate SIP experiment (see 2.3.4). However, the initial pH of the soil slurry was still low (pH 3.6). In order to determine the effect of a higher pH on the methylotrophic microorganisms in an acidic soil the pH was adjusted to a more neutral value around 7. The incubation of in situ pH (‘pH 4 incubation’) and pH-adjusted (‘pH 7 incubation’) soil slurry treatments showed no dramatic changes in terms of pH over the incubation time. A slight increase in ‘pH 4 incubation’ up to a pH of 4.2 at the end was observed. Mean pH values of unsupplemented controls and methanol treatments were 4.0 ± 0.1 for ‘pH 4 incubation’ and 6.9 ± 0.1 for ‘pH 7 incubation’, respectively (data not shown).

In accordance to previously performed substrate SIP experiment (see 3.3.1), only methanol was supplemented. The utilisation of methanol was indicated by the increased formation of CO₂ even after one day of incubation compared to unsupplemented controls (Figure 46C &

D). In general, the CO₂ formation was always higher in ‘pH 7 incubations’ (control and methanol treatments).

No preferred [¹²C]-methanol utilisation was indicated by similar amounts of CO₂ produced in [¹²C]- and [¹³C]-methanol incubations (Figure 46C & D). This is in accordance with the substrate SIP experiment as well as the constant formation of [¹³C]-CO₂ per 1 mM methanol pulse during both incubations. For ‘pH 4 incubation’ a mean carbon recovery of approximately 13.5 % was assumed, which is lowered compared to the methanol incubations of the preliminarily performed substrate SIP experiment, assuming a putatively less active methanol-utilising community. For ‘pH 7 incubation’ the [¹³C]-CO₂ formation was more than 2-fold higher compared to ‘pH 4 incubation’ revealing a mean carbon recovery of approximately 29.67 %.

3.4. Pyrosequencing read yield of 16S rRNA gene, ITS gene sequences and mxaF gene sequences

In order to obtain genetic information the nucleic acids were specifically amplified with different primer sets (see 2.5.7.1 & 2.5.7.6) and achieved amplicon libraries were further sequenced (see 2.5.12.2).

In total, 200’785 sequences of 16S rRNA gene sequences were obtained from pyrosequencing. A total of 105’689 16S rRNA gene sequences were obtained after further processing (i.e., quality filtering, checking for chimeric sequences and clustering based on primer and different barcodes at a family-level cut-off of 90.1 % similarity, see 2.5.13.1) resulting in only 1’492 sequences (i.e., t₀1 of substrate SIP experiment) up to 13’979 sequences (i.e., [¹³C]-methanol treatment of the ‘pH 4 incubation’ of the pH shift SIP experiment) for samples.

139’329 sequences of mxaF were obtained in total from pyrosequencing. After processing (i.e., clustering based on primer and different barcodes at a threshold of 90% similarity, see 2.5.13.1) 113’689 sequences remained, resulting in only 955 sequences (i.e., [¹³C]-methanol incubation of the substrate SIP experiment) up to 14’934 sequences (i.e., [¹³C]-methanol treatment of the ‘pH 7 incubation’ of the pH shift SIP experiment) for each sample.

Due to the huge range of remaining sequence numbers of the different samples, a normalization step was not realized for 16S rRNA gene and mxaF sequence analysis to avoid a larger loss of information.

For ITS gene sequences a total of 237’495 sequences were obtained from pyrosequencing.

The data sets were rarefied to 1’503 sequences per sample (i.e., 99’198 sequences in total)

and after processing (i.e., checking for chimeric sequences and confirming fungal origin of sequence, see 2.5.13.1) a total of 95’065 sequences remained, ranging from 4’246 sequences (i.e., [¹²C]-vanillic acid incubation of the substrate SIP experiment) up to 4’440 sequences (i.e., [¹³C]-xylose incubation of the substrate SIP experiment) for each sample.

3.5. The impact of methanol, multi-carbon substrates and pH