Comparative analysis and identification of putative facultatively methylotrophic

Several methylotrophic organisms are not only restricted to C1 compounds like methanol.

These microorganisms are facultatively methylotrophic and are known to utilise different multi-carbon compounds [Anthony, 1982; Chistoserdova et al., 2009; Semrau et al., 2011;

Kolb & Stacheter, 2013]. The availability of complete genomes as well as diverse metagenomic studies in various environments revealed a hitherto unknown methylotrophic potential of different organisms [Kaneko et al., 2002; Greenberg et al., 2006; Giovannoni et al., 2008; Chistoserdova et al., 2009; Halsey et al., 2012]. Previous studies addressing methylotrophic organisms used typical C1 compounds such as methane or methanol as substrates and thus were not able to draw any conclusion about a broader substrate range of the detected methylotrophs. In addition, descriptions of new methylotrophic species include growth studies with a certain range of possible substrates utilised, but nonetheless these studies provide only hints for in situ relevant situations. The objective of the comparative SIP was to detect methylotrophs by identifying labelled phylotypes in a methanol-treated sample and estimate the potential substrate range of these identified methylotrophs by re-detecting the same phylotypes as labelled in multi-carbon substrate treatments.

In general, mainly the Beijerinckiaceae-affiliated phylotype (OTU_16S 438) was identified as the dominant methylotrophic taxon in the acidic forest soil sample (see 3.7.1.3) when samples where incubated with methanol. Several facultatively methylotrophic species are known for this family in which most cultivated species show only a narrow substrate range. Acetate is utilised by the majority of all cultured facultatively methylotrophic Beijerinckiaceae [Dunfield et al., 2010; Dunfield et al., 2003] and only Methylorosula polaris and Beijerinckia mobilis possess the ability to utilise sugars and polysaccharides [Berestovskaya et al., 2012; Dedysh et al., 2005b]. Interestingly, in all treatments of the substrate SIP experiment with multi-carbon compounds the Beijerinckiaceae-related phylotype was also identified as labelled in

‘heavy’ or ‘middle’ fractions indicating that the taxa comprised by OTU_16S 438 possess the ability to assimilate acetate, sugars and aromatic compounds in the presence of methanol since [¹²C]-methanol was always supplemented (Figure 62). The labelling was not as efficient

as it was observed for methanol indicated by lower LPs and a detected labelling in ‘middle’

fractions (Table A 15 – Table A 18). Thus, a higher competition or slower growth rates on multi-carbon substrates were suggested. Incorporation of [¹³C]-carbon via [¹³C]-CO₂ and thus a cross feeding effect was negligible, since the phylotype OTU_16S 438 was not identified as labelled in both CO₂ treatments (Figure 61C & Figure 62, Table A 19,Table A 20).

Figure 62 Congruently labelled bacterial phylotypes in treatments of both SIP experiments.

Labelled phylotypes (OTUs) in methanol treatments as well as the consistent presence of these phylotypes in the substrate SIP and pH shift SIP experiments (‘ P’ > 5 %, black; ‘ P’ < 5 %, grey). Phylotypes that are only labelled in treatments with multi-carbon substrates are not considered. Additional [¹²C]-methanol supplementation in the substrate SIP experiment is indicated by a cross. Phylogenetic affiliation is indicated by equal colours. ‘H’ and ‘M’

indicate ‘heavy’ and ‘middle’ fraction, respectively. This figure has been published in Morawe et al. 2017.

In order to resolve the putative trophic types that are comprised by the detected Beijerinckiaceae-affiliated phylotype the taxon OTU_16S 438 was further analysed on a species level cut-off value. Nine different phylotypes (A to I) were identified, that revealed three different trophic types including obligatley methylotrophic (phylotype A, B and D), restricted facultatively methylotrophic (phylotype C) and chemoorganotrophic taxa (phylotype E, F, G, H) (see Table 36). All methylotrophic phylotypes (A - D) are affiliated to methylotrophic Beijerinckiaceae and the restricted facultatively methylotrophic phylotype C clustered next to Methylocella (see Figure A 2), emphasising that acetate was assimilated by a Beijerinckiaceae-phylotype affiliated to facultatively methylotrophic representatives. Although

for the family-level phylotype OTU16S 438 CO2 assimilation was not assumed, phylotype I was only detected in the treatments with CO₂ and methanol indicating that CO₂ was assimilated by this taxon, which seems affiliated to Bradyrhiziobiaceae (see Figure A 2). The chemoorganotrophic taxa are affiliated to Bradyrhiziobiaceae (phylotype E), Hyphomicrobiaceae (phylotype F & H), and Roseiarcus fermentans (phylotype G) (see Figure A 2) and are further distinguishable in taxa with a wide substrate range (phylotype E, F, G) including sugars, acetate and vanillic acid and a narrow substrate range (phylotype H) restricted to glucose. It is worth to mention that for the chemoorganotrophic phylotypes the utilisation of methanol is not verified, but is also not excluded, since SIP analysis are unable to resolve the dissimilatory utilisation of methanol that might have occurred. Nevertheless, the detected Beijerinckiaceae-affiliated taxon seems to be clearly distinguishable in taxa that are preferring methanol assimilation or multi-carbon compound assimilation, wherefore the substrate range – C1 and multi-carbon compounds – is one defining factors for their different ecological niches.

Table 36 Putative trophic types comprised by the Beijerinckiaceae-phylotype.

The resolution was done at species level. The ‘X’ means that the taxon was detected in the corresponding fraction (‘H’, ‘heavy’ fraction or ‘M’, ‘middle’ fraction)

substrate SIP experiment pH shift^a MeOH Ace + Glu + Xyl + Van + CO2 + CO2 pH4^b

a Abbrevation for the pH shift SIP experiment.

b Cross indicates [¹²C]-methanol supplementation. ‘H’ and ‘M’ indicate ‘heavy’ and ‘middle’ fraction, respectively.

c [¹³C1]-methanol was supplemented

Another alphaproteobacterial phylotype that was putatively labelled in the methanol incubation as well as in the substrate incubations was the Sphingomonas-affiliated phylotype OTU_16S 449 with a broader substrate range including acetate, sugars and vanillic acid. The Acetobaceraceae-affiliated phylotype OTU_16S 467 that was also labelled in methanol treatments revealed a narrow substrate range including acetate and glucose (Figure 62). The actinobacterial phylotypes OTU16S 652 (Acidimicrobiaceae-affiliated) and OTU16S 703

treatment indicating a delayed labelling. Both phylotypes were also detected in ‘middle’

fractions of vanillic acid treatments. The phylotype OTU_16S 703 was also labelled in ‘middle’

fraction of the acetate treatment. These delayed labelling could be explained by slow growth, utilisation of endogenous C-sources or cross feeding effects which could not be excluded, since both phylotypes were also identified as labelled in both CO₂ incubations (Figure 62).

The verrucomicrobial phylotypes OTU_16S 6 and OTU_16S 18 labelled in methanol treatments were also labelled in treatments with acetate (i.e., for OTU_16S 6) and glucose (i.e., OTU_16S 18) and thus showed the ability to utilise multi-carbon substrates (Figure 62). In addition, both phylotypes were also detected as labelled in both CO₂ incubations not excluding cross feeding effects.

Interestingly, phylotypes that were only labelled in the pH 7 treatments were not detected as labelled in any treatment with the in situ pH 4. These phylotypes were affiliated to Bacteroidetes (i.e., OTU_16S 1045, OTU_16S 1078, OTU_16S 1094, and OTU_16S 1108), and the genera Paracoccus (OTU16S 450), Methylophilus (OTU16S 358) and Terrimicrobium (OTU16S

54) assuming an inhibitory effect of a more acidic pH and thus no utilisation of any multi-carbon substrate at lowered pH. Only the Leifsonia-affiliated phylotype OTU16S 721 that was labelled in both pH shift SIP experiment samples was also labelled in the acetate, glucose and xylose treatments as well as in the CO₂ with methanol treatment of substrate SIP experiment, suggesting a broader substrate range including acetate, sugars and CO₂ (Figure 62).