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Supplementary
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The more, the merrier:
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heterotroph richness stimulates methanotrophic activity.
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Adrian Ho1,#*, Karen de Roy1, Olivier Thas2,3, Jan De Neve2, Sven Hoefman4, Peter
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Vandamme4, Kim Heylen4, and Nico Boon1*.
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1Laboratory
of Microbial Ecology and Technology (LabMET), Faculty of Bioscience
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Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
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2Department
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Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
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3National
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and Applied Statistics, University of Wollongong, NSW 2522, Australia.
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4Laboratory
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Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
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#Current
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KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands.
of Mathematical Modelling, Statistics and Bioinformatics, Faculty of Bioscience
Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics
of Microbiology (LM-UGent), Department of Biochemistry and Microbiology,
address: Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-
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*For
correspondence:
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(A.Ho@nioo.knaw.nl).
Nico
Boon
(Nico.Boon@UGent.be)
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and
Adrian
Ho
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Figure S1: (a) Methane oxidation rate and (b) total cell counts in incubations with the least
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(single heterotroph) and most diverse (ten heterotrophs) heterotrophic population in the
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methanotroph-heterotroph co-cultures as determined from two independent batch
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incubations (mean ± s.d; n=3) performed over approximately three days. Incubation
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containing methanotroph alone served as a reference. Abbreviation; H: heterotroph. H1 to
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H10 denote heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H
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denotes a combination of 10 heterotrophs.
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Figure S2: Stimulation of methane oxidation with increasing heterotroph richness as
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determined from two independent batch incubations. The experimental design required 80
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incubations which were performed in two separate batch incubations (40 incubations per
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batch). In addition, incubations with methanotroph alone (n=3) served as a reference for
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each batch. Subsequently, data from these batch incubations were combined and given as
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the ratio of methane oxidation rates in co-cultures and reference incubation in Figure 1.
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Black and red denote the different batch incubations.
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Figure S3: Methane uptake rate in incubations containing methanotroph in heterotroph
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spent NMS medium (mean ± s.d; n=2). Incubation containing methanotroph in NMS medium
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served as a reference. Abbreviations; H: heterotroph; SM: spent medium. H1 to H10 denote
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heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H denotes a
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combination of 10 heterotrophs.
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Figure S4: Methane uptake rate in incubations containing methanotroph in undiluted LB
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medium, and 0.1X, 0.01X and 0.001X diluted LB in NMS medium (mean ± s.d; n=3).
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Incubation containing methanotroph in NMS medium served as a reference (mean ± s.d;
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n=3).
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Figure S5: Methylomonas methanica growth curve. Mean and standard deviation of
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triplicate measurements.
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Methods and Materials
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M.methanica and heterotroph culturing, and artificial community assembly
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The growth curve for M.methanica was determined in a 1L Schott bottle containing 100 ml
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Nitrate Medium Salts (NMS; Knief and Dunfield, 2005) medium and approximately 20 vol.%
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methane in the headspace. The bottle was capped with a butyl rubber stopper (boiled twice)
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and incubated at 28 ̊C on a shaker (120 rpm). Methane and headspace air was replenished
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every day. The growth curve was followed by measuring the optical density of the culture
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medium at 600 nm. The experimental set up and subsequent sampling was performed
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aseptically. The purity of the culture was checked by plating 100 μl of the culture in a
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Trypticase Soy Agar (TSA; BD, Spark MD, USA) plate, and incubated at 37˚C. The cultures
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were considered pure if no cell colonies formed after five days. Cells were harvested during
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logarithmic phase (after 3-6 days; Figure S5), and enumerated using a flow cytometer (Accuri
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C6, BD Biosciences, Erembodegem, Belgium) as described before (de Roy et al, 2012).
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Ten heterotroph species covering two phyla (Firmicutes and Proteobacteria) and three
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classes of the Proteobacteria (Table 1) were used to assemble the artificial communities. The
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heterotrophs were grown on Luria Bertani (LB) medium plates and incubated at 28˚C for
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three days before cells were collected and suspended in NMS liquid medium. The
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heterotroph cells were not washed before suspended in NMS liquid medium to avoid further
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disruption of the cells. After homogenization by vortex, the cells were enumerated using the
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flow cytometer. Cell culturing was performed aseptically. Purity of the cultures was
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determined by cell and colony morphology. Heterotroph spent medium was prepared by
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filtering the medium through a 0.22 µm sterile filter (Millex®GV, Merck Millipore, Cork,
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Ireland) twice after incubation in NMS medium for three days.
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Methanotroph and heterotoph cell numbers were enumerated using the flow cytometer and
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assembled in equal total starting cell numbers (107 cells ml-1). In incubations consisting of
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more than one heterotroph, the heterotrophs were assembled separately in a larger volume
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as a master-mix, and homogenized by vortex, before distributing an aliquot of the mixture to
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the individual incubation containing the methanotroph. These cells were harvested at
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logarithmic phase (M.methanica) or 3-4 days after plating (heterotrophs), and were largely
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comprised of intact cells (>70%) as indicated by fluorescent dye staining according to de Roy
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et al (2012).
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Experimental set up and methane uptake rate
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Incubation was performed in 120 ml opaque bottles containing 10 ml NMS and
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approximately 20 vol.% methane in the headspace, and capped with butyl rubber stoppers
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(boiled twice). The bottles were incubated on a shaker (120 rpm) at 28°C in the dark. The
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incubation set-up and subsequent sampling were performed aseptically. After incubation,
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the purity of the reference incubation containing the methanotroph alone was confirmed by
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plating on TSA medium plate and incubated at 37 ̊C, and showed no cell colony formation
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after five days.
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Potential methane oxidation rate was determined by linear regression over approximately
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three days (65-67 h). At the end of the incubation, methane concentration was above 11
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vol.%. Methane in the headspace was measured using a compact gas chromatograph
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(Convenant Analytical Solutions, Belgium).
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Statistical analysis
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The data were analyzed with a general linear model with methane oxidation rate as the
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response variable and richness as a continuous regressor. The model also included the batch
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factor (by design) and regressors for the 10 heterotrophs. Because of the large
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multicolinearity among the ten 0/1 indicators for the heterotrophs, these ten indicators
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were replaced by their first nine eigenvectors. This linear transformation does not alter the
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assessment of the effect of richness (primary research question), while removing
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multicolinearity issues. Note that the tenth eigenvector was not included because richness is
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– as per definition – equal to the sum of the ten 0/1 indicator variables.
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The effect of richness was tested in this linear model using a t-test at the 5% level of
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significance. All model assumptions (linearity, additivity, normality, constancy of variance)
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were assessed by means of residual plots and normal QQ-plots.
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References
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De Roy K, Clement L, Thas O, Wang Y, Boon N. (2012). Flow cytometry for fast microbial
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community fingerprinting. Water Res 46: 907-919.
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Knief C, Dunfield PF. (2005). Response and adaptation of different methanotrophic bacteria
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to low methane mixing ratios. Environ Microbiol 7: 1307-1317.
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