The More, the Merrier

1Supplementary

3 The more, the merrier:

4 heterotroph richness stimulates methanotrophic activity.

6Adrian Ho1,#*, Karen de Roy1, Olivier Thas2,3, Jan De Neve2, Sven Hoefman4, Peter Vandamme4,

7Kim Heylen4, and Nico Boon1*.

91Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience

10Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.

112Department of Mathematical Modelling, Statistics and Bioinformatics, Faculty of Bioscience

12Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.

133National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics

14and Applied Statistics, University of Wollongong, NSW 2522, Australia.

154Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology,

16Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium

17#Current address: Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-

18KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands.

20*For correspondence: Nico Boon ([email protected]) and Adrian Ho

21([email protected]).

1 1 2 24Figure S1: (a) Methane oxidation rate and (b) total cell counts in incubations with the least

25(single heterotroph) and most diverse (ten heterotrophs) heterotrophic population in the

26methanotroph-heterotroph co-cultures as determined from two independent batch

27incubations (mean ± s.d; n=3) performed over approximately three days. Incubation

28containing methanotroph alone served as a reference. Abbreviation; H: heterotroph. H1 to

29H10 denote heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H

30denotes a combination of 10 heterotrophs.

32Figure S2: Stimulation of methane oxidation with increasing heterotroph richness as

33determined from two independent batch incubations. The experimental design required 80

34incubations which were performed in two separate batch incubations (40 incubations per

35batch). In addition, incubations with methanotroph alone (n=3) served as a reference for

36each batch. Subsequently, data from these batch incubations were combined and given as

37the ratio of methane oxidation rates in co-cultures and reference incubation in Figure 1.

38Black and red denote the different batch incubations.

40Figure S3: Methane uptake rate in incubations containing methanotroph in heterotroph

41spent NMS medium (mean ± s.d; n=2). Incubation containing methanotroph in NMS medium

42served as a reference. Abbreviations; H: heterotroph; SM: spent medium. H1 to H10 denote

43heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H denotes a

44combination of 10 heterotrophs.

46Figure S4: Methane uptake rate in incubations containing methanotroph in undiluted LB

47medium, and 0.1X, 0.01X and 0.001X diluted LB in NMS medium (mean ± s.d; n=3).

3 2 4 48Incubation containing methanotroph in NMS medium served as a reference (mean ± s.d;

49n=3).

51Figure S5: Methylomonas methanica growth curve. Mean and standard deviation of

52triplicate measurements.

5 3 6 72Methods and Materials

74M.methanica and heterotroph culturing, and artificial community assembly

76The growth curve for M.methanica was determined in a 1L Schott bottle containing 100 ml

77Nitrate Medium Salts (NMS; Knief and Dunfield, 2005) medium and approximately 20 vol.%

78methane in the headspace. The bottle was capped with a butyl rubber stopper (boiled twice)

79and incubated at 28 C on a shaker (120 rpm). Methane and headspace air was replenished

80every day. The growth curve was followed by measuring the optical density of the culture

81medium at 600 nm. The experimental set up and subsequent sampling was performed

82aseptically. The purity of the culture was checked by plating 100 μl of the culture in a

83Trypticase Soy Agar (TSA; BD, Spark MD, USA) plate, and incubated at 37˚C. The cultures

84were considered pure if no cell colonies formed after five days. Cells were harvested during

85logarithmic phase (after 3-6 days; Figure S5), and enumerated using a flow cytometer (Accuri

86C6, BD Biosciences, Erembodegem, Belgium) as described before (de Roy et al, 2012).

88Ten heterotroph species covering two phyla (Firmicutes and Proteobacteria) and three

89classes of the Proteobacteria (Table 1) were used to assemble the artificial communities. The

90heterotrophs were grown on Luria Bertani (LB) medium plates and incubated at 28˚C for

91three days before cells were collected and suspended in NMS liquid medium. The

92heterotroph cells were not washed before suspended in NMS liquid medium to avoid further

93disruption of the cells. After homogenization by vortex, the cells were enumerated using the

94flow cytometer. Cell culturing was performed aseptically. Purity of the cultures was

95determined by cell and colony morphology. Heterotroph spent medium was prepared by

7 4 8 96filtering the medium through a 0.22 µm sterile filter (Millex®GV, Merck Millipore, Cork,

97Ireland) twice after incubation in NMS medium for three days.

99Methanotroph and heterotoph cell numbers were enumerated using the flow cytometer and

100assembled in equal total starting cell numbers (107 cells ml-1). In incubations consisting of

101more than one heterotroph, the heterotrophs were assembled separately in a larger volume

102as a master-mix, and homogenized by vortex, before distributing an aliquot of the mixture to

103the individual incubation containing the methanotroph. These cells were harvested at

104logarithmic phase (M.methanica) or 3-4 days after plating (heterotrophs), and were largely

105comprised of intact cells (>70%) as indicated by fluorescent dye staining according to de Roy

106et al (2012).

108Experimental set up and methane uptake rate

110Incubation was performed in 120 ml opaque bottles containing 10 ml NMS and

111approximately 20 vol.% methane in the headspace, and capped with butyl rubber stoppers

112(boiled twice). The bottles were incubated on a shaker (120 rpm) at 28°C in the dark. The

113incubation set-up and subsequent sampling were performed aseptically. After incubation,

114the purity of the reference incubation containing the methanotroph alone was confirmed by

115plating on TSA medium plate and incubated at 37 C, and showed no cell colony formation

116after five days.

118Potential methane oxidation rate was determined by linear regression over approximately

119three days (65-67 h). At the end of the incubation, methane concentration was above 11 vol.

9 5 10 120%. Methane in the headspace was measured using a compact gas chromatograph

121(Convenant Analytical Solutions, Belgium).

123Statistical analysis

125The data were analyzed with a general linear model with methane oxidation rate as the

126response variable and richness as a continuous regressor. The model also included the batch

127factor (by design) and regressors for the 10 heterotrophs. Because of the large

128multicolinearity among the ten 0/1 indicators for the heterotrophs, these ten indicators

129were replaced by their first nine eigenvectors. This linear transformation does not alter the

130assessment of the effect of richness (primary research question), while removing

131multicolinearity issues. Note that the tenth eigenvector was not included because richness is

132– as per definition – equal to the sum of the ten 0/1 indicator variables.

133The effect of richness was tested in this linear model using a t-test at the 5% level of

134significance. All model assumptions (linearity, additivity, normality, constancy of variance)

135were assessed by means of residual plots and normal QQ-plots.

11 6 12 144References

146De Roy K, Clement L, Thas O, Wang Y, Boon N. (2012). Flow cytometry for fast microbial

147community fingerprinting. Water Res 46: 907-919.

149Knief C, Dunfield PF. (2005). Response and adaptation of different methanotrophic bacteria

150to low methane mixing ratios. Environ Microbiol 7: 1307-1317.

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