<p> 1Supplementary</p><p>2</p><p>3 The more, the merrier: </p><p>4 heterotroph richness stimulates methanotrophic activity. </p><p>5</p><p>6Adrian Ho1,#*, Karen de Roy1, Olivier Thas2,3, Jan De Neve2, Sven Hoefman4, Peter Vandamme4,</p><p>7Kim Heylen4, and Nico Boon1*.</p><p>8</p><p>91Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience</p><p>10Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.</p><p>112Department of Mathematical Modelling, Statistics and Bioinformatics, Faculty of Bioscience</p><p>12Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium.</p><p>133National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics</p><p>14and Applied Statistics, University of Wollongong, NSW 2522, Australia.</p><p>154Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology,</p><p>16Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium</p><p>17#Current address: Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-</p><p>18KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands.</p><p>19</p><p>20*For correspondence: Nico Boon ([email protected]) and Adrian Ho</p><p>21([email protected]).</p><p>22</p><p>23</p><p>1 1 2 24Figure S1: (a) Methane oxidation rate and (b) total cell counts in incubations with the least</p><p>25(single heterotroph) and most diverse (ten heterotrophs) heterotrophic population in the</p><p>26methanotroph-heterotroph co-cultures as determined from two independent batch</p><p>27incubations (mean ± s.d; n=3) performed over approximately three days. Incubation</p><p>28containing methanotroph alone served as a reference. Abbreviation; H: heterotroph. H1 to</p><p>29H10 denote heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H</p><p>30denotes a combination of 10 heterotrophs.</p><p>31</p><p>32Figure S2: Stimulation of methane oxidation with increasing heterotroph richness as</p><p>33determined from two independent batch incubations. The experimental design required 80</p><p>34incubations which were performed in two separate batch incubations (40 incubations per</p><p>35batch). In addition, incubations with methanotroph alone (n=3) served as a reference for</p><p>36each batch. Subsequently, data from these batch incubations were combined and given as</p><p>37the ratio of methane oxidation rates in co-cultures and reference incubation in Figure 1.</p><p>38Black and red denote the different batch incubations.</p><p>39</p><p>40Figure S3: Methane uptake rate in incubations containing methanotroph in heterotroph</p><p>41spent NMS medium (mean ± s.d; n=2). Incubation containing methanotroph in NMS medium</p><p>42served as a reference. Abbreviations; H: heterotroph; SM: spent medium. H1 to H10 denote</p><p>43heterotrophs 1 to 10 (heterotroph designation is given in Table 1), while 10H denotes a</p><p>44combination of 10 heterotrophs.</p><p>45</p><p>46Figure S4: Methane uptake rate in incubations containing methanotroph in undiluted LB</p><p>47medium, and 0.1X, 0.01X and 0.001X diluted LB in NMS medium (mean ± s.d; n=3).</p><p>3 2 4 48Incubation containing methanotroph in NMS medium served as a reference (mean ± s.d;</p><p>49n=3).</p><p>50</p><p>51Figure S5: Methylomonas methanica growth curve. Mean and standard deviation of</p><p>52triplicate measurements.</p><p>53</p><p>54</p><p>55</p><p>56</p><p>57</p><p>58</p><p>59</p><p>60</p><p>61</p><p>62</p><p>63</p><p>64</p><p>65</p><p>66</p><p>67</p><p>68</p><p>69</p><p>70</p><p>71</p><p>5 3 6 72Methods and Materials</p><p>73</p><p>74M.methanica and heterotroph culturing, and artificial community assembly</p><p>75</p><p>76The growth curve for M.methanica was determined in a 1L Schott bottle containing 100 ml </p><p>77Nitrate Medium Salts (NMS; Knief and Dunfield, 2005) medium and approximately 20 vol.% </p><p>78methane in the headspace. The bottle was capped with a butyl rubber stopper (boiled twice)</p><p>79and incubated at 28 C on a shaker (120 rpm). Methane and headspace air was replenished </p><p>80every day. The growth curve was followed by measuring the optical density of the culture </p><p>81medium at 600 nm. The experimental set up and subsequent sampling was performed </p><p>82aseptically. The purity of the culture was checked by plating 100 μl of the culture in a </p><p>83Trypticase Soy Agar (TSA; BD, Spark MD, USA) plate, and incubated at 37˚C. The cultures </p><p>84were considered pure if no cell colonies formed after five days. Cells were harvested during </p><p>85logarithmic phase (after 3-6 days; Figure S5), and enumerated using a flow cytometer (Accuri</p><p>86C6, BD Biosciences, Erembodegem, Belgium) as described before (de Roy et al, 2012). </p><p>87</p><p>88Ten heterotroph species covering two phyla (Firmicutes and Proteobacteria) and three </p><p>89classes of the Proteobacteria (Table 1) were used to assemble the artificial communities. The</p><p>90heterotrophs were grown on Luria Bertani (LB) medium plates and incubated at 28˚C for </p><p>91three days before cells were collected and suspended in NMS liquid medium. The </p><p>92heterotroph cells were not washed before suspended in NMS liquid medium to avoid further</p><p>93disruption of the cells. After homogenization by vortex, the cells were enumerated using the </p><p>94flow cytometer. Cell culturing was performed aseptically. Purity of the cultures was </p><p>95determined by cell and colony morphology. Heterotroph spent medium was prepared by </p><p>7 4 8 96filtering the medium through a 0.22 µm sterile filter (Millex®GV, Merck Millipore, Cork, </p><p>97Ireland) twice after incubation in NMS medium for three days.</p><p>98</p><p>99Methanotroph and heterotoph cell numbers were enumerated using the flow cytometer and</p><p>100assembled in equal total starting cell numbers (107 cells ml-1). In incubations consisting of </p><p>101more than one heterotroph, the heterotrophs were assembled separately in a larger volume </p><p>102as a master-mix, and homogenized by vortex, before distributing an aliquot of the mixture to</p><p>103the individual incubation containing the methanotroph. These cells were harvested at </p><p>104logarithmic phase (M.methanica) or 3-4 days after plating (heterotrophs), and were largely </p><p>105comprised of intact cells (>70%) as indicated by fluorescent dye staining according to de Roy </p><p>106et al (2012). </p><p>107</p><p>108Experimental set up and methane uptake rate</p><p>109</p><p>110Incubation was performed in 120 ml opaque bottles containing 10 ml NMS and </p><p>111approximately 20 vol.% methane in the headspace, and capped with butyl rubber stoppers </p><p>112(boiled twice). The bottles were incubated on a shaker (120 rpm) at 28°C in the dark. The </p><p>113incubation set-up and subsequent sampling were performed aseptically. After incubation, </p><p>114the purity of the reference incubation containing the methanotroph alone was confirmed by </p><p>115plating on TSA medium plate and incubated at 37 C, and showed no cell colony formation </p><p>116after five days. </p><p>117</p><p>118Potential methane oxidation rate was determined by linear regression over approximately </p><p>119three days (65-67 h). At the end of the incubation, methane concentration was above 11 vol.</p><p>9 5 10 120%. Methane in the headspace was measured using a compact gas chromatograph </p><p>121(Convenant Analytical Solutions, Belgium). </p><p>122</p><p>123Statistical analysis</p><p>124</p><p>125The data were analyzed with a general linear model with methane oxidation rate as the </p><p>126response variable and richness as a continuous regressor. The model also included the batch </p><p>127factor (by design) and regressors for the 10 heterotrophs. Because of the large </p><p>128multicolinearity among the ten 0/1 indicators for the heterotrophs, these ten indicators </p><p>129were replaced by their first nine eigenvectors. This linear transformation does not alter the </p><p>130assessment of the effect of richness (primary research question), while removing </p><p>131multicolinearity issues. Note that the tenth eigenvector was not included because richness is</p><p>132– as per definition – equal to the sum of the ten 0/1 indicator variables. </p><p>133The effect of richness was tested in this linear model using a t-test at the 5% level of </p><p>134significance. All model assumptions (linearity, additivity, normality, constancy of variance) </p><p>135were assessed by means of residual plots and normal QQ-plots. </p><p>136</p><p>137</p><p>138</p><p>139</p><p>140</p><p>141</p><p>142</p><p>143</p><p>11 6 12 144References</p><p>145</p><p>146De Roy K, Clement L, Thas O, Wang Y, Boon N. (2012). Flow cytometry for fast microbial </p><p>147community fingerprinting. Water Res 46: 907-919.</p><p>148</p><p>149Knief C, Dunfield PF. (2005). Response and adaptation of different methanotrophic bacteria </p><p>150to low methane mixing ratios. Environ Microbiol 7: 1307-1317.</p><p>13 7 14</p>