Overview of the experimental design
The design is a split-split plot design with repeated measures. Cx (x=1-5) represents the cycle of (co)-cultivation, where n represents the total amount of headspace measurements available for each cycle.
DGGE pattern among the cycles
16S rRNA gene DGGE was performed as described in materials & methods. Using BioNumerics (Applied Maths, version 5.1) band classes were assigned. Only the most abundant band class of a pure strain loaded on the gel was selected as “representative” band class, hence correcting for possible ghost bands. Samples were color-coded according to the cycle they belonged to. R45002 represents Methylomonas methanica NCIMB 11130T. +8HET refers to co-cultivation incubations. 8HET as such are lanes from the negative control of the 8 non-MOB incubated without the MOB. Cycle “none” refers to pure cultures loaded in the lane. Color codes (Column “Code”) are depicting the (co-) incubation cycle (column “Cycle”). In the column MOB “NOMOB” refers to lanes where amplicon of the (pure) cultures of the non-MOB partners were loaded. Fuzzy clustering was performed using the Jaccard distance (aware of band intensity) and UPGMA linkage.
Methane removal profiles
CH4(t=t)/CH4(t=t0) profiles for all experimental conditions and cycles. Observation points represent average CH4(t=t)/CH4(t=t0) of either triplicate (MOB alone) or quadruplicate (MOB with heterotrophs) measurements, except for the heterotroph control incubation without methanotrophs, where only one biological replicate was used. A dashed horizontal red line represents 25% methane removal from the initial concentration. Shaded areas represent 95% confidence intervals on the observations.
Box-and-whisker plots for methane oxidation ratio's (MOR) for each cycle per treatment
MOR is expressed as mmol CH4 oxidized per liter of broth per hour. MOR is corrected for losses during incubation by means of a negative control with only heterotrophs (HET). LMG: pure culture cultivation of Methylosinus sp. LMG 26262. LMG+HET: co-cultivation of Methylosinus sp. LMG26262 with 8 non-MOB partners as described in materials & methods. R: pure culture cultivation of Methylomonas methanica NCIMB 11130T. R+HET: co-cultivation of Methylomonas methanica NCIMB 11130T with 8 non-MOB partners as described in materials & methods. Black dots in the boxplot represent the median MOR.
Δ designates increase or removal between beginning (t0) and end (tend) of a cycle, where the amount of protein at t0was calculated based upon the 10% v/v transfer at the end of cycle 3.
Comparative genomics of C1 metabolism
Microsoft Excel spreadsheet of C1 modules compiled from Chistoserdova (2011) and RAST subsystems/scenarios for the metabolic processes. “?” indicates that it is unclear if a certain function is correctly assigned or (in)complete. “Maybe” indicates that a gene was found that could be involved in the functionality though it is not clear if it is enough for the functionality or the correct gene. Individual gene names were given when appropriate.
Raw data of the methane oxidation ratios
Methane oxidation ratios (MOR) were calculated by linear regression as described in material and methods.
Protein and CO2 concentrations dataset
Columns are as follows: 1 - Bottle number = number that was assigned to the bottle during incubation 2 - Cycle = co-cultivation cycle 3 - Content = microbial composition 4 - CycleCont = code incorporating the information on Cycle and Content 5 - proteinmgpermL = mg protein formed per mL of broth 6 - proteinmmolC = mg protein formed per mL of broth converted to mmol carbon (Rouwenhorst et al., (1991) Journal of Biochemical and biophysical methods 22 (2) 119-128) 7 - mmolCO2permL = mmol of CO2 measured per mL of culture at the end of the cycle 8 - CO2protmmolratio = ratio of columns 6 and 7 9 - mmolCO2formed = total amount (mmol) of CO2 formed for the whole culture volume as compared to the start of the cycle 10 - mmolCO2formedpermL = 9 expressed per mL of culture 11 - CO2fdProtmmolratio = ratio of 10 and 6 12 - CO2fdProtmgratio = ratio of 9 and 5