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Supplementary Material Sample details


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Supplementary Material

  1. Sample details

Samples of the liquid portion of the forestomach contents of three wild male Eastern Grey kangaroos (M. giganteus) were collected under a Queensland Parks and Wildlife Scientific Purposes Permit (permit number W0/001397/00/SAA). Samples were collected in the field near Milmerran, Queensland from animals which had been euthanized as part of the regular annual cull. The forestomach contents were immediately mixed with sterile, anaerobic glycerol to give a final glycerol concentration of 30-40% (v/v). These samples were placed on ice for short term transport, before being frozen on dry ice for transport back to the laboratory, where they were transferred to storage at -20 °C. Samples of rumen fluid from four Bos taurus cattle that had been fed a diet consisting primarily of whole barley grain for 110 days were collected immediately after slaughter at Highchester Abattoir, Beaudesert, Qld, Australia. Whole digesta samples were mixed with glycerol, transported and stored as described above. Samples of rumen fluid of grass-fed cattle were collected from three rumen fistulated B. taurus x B. indicus cattle housed at the Centre for Advanced Animal Studies at Gatton, Queensland. Liquid digesta was mixed with sterile anaerobic glycerol and handled as described above. During collection of all samples, a portion of liquid digesta was also collected into separate bottles which did not contain glycerol. These bottles were transported on ice and frozen at the earliest opportunity. This digesta was used for preparation of media for the incubation experiment.
The kangaroo forestomach sample used for RNA-SIP was taken from an archive of samples that were previously collected and frozen as described above (Ouwerkerk et al., 2005; Ouwerkerk et al., 2009)


  1. Summary of samples used in the experiments

Experiment 1 – Isotope tracing to compare the fates of carbon dioxide/bicarbonate in the bovine rumen and kangaroo forestomach


Animal

Diet

Number of animals sampled

Isotope tracer treatments

Incubation sampling time points (hours)

Macropus giganteus (eastern grey kangaroo)

Mixed pasture

3

(1) 13C (5% label)
(2) 12C (no label)

0, 3, 6, 24, 48, 168

Bos indicus x Bos taurus

Mixed pasture

3

(1) 13C (5% label)
(2) 12C (no label)

0, 3, 6, 24, 48, 168

Bos taurus

Barley ration

4

(1) 13C (5% label)
(2) 12C (no label)

0, 3, 6, 24, 48, 168


Experiment 2 – RNA stable isotope probing to identify bacteria associated with metabolism of carbon dioxide/bicarbonate and hydrogen.


Animal

Diet

Number of animals sampled

Isotope tracer treatments

Incubation sampling time point (hours)

Macropus giganteus (eastern grey kangaroo)

Mixed pasture

1

(1) 13C (99% label) + hydrogen
(2) 12C (no label)
(3) 13C (99% label) without hydrogen

16


3. Media compositions

All media used were designed to approximately simulate the pH and major ion concentrations of the bovine rumen and kangaroo forestomach. In the isotope tracer experiments, the composition of the basal salt solution was identical for each of the three systems that were investigated (kangaroo forestomach, grass-fed bovine rumen and grain-fed bovine rumen). The only difference in the media used for each of the three systems was the type of gut fluid that was added. All media were adjusted to pH 7.0 prior to autoclaving.



Digesta based media for isotope tracing experiments Per litre
Clarified rumen fluid or kangaroo forestomach fluid 330 ml

NaCl 100 mg

(NH4)2SO4 50 mg

CaCl2 5 mg

MgSO4 5 mg

K2HPO4 50 mg

NiCl2 x 6 H2O 10 mg

Ascorbic acid 0.5 mg

Myo-inositol 0.5 mg

Niacinamide 0.5 mg

Choline chloride 0.5 mg

Trace Element Solution* 10.0 mL

Resazurin 0.02 mg

NaHCO3 10.5 g


Minimal Carbon Anaerobic Salts (MCAS) medium Per litre
NaCl 3.6 g

KH2PO4 50 mg

(NH4)2SO4 50 mg

CaCl2 5 mg

MgSO4 5 mg

K2HPO4 50 mg

Trace Element Solution* 10.0 mL

Cysteine-HCl 0.22 g

Resazurin 0.02 mg

NiCl2 x 6 H2O 10 mg

Ascorbic acid 0.5 mg

Myo-inositol 0.5 mg

Niacinamide 0.5 mg

Choline chloride 0.5 mg



*Trace element solution per litre
MgSO4.H2O 3 g

MnSO4.H2O 0.5 g

NaCl 1 g

FeSO4.7H2O 0.1 g

CoCl2.6H2O 0.1 g

CaCl2 0.1 g

ZnSO4.7H2O 0.1 g

CuSO4.5H2O 10 mg

AlK(SO4)2.12H2O 10 mg

H3BO3 10 mg

Na2MoO4.2H2O 10 mg

NiSO4.6H2O 30 mg

Na2SeO3 20 mg

Na2WO4.2H2O 20 mg



4. Gas Chromatography - Isotope ratio mass spectrometry

Determination of carbon stable isotope ratios relative to the international standard Vienna Peedee Belemnite (δ13CVPDB) (Coplen et al., 2006) was conducted using an Agilent 7890A GC system (Agilent Technologies, Santa Clara, CA, USA) coupled to an Isoprime GC5 oxidation furnace and an Isoprime 100 isotope ratio mass spectrometer (Isoprime, Cheadle, UK). For both headspace gases and VFAs, the gases eluting from the chromatographic column were split into two streams. One of these was directed into an Agilent 5975C inert mass spectrometer detector (MSD), for sample identification and quantification, while the other was directed through the GC5 furnace held at 990 °C to oxidise all carbon species into CO2. The CO2 exiting the furnace was then directed into the IRMS for the determination of δ13C. The IRMS was calibrated using laboratory standard gas Labgas 50:50, which is a 50:50 mix of CH4 and CO213CVPDB of -40.1‰ and -18.8‰ respectively) and international standard NBS19 (δ13CVPDB +1.95‰).


For analysis of gas headspace samples, the gas chromatography column was a 0.5 mm × 30 m Varian CP7352 (Agilent). Twenty microlitres of gas was injected into the system manually via a split/splitless injector at a 20:1 split ratio. The carrier gas was helium at a constant flow rate of 1.3 mL/min and the column was held at a constant temperature of 40 °C. A standard curve for methane concentration was constructed by performing 5 μL, 10 μL, 20 μL, 30 μL, 40 μL and 50 μL injections of a standard 2.5% methane sample and plotting the resulting major beam area obtained from the IRMS.
Stable isotope analysis of VFAs was conducted by the method of Morrison et al. (2004), The gas chromatography column was a 250 μm × 30 m VF-WAXms (Agilent). One μL of aqueous solution containing VFAs was injected manually at a split ratio of 2:1. The carrier gas was helium at a flow rate of 1.1 mL/min. Chromatographic separation of VFAs was achieved using a temperature program which ramped the column oven from 70 °C to 180 °C over 14 minutes. The concentrations of each VFA in the samples were determined using a series of VFA standard solutions each of which contained 4.0 mM of the internal standard 3-methyl valeric acid. These standards were used to construct calibration curves using the ratio of response (peak area) obtained from the quadrupole MSD for each VFA to the response for the internal standard.
Table S1. Composition of VFA standard solutions


VFA

Standard 1

Standard 2

Standard 3

Response factor

acetic acid

5.521 mM

52.51 mM

105.02 mM

0.345

propionic acid

1.339 mM

13.39 mM

26.78 mM

0.481

iso-butyric acid

0.535 mM

5.35 mM

10.70 mM

0.679

n-butyric acid

0.545 mM

5.45 mM

10.90 mM

0.678

iso-valeric acid

0.455 mM

4.55 mM

9.10 mM

0.832

n-valeric acid

0.455 mM

4.55 mM

9.10 mM

0.881

3-methyl-valeric acid (internal standard)

3.986 mM

3.986 mM

3.986 mM

1.000


5. Resolution of 13C-labelled RNA by isopycnic centrifugation

13C-labelled RNA was resolved from unlabelled RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) using a protocol adapted from Whiteley et al (2007). Gradients were formed in 5.1 polyallomer ultracentrifuge tubes (Beckman Coulter, Gladesville, NSW ), which contained 4.083 mL of a 1.99 g/mL CsTFA solution (GE Life Science), 173.9 μL deionised formamide and 840.2 μL of RNA solution containing a total of 600 ng of RNA. Control gradients were loaded with 300 ng each of 13C and 12C-­E coli RNA prepared as described above. Tubes were centrifuged in an NVT-100 rotor in an Optima L-XP ultracentrifuge (Beckman Coulter, Gladesville, NSW ) at 53,800 rpm and 20 °C for 48-64 hours. After centrifugation, each gradient tube was divided into 20 fractions by using a gradient fractionator (Isco) to collect fluid at a controlled rate from the bottom of the tube. The density of the solution in each fraction of the control gradient was determined using a digital refractometer (Reichert, USA) to verify correct formation of gradients during the centrifuge run. The densities of fractions of the sample gradients were not measured to prevent carryover of RNA and cross-contamination of samples. The RNA was recovered from each gradient fraction by precipitation in isopropanol using certified nucleic acid–free glycogen (Life Technologies, Mulgrave, Vic, Australia) as a carrier, and resuspended in 30 μL nuclease free water. Profiles illustrating the quantity of total RNA recovered from each gradient fraction are presented in figure S1.
6. 13C labelling of E. coli controls

As a control to allow the verification of gradient formation and separation of 13C labelled (‘heavy’) and unlabelled (‘light’) RNA, E. coli strain YE263, originally isolated from kangaroo foregut contents (Ouwerkerk et al., 2009), was grown to stationary phase in M9 minimal medium (Sambrook et al., 1989) supplemented with trace elements and vitamins (Joblin, 2005) plus 0.4% (w/v) of either 12C or 13C6 glucose (Sigma-Aldrich, St. Louis, USA). Cultures were passaged three times in this medium, then cells were recovered by centrifugation, and RNA was extracted as described below.


7. Comparison of gradient fractions by denaturing gradient gel electrophoresis

DGGE was used for preliminary comparison of the structure of bacterial communities represented in the ‘heavy’ and ‘light’ fractions of CsTFA gradients. For each gradient, 20 pg RNA recovered from each of the three fractions corresponding to the ‘heavy’ RNA peak of the control gradient and 20 pg from each of the three fractions corresponding to the ‘light’ RNA peak of the control gradient was reverse transcribed using the Thermoscript reverse transcriptase kit (Invitrogen) according to the manufacturer’s instructions. The resulting cDNA solution was used as template for amplification of the V3 region of the 16S rRNA gene using the forward primer 341F-GC (5’-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’) which includes a 5’ GC-clamp (indicated in bold) and reverse primer 534R (ATTACCGCGGCTGCTGG) (Muyzer et al., 1993). Thermocycling was performed according to the following; initial denaturation at 95 °C for 5 min followed by 5 cycles of 30 s denaturation at 95 °C, 10 s annealing at 65 °C and 30 s elongation at 72 °C. This was followed by a touchdown phase consisting of nineteen cycles of 95 °C for 30 s, annealing for 10 s (decreasing 0.5 °C per cycle from 65 °C) and 72 °C for 30 s. The final phase consisted of 11 cycles of 95 °C for 30 s, 55 °C for 10 s and 72 °C for 30 s, with a final elongation step of 72 °C for 10 min.


PCR products were purified using the Qiagen PCR cleanup kit (Qiagen, Doncaster, Victoria) according to the manufacturer’s instructions and quantified using a Nanodrop 8000 (Thermo Scientific, DE, USA). A 20 ng subsample of this product was then loaded into each well of a denaturing gradient gel. The PCR products were separated by electrophoresis on an 8% (wt/vol) polyacrylamide gel with a 30 to 60% urea/formamide gradient as described previously (Martínez et al., 2012).
Representative denaturing gradient gels illustrating the changes in the distribution of bacterial 16S amplicons between ‘heavy’ and ‘light’ gradient fractions as isotope label was incorporated are presented in figures S2 and S3.
8. PCR for production of barcoded amplicons for 454 sequencing

PCR for generation of barcoded amplicons was performed as described previously (Gulino et al., 2013) using Phusion high fidelity DNA polymerase (Thermo Fisher, Waltham, MA, USA), and barcoded forward primer 341F (5’-Fusion A-Barcode-CCTACGGGAGGCAGCAG-3’) (Watanabe et al., 2001) and reverse primer 787R (5’-Fusion B–CTACCAGGGTATCTAAT -3’) (Baker et al., 2003) (Fusion sequences and barcodes available at http://www.roche.com.au). One set of barcoded primers was used for each cDNA sample. This produced a total of six barcoded PCR products, designated 12C+H ‘heavy’, 12C+H ‘light’, 13C+H ‘heavy’, 13C+H ‘light’, 13C-H ‘heavy’ and 13C-H ‘light’. The PCR products were visualised on a 2% agarose gel, and bands corresponding to the correct sized product were excised and purified using the QIAquick gel extraction kit. Three hundred nanograms of the purified product was submitted to the Australian Genome Research Facility, University of Queensland, for 454-sequencing.


9. Statistical analysis of pyrosequencing data to identify OTUs significantly associated with CO2/H2 utilisation

To normalise differences in the total number of sequences obtained for each gradient fraction (sampling depth), the OTU table containing the number of sequences per OTU for each fraction was resampled using the rarefaction function of QIIME to produce a new table containing 10,000 sequences per sample. A 2x2 contingency table was then constructed for each OTU consisting of the numbers of sequences in the ‘heavy’ and ‘light’ fractions recovered from the control incubation (12C + H) and the heavy-isotope treated incubation (13C + H). The X2 statistic was calculated for each OTU, and used to obtain a p-value which indicated how significant the difference between the control and isotope-treated incubations was in the distribution of sequences between the ‘heavy’ and ‘light’ fractions. OTUs with significantly (p<0.001) increased numbers of sequences in the ‘heavy’ fraction of the isotope-treated incubation relative to the control incubation were considered representative of organisms that incorporated the labelled CO2. Construction of contingency tables and X2 analysis was conducted in R studio 2.15 (Bates et al., 2012). An identical analysis was conducted to compare the heavy isotope and hydrogen treated incubation with the heavy isotope incubation performed without hydrogen. This was done to identify organisms that were able to incorporate the 13C-label but did not require high concentrations of hydrogen to do so. These OTUs were removed from the final list of putative hydrogen/CO2 metabolising organisms.


References

Baker GC, Smith JJ, Cowan DA (2003). Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55: 541-55.


Bates D, Chambers J, Dalgaard P, Falcon S, Gentleman R, Hornik K et al. (2012). R Foundation for Statistical Computing: Vienna, Austria.
Coplen TB, Brand WA, Gehre M, Gröning M, Meijer HAJ, Toman B et al (2006). New Guidelines for δ13C Measurements. Analytical Chemistry 78: 2439-2441.
Gulino L, Ouwerkerk D, Kang A, Maguire AJ, Kienzle M, Klieve AV (2013). Shedding the light on the macropod foregut using 454-amplicon pyrosequencing. PLos ONE 8: e1463.
Joblin KN (2005). Methanogenic archaea. In: Makkar HPS and McSweeney CS (eds). Methods in gut microbial ecology for ruminants. Springer: Netherlands.
Martínez ED, Turnbull KE, Quigley SP, Streeter SJ, Swain A, Klieve AV et al (2012). Liquid-phase denaturant gradient gel electrophoresis profiles of rumen bacteria from Brahman cross steers selected into two groups on the basis of post-weaning liveweight gain on low crude protein pasture. Animal Production Science 52: 647-652.
Morrison DJ, Cooper K, Waldron S, Slater C, Weaver LT, Preston T (2004). A streamlined approach to the analysis of volatile fatty acids and its application to the measurement of whole-body flux. Rapid communications in mass spectrometry 18: 2593-2600.
Muyzer G, de Waal EC, Uitterlinden AG (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695-700.
Ouwerkerk D, Klieve AV, Forster RJ, Templeton JM, Maguire AJ (2005). Characterization of culturable anaerobic bacteria from the forestomach of an eastern grey kangaroo, Macropus giganteus. Letters in Applied Microbiology 41: 327-333.
Ouwerkerk D, Maguire AJ, McMillen L, Klieve AV (2009). Hydrogen utilising bacteria from the forestomach of eastern grey (Macropus giganteus) and red (Macropus rufus) kangaroos. Animal Production Sciences 49: 1043-1051.
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning - A Laboratory Manual. Cold Spring Harbour Laboratory Press: New York.
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Whiteley AS, Thomson B, Lueders T, Manefield M (2007). RNA stable-isotope probing. Nature Protocols 2: 838-844.

Titles and legends to supplementary figures
 Figure S1. RNA recovery from CsTFA gradients prepared with total RNA extracted from kangaroo forestomach material that had been incubated for 3 hours, 16 hours or 7 days in the presence of ▲ unlabelled CO2/HCO3- and H2, ■ 99 atom % 13CO2/H13CO3- and H2 or ● 99 atom % 13CO2/H13CO3- without H2. □ Gradient controls consisted of an equimolar mix of unlabelled (‘light’) RNA and labelled (‘heavy’) RNA extracted from E. coli grown in the presence of 12C or 13C6 glucose. While incubation of forestomach material in the presence of 13CO2/H13CO3- and H2 for 3 or 16 hours did not result in an observable increase in buoyant density of RNA relative to controls incubated in the presence of unlabelled CO2/HCO3- and H2, incubation for 7 days resulted in complete incorporation of 13C label. On the basis of these observations 16 hours was chosen as the maximum incubation time that resulted in specific incorporation of the isotope label by primary utilisers of the CO2/HCO3- substrate.
Figure S2. Denaturing gradient gel comparing CsTFA gradient fractions for the three incubation treatments after three hours incubation. No major differences between the ‘heavy’ and ‘light’ gradient fractions could be observed. (L – DGGE reference ladder, 12C + H2 – kangaroo forestomach content incubated in the presence of H2 and unlabelled (12C) CO2/HCO3-, 13C+H2 - kangaroo forestomach content incubated in the presence of H2 and labelled (13C) CO2/HCO3-, 13C (no H2)- kangaroo forestomach content incubated in the presence of labelled (13C) CO2/HCO3, without H2)
Figure S3. Denaturing gradient gel comparing CsTFA gradient fractions for the three incubation treatments after 16 hours incubation. Several instances of 13C labelling of specific organisms, represented by bands that appear in the ‘heavy’ fractions of the isotope labelled treatment while appearing only in the ‘light’ fractions of the no-label control incubation, can be observed (bands indicated in boxes). (L – DGGE reference ladder, 12C + H2 – kangaroo forestomach content incubated in the presence of H2 and unlabelled (12C) CO2/HCO3-, 13C+H2 - kangaroo forestomach content incubated in the presence of H2 and labelled (13C) CO2/HCO3-, 13C (no H2)- kangaroo forestomach content incubated in the presence of labelled (13C) CO2/HCO3, without H2)
Figure S4 Mean (± SE) changes relative to time 0 in concentration and δ13CVDPB of butyrate (a,b) and valerate (c,d) in in vitro gut content fermentations spiked with labelled (13C) HCO3-. ▲ Bovine rumen fluid (grain-fed) (n=4) ● Bovine rumen fluid (grass-fed) (n=3) ■ Kangaroo forestomach content (n=3).
Figure S5. Mean (± SE) changes relative to time 0 in concentration and δ13CVDPB of acetate (a,b), propionate (c,d), butyrate (e,f) and valerate (g,h) in in vitro gut content fermentations spiked with unlabelled (12C) HCO3-. ▲ Bovine rumen fluid (grain-fed) (n=4) ● Bovine rumen fluid (grass-fed) (n=3) ■ Kangaroo forestomach content (n=3).




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