Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17 h After Birth and Raised Aseptically

ENVIRONMENTAL MICROBIOLOGY

Methanogen Colonisation Does Not Significantly AlterAcetogen Diversity in Lambs Isolated 17 h After Birthand Raised Aseptically

Emma J. Gagen & Pascale Mosoni & Stuart E. Denman &

Rafat Al Jassim & Christopher S. McSweeney &

Evelyne Forano

Received: 16 January 2012 /Accepted: 3 February 2012 /Published online: 2 March 2012# Springer Science+Business Media, LLC 2012

Abstract Reductive acetogenesis is not competitive withmethanogenesis in adult ruminants, whereas acetogenic bacte-ria are the dominant hydrogenotrophs in the early rumenmicrobiota. The ecology of hydrogenotrophs in the developingrumen was investigated using young lambs, raised in sterileisolators, and conventional adult sheep. Two lambs were bornnaturally, left with their dams for 17 h and then placed into asterile isolator and reared aseptically. They were inoculatedwith cellulolytic bacteria and later with Methanobrevibactersp. 87.7 to investigate the effect of methanogen establishmenton the rumen acetogen population since they lacked cultivablerepresentatives of methanogens. Putative acetogens were in-vestigated by acetyl-CoA synthase and formyltetrahydrofolate

synthetase gene analysis and methanogens by methyl coen-zyme reductase A gene analysis. Unexpectedly, a low abun-dant but diverse population of methanogens (predominantlyMethanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to thecommunity structure in conventional sheep. In contrast, poten-tial acetogen diversity in isolated lambs and conventionalsheep was different. Potential acetogens affiliated betweenthe Lachnospiraceae andClostridiaceae in conventional sheepand with the Blautia genus and the Lachnospiraceae inisolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affectacetogen diversity.

Introduction

Reductive acetogens (hereafter referred to as acetogens)have received considerable attention in recent years as ahydrogenotrophic population that may play a role in reduc-ing ruminal methanogenesis [1–6]. Naturally, reductive ace-togenesis is not a significant hydrogen sink in the rumen.However, in the absence of methanogenesis, acetogens cancontribute significantly to hydrogen capture and can sustaina functional rumen [7]. In order to better understand aceto-genesis as a potential alternative hydrogenotrophic pathwayin the rumen, the natural rumen acetogen population must beidentified, characterised and investigated in response to thepresence of methanogens. The developing rumen provides aunique environment to study these types of interactions asacetogens are thought to be the dominant hydrogenotrophicpopulation in the developing rumen before methanogenestablishment, but proportionally decrease in numbers asmethanogens colonise the rumen during the first week of

Emma J. Gagen and Pascale Mosoni contributed equally to themanuscript

Electronic supplementary material The online version of this article(doi:10.1007/s00248-012-0024-z) contains supplementary material,which is available to authorized users.

E. J. Gagen : S. E. Denman : C. S. McSweeneyCSIRO Livestock Industries,St. Lucia, QLD 4067, Australia

E. J. Gagen :R. Al JassimSchool of Animal Studies, The University of Queensland,Gatton, QLD 4343, Australia

P. Mosoni : E. ForanoINRA, Unité de Microbiologie, UR454,Centre de Clermont-Ferrand/Theix,63122 Saint-Genès-Champanelle, France

E. J. Gagen (*)Department of Microbiology, University of Regensburg,Universitaetstrasse 31,93053 Regensburg, Germanye-mail: [email protected]

Microb Ecol (2012) 64:628–640DOI 10.1007/s00248-012-0024-z

http://dx.doi.org/10.1007/s00248-012-0024-z

life [2]. Characterizing the naturally resident acetogen pop-ulation present during early rumen development is difficultand in vitro studies can only mimic the rumen to a limiteddegree. An animal model has been designed that involvesthe placement of a lamb into an aseptic isolator soon afterbirth (~17 h) thus enabling the development and mainte-nance of their initial rumen population, free from specificfunctional groups of cultivable microorganisms (cellulolyticbacteria and methanogens) and eukaryotes (protozoa andfungi) present in adult sheep [8]. Such animal models arereared in the same sterile conditions as those used forgnotobiotic animals [9–11] and together with gnotoxenicand meroxenic lambs have been useful in investigations onthe role of cellulolytic bacteria, fungi, methanogens andacetogens in the rumen [7–9, 11–16]. In the present study,lambs that were raised aseptically 17 h after birth will bereferred to as ‘isolated lambs’. To date, all studies involvingaseptically reared ruminants have relied on traditional culti-vation techniques to investigate microbial populations, de-spite known limitations with cultivation techniques [17]. Toour knowledge, there is no literature describing the rumenbacterial diversity of newborn lambs using molecular tech-niques. The aim of the present study was to use molecularmethods to further investigate microbial populations in veryyoung lambs, and specifically, evaluate acetogen diversitybefore and after methanogen establishment. The lambs wereinoculated with cellulolytic bacteria to enable the consump-tion of hay, thus providing a functioning fibrolytic rumen.The microbial diversity of isolated lambs (corresponding tothat of a 17-h-old lamb microbiota) was also compared withthat of a mature sheep microbiota.

Methods

Two INRA 401 lambs were obtained as previously described[10, 11]. The lambs were born naturally, left with their damsfor 17 h and then placed into a sterile isolator and rearedaseptically, i.e. in the same sterile conditions as those usedfor gnotobiotic lambs. As there is no appropriate term toqualify such lamb models [18], they were simply called ‘iso-lated lambs’. Isolated lambs were fed ultra-high-temperaturesterilized milk from cows until they were 10 weeks old, andafter 5 weeks, they also had access to a ration of dehydratedalfalfa hay (Fig. 1) in the form of 7-mm pellets (SAFE, Augy,France) sterilized by γ irradiation (4 Mrad, Ionisos, Dagneux,France). After 10 weeks of age, the animals were fed twicedaily with pellets only (Fig. 1). Each lamb was fitted with apermanent rumen plastisol cannula (diameter, 2.5 cm) at16 weeks (Fig. 1). Two, 2-year-old Texel sheep fed the samediet as the mature lambs were used for comparative purposes.They were cannulated as described previously [19]. The

experimental protocol had been reviewed and validated bythe local ethics committee before beginning the trial.

Inoculation of Isolated Lambs with Cellulolytic Bacteriaand Methanogens

Eight days after birth, isolated lambs were inoculated on3 days consecutively with 5 ml (109 cells ml−1) of a mixtureof 2-day-old pure cultures grown on cellulose of each of:Ruminococcus flavefaciens FD1 (from Department of Ani-mal Science, Urbana-Champain, IL, USA) and 007 (fromRowett Research Institute, Aberdeen, UK), Ruminococcusalbus 7 (ATCC 27210) and 8 (from USDA-NCAUR, Peoria,IL, USA), and Fibrobacter succinogenes S85 (ATCC19169) and 98 (from Lethbridge Research Station, Alberta,Canada), see Fig. 1. The microbial inocula were directlyintroduced into the rumen via a stomach tube. This inocu-lation is necessary because at 17 h of age cellulolytics werenot detected as previously shown [11, 16] and under theseconditions, the animals could not be weaned from a milk toa lignocellulosic based diet [14]. At 5 months of age(150 days), isolated lambs were further inoculated with apure culture of the ruminal methanogenMethanobrevibactersp. 87.7 (Fig. 1), directly through the cannula. Mbb. sp. 87.7was previously isolated from a young lamb at INRA andused in this study because it had been found to establisheasily in aseptically-raised lambs [7, 11].

Rumen Sampling

Rumen contents were withdrawn from isolated lambs bystomach tubing (before fistulation) or through the cannula(after fistulation) before morning feeding (T0) and 3 h after(T3). Three samples of 30 g were collected each time andimmediately frozen at −20°C. Fresh samples were used formicrobial enumerations. For molecular and short-chain fattyacid (SCFA) analyses, samples were collected on 3 days

Figure 1 Overview of lamb trial. The lambs were initially fed steri-lised cows milk (open area) and then sterilised alfalfa hay (shadedarea). Rumen contents were withdrawn before feeding and 3 h afterfeeding on 3 days in 1 week for sampling period 1 and on 3 days over a3-week period for sampling period 2. The lambs were 130 days old and200 days old in the first and second sampling periods, respectively

Early Colonising Rumen Acetogens and Methanogens 629

over a 1-week period during sampling period 1 (pre-inocula-tion with methanogens, Fig. 1), and on 3 days over a 3-weekperiod, during sampling period 2 (post-inoculation withMbb.sp. 87.7, Fig. 1). Sampling and molecular analyses wereperformed in the same way with the two conventional sheepafter a 4-week adaptation to the diet.

Enumeration of Microbial Communities

The presence of protozoa, fungi, cellulolytic bacteria andmethanogens in the rumen of isolated lambs initially wasinvestigated by phase-contrast microscopy, microbialgrowth on cellulose culture medium or microbial growthon methanogen culture medium respectively (see below).Protozoa and fungi were never detected by phase contrastmicroscopy throughout the experiment. Cellulolytic bacteriawere enumerated, based on degradation of a cellulose filterpaper strip in each tube, in a liquid medium containing 40%clarified rumen fluid [20] and a cellulose filter paper strip ineach tube. Reductive acetogens were cultured as describedpreviously [2] in AC-11 medium [21] including 50 mM ofmethanogen inhibitor bromoethanesulfonate. Reductive ace-togenic activity was evidenced by hydrogen consumptionand acetate production in cultures provided with 202 kPaH2/CO2 (80:20). Methanogens were cultured in Balch me-dium [22] including antibiotics cephalothin (6.7 μg ml−1)and clindamycin (1.7 μg ml−1) to inhibit growth of non-methanogenic hydrogenotrophs. Methanogenesis wasevidenced by hydrogen consumption and methane produc-tion in cultures provided with 202 kPa H2/CO2 (80:20). Allcultures were incubated for 2 to 3 weeks at 39°C. Enumer-ation was carried out in triplicate. Cell densities were cal-culated using the most probable number (MPN) method ofClarke and Owens [23].

Chemical Analyses

SCFA and gas concentrations were determined by gas chro-matography (DI700 chromatograph; Delsi Instruments) us-ing previously described methods [7]. Acetate concentrationin culture supernatant was determined using a commercialkit (Boehringer-Mannheim, SA, Meylan, France).

Calculation of Reducing Equivalents and PotentialReductive Acetogenesis

The reducing equivalent (2H) recovery was calculated fromSCFA data after methanogen inoculation, as described pre-viously [7, 24] using the stoichiometry of rumen fermenta-tion [25] as follows:

2H recovery ¼ 2H accepted=2H released; w i t h 2Hreleased ¼ 2Aþ P þ 4Bþ 3V and 2H accepted ¼ 2P þ 2

Bþ 4V þ 4M , where A is acetate, P is propionate, B isbutyrate, V is valerate and M is methane.

The expected methane production in rumen contents oflambs inoculated withMbb. sp. 87.7 was calculated from therelat ionship between methane and SCFAs [25]:M ¼ 0:45A� 0:25P þ 0:40B

The potential proportional contribution of reductive ace-togenesis (X) to rumen fermentations was calculated usingthe method of Faichney et al. [24] as follows:

X ¼ 1:8A� 1:1P þ 1:6B� 1:3V � 4Mð Þ= 5:8Aþ 11:6Bð Þ

DNA Extraction

Genomic DNAwas extracted from rumen samples collectedin sampling period 1 and sampling period 2 (Fig. 1) accord-ing to the method of Yu and Morrison [26] with minormodifications for relatively solid samples performed as out-lined by Mosoni et al. [27]. Genomic DNA from Mbb. sp.87.7 was isolated from a 5-day old culture grown on Balchmedium [22] according to the method outlined by Mosoni etal. [27]. DNA concentrations were estimated by absorbanceat 260 nm and DNA integrity was confirmed by agarose gelelectrophoresis [28].

Quantitative Real-Time PCR (qPCR)

The cellulolytic species R. albus, R. flavefaciens and F.succinogenes were quantified in isolated lambs by qPCRaccording to the method of Mosoni et al. [27].

Methanogens from the genera Methanobrevibacter,Methanosphaera and Methanobacterium were quantifiedin samples from isolated lambs by qPCR according to themethod of Ohene-Adjei et al. [29] and using a Mastercy-cler® ep Realplex 2S and Realplex version 2.0 software(Eppendorf, Hamburg, Germany). qPCRs (20 μl reactions)contained 0.3 μM of forward primer MB 1174 F and reverseprimer Arch 1406-1389R [29], 1× IQ SYBR green Super-mix (Bio-Rad, Hercules, California, USA) and 50 to 100-ngtemplate DNA. qPCR cycling was as follows: enzyme acti-vation at 98°C for 30 s, 45 cycles of denaturation at 95°C for15 s, annealing at 63°C for 15 s and extension at 72°C for30 s with fluorescence measured at the end of each cycle,followed by dissociation curve analysis (65–95°C increas-ing at a rate of 0.1°C s−1 with continuous fluorescencemeasurement). Negative controls were run in each assayby omitting DNA template. qPCRs were performed in trip-licate and results were expressed as rrs copies per μg DNA.A standard curve was generated from 103 to 108 copies ofMbb. sp. 87.7 rrs that had been amplified using forwardprimer Arch 344 F, reverse primer Arch 1406-1389R andthe conditions described by Ohene-Adjei et al. [29].

630 E. J. Gagen et al.

Statistical Analyses

In tables, results are given as mean ± standard error. Stu-dent’s t test was used to compare SCFAs as well as numbersof cellulolytic bacteria and numbers of methanogens inisolated lambs pre- and post-inoculation with Mbb. sp.87.7. Differences were considered significant at p<0.05.

Total Bacterial Diversity

Bacterial rrs were amplified in 50 μl reactions containingfinal concentrations 1× PCR buffer (10× buffer stock con-sists of 200 mM Tris–HCl pH 8.4 and 500 mM KCl), 3 mMMgCl2, 0.2 mM dNTPs, 0.14 μM of primers 27f and 1492r[30], 1 U Platinum® Taq DNA Polymerase (Invitrogen,Carlsbad, California, USA) and between 50 and 100 ng oftemplate DNA. PCR conditions were: initial denaturation at95°C for 5 min; 25 cycles of denaturation at 95°C for 15 s,annealing at 55°C for 25 s, extension at 72°C for 2 min; andfinal extension at 72°C for 7 min. For clone library con-struction, PCR products were amplified individually fromextracted DNA samples [n06 from isolated lambs pre-inoculation with methanogens (three samples taken 3 h afterfeeding from two lambs); n06 from isolated lambs post-inoculation with methanogens (three samples taken 3 h afterfeeding from two lambs); n06 from conventional sheep(three samples taken 3 h after feeding from two sheep)],then purified and pooled on an equal concentration basis,according to source. Amplicons were cloned using thepGEM-T Easy ® vector (Promega Corporation, Madison,Wisconsin, USA) and TOP 10 electrocompetent cells(Invitrogen, Carlsbad, California, USA) according tomanufacturer’s instructions. For each library, cloneswere grouped according to restriction fragment lengthpolymorphism (RFLP) pattern after digestion with MboI (Fermentas, Hanover, Maryland, USA) according tothe manufacturer’s instructions followed by visualisation bygel electrophoresis [28]. One clone from every RFLP groupand a second clone for those RFLP groups containing morethan one clone were sequenced with 27f primer using theBigDye ® Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems Inc., Foster City, California, USA) according tomanufacturer’s instructions. Sequences were determined us-ing an ABI3130xl Genetic Analyzer (Applied BiosystemsInc.). Potentially chimeric sequences determined using thechimera detection program at the Ribosomal Database ProjectII [31] and sequences shorter than 500 bp were removed fromfurther analysis. Partial rrs sequences were aligned using theNAST aligner at Greengenes [32] and classified using theHugenholtz taxonomy at the Greengenes database [32].Sequences were grouped into operational taxonomic units(OTUs) using MOTHUR [33] and a distance ≤0.025 in orderto group sequences at an approximate species level [34].

Partial rrs sequences recovered in this study have been sub-mitted to the Genbank database under the accession numbersHQ259770 to HQ259862 (n055 from isolated lambs pre-inoculation with Mbb. sp. 87.7; n047 from isolated lambspost-inoculation withMbb. sp. 87.7; n038 from conventionalsheep).

Functional Gene Analyses

Potential acetogen diversity was assessed in isolated lambspre- and post-inoculation with Mbb. sp. 87.7 and in conven-tional sheep, by analysis of formyltetrahydrofolate synthetasegenes (fhs) and acetyl-CoA synthase genes (acsB). Methano-gen diversity was assessed in the same samples by analysis ofmethyl coenzyme M reductase A genes (mcrA). For libraryconstruction, PCR products were amplified individually fromextracted DNA samples (same samples as those used forexamining bacterial diversity, n06 from isolated lambs pre-inoculation with methanogens; n06 from isolated lambs post-inoculation with methanogens; n06 conventional sheep), thenpurified and pooled on an equal concentration basis, accordingto source. LIBSHUFF inMOTHUR [33] was used to comparethe similarity of libraries from the three sample groups, foreach gene. MOTHUR [33] was also used for clusteringsequences into OTUs and rarefaction analyses.

fhs libraries were constructed using the protocol and pri-mers of Leaphart and Lovell [35]. Deduced amino acidsequences of formyltetrahydrofolate synthetase (FTHFS)were aligned in ARB [36] with publicly available sequencesand grouped into OTUs at a distance of ≤0.025 to clustersimilar sequences while separating those from distinct species[37]. Neighbour joining and maximum likelihood trees wereconstructed as outlined previously [37] using ARB [36] andRaxML [38], respectively, and fhs sequences from namedspecies only were also included to provide the tree framework.Sequences were assessed for similarity to FTHFSs from au-thentic acetogens using the homoacetogen similarity (HS)score of Henderson et al. [39]. acsB libraries were constructedusing the protocol and primers of Gagen et al. [37]. Deducedamino acid sequences of acetyl-CoA synthase (ACS) werealigned in ARB [36] with publicly available sequences andgrouped into OTUs at a distance of ≤0.035 to cluster similarsequences while separating those from distinct species [37].ACS trees were constructed as for FTHFS sequences andsequences from named species were also included to givethe tree framework. mcrA libraries were constructed usingthe primers and protocol of Luton et al. [40]. Deduced mcrAamino acid sequences were aligned in ARB [36] with publiclyavailable mcrA sequences. Trees of deduced amino acidsequences from mcrA were constructed as for FTHFS andACS sequences also including selected mcrA sequences fromnamed species and from one environmental clone (accession:AAL29268) to provide an appropriate tree framework.


Deduced mcrA amino acid sequences were grouped intoOTUs at a distance of ≤0.025. While OTUs for mcrA aminoacid sequences do not strictly correspond to a single species,this distance was chosen to group similar mcrA sequenceswhile separating sequences from the closest distinct species(Methanobrevibacter millerae and Methanobrevibacter gott-schalkii which share 96.5% amino acid identity across theamplified region of mcrA).

Putative fhs sequences recovered in this study have beensubmitted to the Genbank database under the accession numb-ers HQ186422 to HQ186631 (n081 from isolated lambs pre-inoculation with Mbb. sp. 87.7; n063 from isolated lambspost-inoculation withMbb. sp. 87.7; n065 from conventionalsheep). Putative acsB sequences have been deposited at theGenbank under accession HQ186632 to HQ186736 (n034from isolated lambs pre-inoculation withMbb. sp. 87.7; n031from isolated lambs post-inoculation with Mbb. sp. 87.7;n040 from conventional sheep). Putative mcrA sequenceshave been deposited at the Genbank under accession numbersHQ186309 to HQ186421 (n033 from isolated lambs pre-inoculation with Mbb. sp. 87.7; n038 from isolated lambspost-inoculation withMbb. sp. 87.7; n041 from conventionalsheep; n01 from Mbb. sp. 87.7).

Results

Isolated Lamb Model

The lamb model is presented in detail in the “Methods” andthe animal trial is outlined in Fig. 1. Prior to inoculation withcellulolytic species, cellulolytic microorganisms were notdetected in isolated lambs using a MPN approach. Sixtydays after inoculation with cellulolytic strains, cellulolyticbacteria were enumerated at 109 cells ml−1 and total bacteriaat 1011 cells ml−1. Successful establishment of R. albus, R.flavefaciens and F. succinogenes was later confirmed byqPCR (Table 1). Prior to inoculation with Methanobrevi-bacter sp. 87.7, acetogens were enumerated in the twoisolated lambs at 8×107 and 5×108 cells ml−1, respectively,

and methanogens were not detected using an MPN approach.However, methanogens were detected at low levels in isolatedlambs pre-inoculationwithMbb. sp 87.7, using qPCR (Table 1).After inoculation with Mbb. sp. 87.7, methanogens were enu-merated at between 107 and 108 cells ml−1 in isolated lambs bycultural methods, and quantification of 16S rRNA genes (rrs)also showed a significant increase in their numbers (p<0.0001,Table 1). Protozoa and fungi were not detected by phasecontrast microscopy throughout the experiment. After cannu-lation, feed intake stabilised at about 1.2 kg day−1 and SCFAanalysis revealed effective fermentation of alfalfa hay. Nosignificant differences in concentrations of the major SCFAs(acetate, butyrate and propionate) were found before and aftermethanogen inoculation; however, acetate as a proportion oftotal SCFAs increased post methanogen inoculation (Table 2).The Ac/Pr ratio in isolated lambs was lower 3 h after feedingthan before feeding, as previously observed in gnotobioticanimal models [7].

Hydrogen (2H) recovery, calculated from SCFA data asdescribed by Demeyer and Van Nevel [25] was estimated at90% in isolated lambs after inoculation of Mbb. sp. 87.7(Table 2), indicating the effectiveness of the inoculatedmethanogen in capturing hydrogen and suggesting balancedfermentations. The calculated 2H recovery before themethanogen inoculation was found at about 20% (Table 2).Reductive acetogenesis contribution to overall ruminal fer-mentation was estimated using the equation of Faichney etal. [7, 24] and was found at 26% in lambs lacking methaneproduction and was negligible or null post methanogeninoculation.

Total Bacterial Diversity in Isolated Lambsand Conventional Sheep

Partial rrs sequences for bacteria from isolated lambs pre-and post-inoculation withMbb. sp. 87.7 grouped into 28 and32 OTUs, respectively. Both libraries were dominated byFirmicutes sequences (75% of sequences from isolatedlambs pre-inoculation and 84.5% of sequences from isolatedlambs post-inoculation with Mbb. sp. 87.7, Table 3).Sequences from the order Clostridiales comprised the great-est proportion within the Firmicutes and unclassified Clos-tridiales, Ruminococcus and Butyrivibrio were the threelargest groups within this order, in isolated lambs pre- andpost-inoculation with Mbb. sp 87.7. Of the cellulolyticstrains inoculated to isolated lambs, only R. albus wasdetected in rrs libraries and R. albus rrs sequences com-prised 2.1% and 3.7% of total rrs sequences in isolatedlambs pre- and post-inoculation with methanogens respec-tively. Bacteroidetes sequences were the only other phylumdetected in isolated lambs and most Bacteroidetes sequencesoriginated from the Bacteroidaceae, both pre- and post-inoculation with Mbb. sp. 87.7 (Table 3). Partial rrs

Table 1 Log 10 copies of rrs per microgram of DNA for cellulolyticbacteria or methanogens in isolated lambs pre- and post-inoculationwith Mbb. sp. 87.7

Inoculation withMbb. sp. 87.7

F. succinogenes R. flavefaciens R. albus Methanogens

Pre- 3.86±0.14a 4.69±0.34a 7.88±0.04a 3.43±0.15a

Post- 4.39±0.32a 4.94±0.42a 8.09±0.09a 6.02±0.15b

Numbers in the same column followed by different lowercase letterswere significantly different (p<0.0001). The difference for R. albuswas not quite significant (p00.057). For both sampling periods, n012(three samples taken from two lambs both before and after feeding)


sequences from conventional sheep grouped into 33 OTUsand bacterial diversity was much greater than in isolatedlambs (Table 3). There were approximately equal propor-tions of Bacteroidetes and Firmicutes sequences in conven-tional sheep (47.91% and 49.35% of sequences respectively,Table 3) and the two largest sequence groups originatedfrom the Prevotellaceae (30.13% of sequences) and unclas-sified Clostridiales (27.40% of sequences). Proteobacterialrrs sequences were also detected in conventional sheep, at alow frequency (2.74% of sequences).

Potential Acetogen Diversity in Isolated Lambsand Conventional Sheep

Deduced amino acid sequences of fhs from isolated lambspre- and post-inoculation with Mbb. sp. 87.7 grouped intoseven and nine OTUs, respectively. Both libraries shared anOTU comprised of sequences that demonstrated≥98.3%amino acid identity to FTHFS from Eubacterium limosum(OTU 2, Fig. 2 and Online Resource 1). Other OTUs werenovel and clustered near the Lachnospiraceae and demon-strated high (>90%) HS scores (OTUs 3, 4 and 5, Fig. 2 andOnline Resource 1). The largest OTU from isolated lambscontained sequences that probably originated from non-acetogens (≥97.7% amino acid identity to FTHFS from thenon-acetogen Clostridium bolteae and with low (≤65%) HSscores, see OTU 1, Fig. 2 and Online Resource 1). Therewas no significant difference between community structureof fhs libraries (deduced amino acids) from isolated lambspre- and post-inoculation withMbb. sp. 87.7 (significance ofthe ΔCXY score, LIBSHUFF, >0.05).

Deduced amino acid sequences of acsB from isolatedlambs pre- and post-inoculation with Mbb. sp. 87.7 clus-tered into eight and five OTUs, respectively. Commonsequences to the two libraries included ACS identical tothat from E. limosum (OTU 8, Fig. 3 and OnlineResource 2), ACS identical to one copy in Ruminococcusobeum (OTU 3, Fig. 3 and Online Resource 2) and novelsequences that clustered with the Ruminococcaceae/Blau-tia group (OTUs 1, 2, 5 and 7, Fig. 3 and OnlineResource 2). One OTU that affiliated with the Blautiagroup (OTU 1, Fig. 3 and Online Resource 2) and twoOTUs that clustered between the Lachnospiraceae andClostridiaceae (OTU 4 and 6, Fig. 3 and Online Resource 2)were detected in isolated lambs pre-inoculation with Mbb.sp. 87.7 but not in the lambs post-inoculation with Mbb.sp. 87.7. Accordingly, the acsB library (deduced aminoacids) from isolated lambs post-inoculation with Mbb.sp. 87.7 was a subset of the library from the lambs pre-inoculation with Mbb. sp. 87.7.

Conventional sheep housed a much greater diversity ofFTHFS sequences (grouped into 34 OTUs) than isolatedlambs and none of the FTHFS sequences that had beenT

able

2Rum

inal

SCFA

concentrations

andpercentagesof

themainSCFA

sin

isolated

lambs

pre-

andpo

st-ino

culatio

nwith

Mbb.sp.87

.7

Inoculationwith

Mbb.sp.

87.7

Tim

eSCFA

concentration(m

M)

2Hrecovery

(%)

Reductiv

eacetog

enesis

(%)

Total

Acetate

Propion

ate

n-Butyrate

Isob

utyrate

n-Valerate

Isov

alerate

Caproate

Pre-

T0

62.9±9.5a

49.0±7.3a

6.1±1.3a

4.8±0.8a

0.23

±0.02

a1.12

±0.19

a0.25

±0.03

a1.39

±0.21

a20

26(78.1±0.9a)

(9.4±0.6a)

(7.7±0.8a)

Pre-

T3

123.2±3.1x

96.3±2.6x

15.7±0.6x

7.2±0.2x

0.25

±0.02

x1.66

±0.18

x0.17

±0.02

x1.90

±0.21

x22

26(78.2±0.3x)

(12.8±0.3x)

(5.8±0.2x)

Post-

T0

51.1±5.7a

41.6±4.5a

5.0±0.9a

3.2±0.4a

0.27

±0.02

a0.49

±0.08

b0.24

±0.03

a0.34

±0.05

b88

<1%

(81.6±0.5b)

(9.5±0.7a)

(6.3±0.5a)

Post-

T3

131.2±11.0

x10

4.5

±8.7

x16

.4±1.6x

7.7±0.9x

0.36

±0.02

y1.17

±0.13

y0.24

±0.05

x0.91

±0.12

y91

0(79.7±0.4y)

(12.4±0.6x)

(5.8±0.3x)

Num

bersin

thesamecolumnfollo

wed

bydifferentlow

ercase

letters(a/b

forT0samples

orx/yforT3samples)weresign

ificantly

different(p<0.05

).For

each

samplingperiod

andcollectiontim

e,n06(three

samples

takenfrom

twolambs).Percentages

ofthemainSCFA

s(acetate,prop

ionate

andbu

tyrate)to

totalaregivenin

parentheses.Reducingequivalent

(2H)recovery

andpo

tential

redu

ctiveacetog

enesiswerecalculated

from

theSCFA

values

asdescribedin

“Metho

ds”


detected in isolated lambs, were also found in conventionalsheep. Accordingly, community structure of the fhs library(deduced amino acids) from conventional sheep was differ-ent to both those from isolated lambs (significance of theΔCXY score, LIBSHUFF, <0.0001). The largest FTHFSOTU from conventional sheep contained sequences thatprobably originated from non-acetogens as indicated by alow HS score and placement in the lower half of the FTHFStree (OTU 12, Fig. 2 and Online Resource 1). Exceptfor two OTUs with reasonably high HS scores (88–90%) all recovered FTHFS sequences in the lower halfof the FTHFS tree (see Online Resource 1) probablyoriginated from non-acetogens. A diversity of FTHFSsequences from conventional sheep clustered in the tophalf of the FTHFS tree between the Lachnospiraceaeand Clostridiaceae and demonstrated reasonably high(80–90%) and high (>90%) HS scores (see Online Re-source 1) and probably originated from novel acetogens.Rarefaction analyses indicated that there is likely moreFTHFS diversity in conventional sheep to be uncoveredwith deeper sequencing, unlike the isolated lambs.

ACS sequences from conventional sheep clustered into17 OTUs and all sequences were distinct from those foundin cultured species to date. Some distantly affiliated withthe Ruminococcaceae/Blautia group (OTUs 10 and 24, 25,Fig. 3 and Online Resource 2) and the Lachnospiraceae(OTUs 15, 17 and 23, Fig. 3 and Online Resource 2). Asingle sequence clustered with those from acetogens in theEubacteriaceae (OTU 18, Fig. 3 and Online Resource 2)and all other sequences placed phylogenetically betweensequences from acetogens in the Lachnospiraceae andClostridiaceae similarly to placement for novel FTHFSsequences (see Online Resources 1 and 2). None of theACS sequences that had been detected in isolated lambswere also found in conventional sheep and communitystructure of the ascB library (deduced amino acids) fromconventional sheep was significantly different to bothlibraries from isolated lambs (significance of the ΔCXY

score, LIBSHUFF, <0.0001). Rarefaction analyses indicat-ed that there is likely more ACS diversity in conventionalsheep to be uncovered with deeper sequencing, unlike theisolated lambs.

Table 3 Broad level total bac-terial diversity in isolated lambsand conventional sheep

Partial rrs sequences were clas-sified according to the Hugen-holtz classification at theGreengenes database [32] andproportions determined based onRFLP pattern of all clones ineach library

Percentage of clones in each library

Phylum Family level classification Isolated lambspre-inoculationwith methanogens

Isolated lambspost-inoculationwith methanogens

Conventionallyraised sheep

Bacteroidetes Bacteroidaceae 19.6 16.0 1.4

Bacteroidetes Barnesiella 3.1 0.0 0.0

Bacteroidetes Parabacteroidaceae 2.1 0.0 0.0

Bacteroidetes Prevotellaceae 0.0 0.0 30.1

Bacteroidetes Rikenellaceae 0.0 0.0 4.1

Bacteroidetes Tannerellaceae 0.0 0.0 4.1

Bacteroidetes Uncl. Bacteroidetes 0.0 0.0 8.2

Firmicutes Ruminococcus 27.8 30.9 1.4

Firmicutes Acholeplasmatales 0.0 0.0 1.4

Firmicutes Acidominococcaceae 5.2 8.6 5.5

Firmicutes Butyrivibrio 11.3 18.5 0.0

Firmicutes Catenibacterium 1.0 0.0 0.0

Firmicutes Clostridiaceae 0.0 0.0 2.7

Firmicutes Desulfotomaculum 1.0 3.7 0.0

Firmicutes Enterococcus 1.0 3.7 0.0

Firmicutes Faecalibacterium 0.0 3.7 1.4

Firmicutes Johnsonella 8.2 1.2 0.0

Firmicutes Lachnospiraceae 0.0 0.0 1.4

Firmicutes Peptostreptococcacea 0.0 0.0 5.5

Firmicutes Streptococcaceae 2.1 1.2 0.0

Firmicutes Uncl.Clostridiales 16.5 12.3 27.4

Firmicutes Uncl. Lactobacillales 1.0 0.0 0.0

Firmicutes Uncl. Mollicutes 0.0 0.0 2.7

Proteobacteria Desulfovibrionaceae 0.0 0.0 1.4

Proteobacteria Roseospira 0.0 0.0 1.4


Diversity of Methanogens in Isolated Lambsand Conventional Sheep

There was a broad diversity of deduced mcrA amino acidsequences (15 OTUs) present in isolated lambs pre-inoculation withMbb. sp. 87.7. Sequences in one OTU wereidentical to mcrA from Mbb. sp. 87.7 (OTU 11, Fig. 4 andOnline Resource 3) and those in three other OTUs (OTU 9,OTU 8 and OTU 6, Fig. 4) showed >97.5% amino acididentity to mcrA from M. millerae. Other mcrA genesdetected in isolated lambs were distinct from those foundin cultured methanogens to date, though most affiliated withthe genusMethanobrevibacter (see Online Resource 3). OneOTU clustered with sequences from a deep-branching noveluncultured methanogen lineage that demonstrates low(<65%) mcrA amino acid identity to mcrA of methanogensin other orders (OTU 5 Fig. 4 and Online Resource 3). Twoother OTUs distantly affiliated with the mrtA-like mcrA

gene of Methanosphaera stadtmanae (OTUs 7 and 10,Fig. 4 and Online Resource 3). The diversity of mcrAgenes recovered from isolated lambs post-inoculationwith Mbb. sp. 87.7 was much more restricted (fiveOTUs) and all sequences affiliated with the Methano-brevibacter genus. Almost all sequences in the largestOTU from isolated lambs post-inoculation with Mbb. sp.87.7 were identical at the amino acid level to mcrA ofthe inoculated methanogen (OTU 11, Fig. 4 and OnlineResource 3) though five sequences in this OTU showedslightly lower amino acid identity (98.4% and 99.2%) tothe inoculated strain. The mcrA library (deduced aminoacids) from isolated lambs post-inoculation with Mbb.sp. 87.7 was a subset of the library from the lambs pre-inoculation with Mbb. sp. 87.7. Relative to total mcrAsequences, the percentage of sequences in OTU 11,corresponding to the mcrA from Mbb. sp 87.7, increasedfrom 6% in isolated lambs pre-inoculation to 80% in

0102030405060708091011121314151617181920212223242526272829303132333435363738394041424344

0102030405060708091011121314151617181920212223242526272829303132333435363738394041424344

OTU OTU

A: Pre-inoculation with methanogens

B: Post-inoculation with methanogens

C: Conventionally raised sheep

65100 **98 **93 **100 **5690 **5893 **88 **90 **558590 **6651606353 93 **73 **636088 **93 **95 **84 **95 **80 **91 **76 **5585 **9085 **605890 **6395 **73 **90 **8860

HS score

Figure 2 Heatmap analysis of deduced FTHFS amino acid sequencesfrom isolated lambs and conventional sheep. Analysis performed forOTUs at ≤0.025 for sequences from; A: isolated lambs pre-inoculationwith Mbb. sp. 87.7; B: isolated lambs post-inoculation with Mbb. sp.87.7; and C: conventional sheep. The scale bar indicates relative

abundance of each OTU within a sample group. OTUs have beenarbitrarily numbered. Average HS scores for OTUs have been indicatedand asterisks indicate OTUs that also place phylogenetically in the tophalf of the FTHFS tree (refer to Online Resource 1)


isolated lambs post-inoculation. This result indicatedthat the 1,000-fold increase in methanogen rrs copynumber post-inoculation was due to the colonization ofthe ecosystem by strain 87.7 (see Fig. 4 and OnlineResource 3).

mcrA sequences from conventional sheep (17 OTUs)affiliated mainly with the Methanobrevibacter thoughOTUs that affiliated with the deep-branching novel un-cultured methanogen lineage (OTUs 5, 20, 21, 22,Fig. 4) and with the mrtA-like gene of Msp. stadtmanaewere also detected (see Online Resource 3). Communitystructure of the mcrA library (deduced amino acids) fromconventional sheep was not significantly different to thatfrom isolated lambs pre-inoculation with Mbb. sp. 87.7(significance of the ΔCXY score, LIBSHUFF, >0.05).Rarefaction analysis indicated that there may be furthermethanogen diversity in both conventional sheep andisolated lambs (pre-inoculation with Mbb. sp 87.7.) tobe uncovered with deeper sequencing.

Discussion

This study is the first reported molecular diversity analysis ofrumen microorganisms in lambs raised aseptically (lambs iso-lated 17 h after birth) and it reveals aspects of this animalmodel [7, 9–11, 14, 41] and rumen colonisation that have notpreviously been revealed using cultivation techniques alone.

Firstly, an unexpected finding of this study was the presence ofa low-abundant but diverse population of methanogens in thedeveloping rumen of lambs that had been isolated at 17 h. Theearliest reported age at which methanogens have been found inthe rumen is 30 h [2], though it is generally accepted thatmethanogens colonize the developing rumen (both bovineand ovine) 3 to 4 days after birth and establish at numberssimilar to those in adults, from the first week onwards [9, 14,42]. All previous observations about methanogen colonizationin the rumen have been made using cultivation techniques todetermine the presence or absence of hydrogenotrophic metha-nogens or methane production in rumen samples from veryyoung lambs [2, 14, 42]. Using cultivation-based techniques inthe present study, methanogens were also not detected inisolated lambs. However, cultivation-independent techniques,which are more sensitive than cultivation-based techniques,revealed their presence.

Although methanogens were present at quite low numb-ers (<104 rrs copies per microgram of DNA) the diversity ofmcrA sequences in these lambs was not significantly dis-similar to that found in the mature rumen of conventionalsheep and consisted of predominantly Methanobrevibacterspp., in agreement with previous reports for the ovine rumen[43–45]. That methanogen diversity was not greatly differ-ent in the rumen of lambs isolated at 17 h and the rumen of2-year old conventional sheep is particularly interestingbecause the bacterial population present in the rumen froma very young age is different to that found in the rumen of

01020304050607080910111213141516171819202122232425

OTU01020304050607080910111213141516171819202122232425

OTUB/RB/RB/RUB/RUB/REubUB/RUUUULachULachEubUUUULachB/RB/R

Classification




Figure 3 Heatmap analysis of deduced ACS amino acid sequencesfrom isolated lambs and conventional sheep. Analysis performed forOTUs at ≤0.035 for sequences from: A: isolated lambs pre-inoculationwith Mbb. sp. 87.7; B: isolated lambs post-inoculation with Mbb. sp.87.7; and C: conventional sheep. The scale bar indicates relative abun-dance of each OTU within a sample group. OTUs have been arbitrarily

numbered. Family level classifications of sequences based on phyloge-netic placement (refer to Online Resource 2) are: B/R: Blautia/Rumino-coccaceae group; Lach: Lachnospiraceae; U: placing between theBlautia/Ruminococcaceae and Lachnospiraceae groups; Eub:Eubacteriaceae


older animals [14], a finding confirmed in this study withbroad-level bacterial rrs analyses (Table 3), and at 17 h therumen is also devoid of fungi and protozoa. Perhaps, unlikethe bacterial population, methanogens that are acquired byruminants from a very young age are maintained throughoutrumen development and life. A similar hypothesis has beensuggested by Skillman et al. [44] who reported that thepredominant methanogens in mature ruminants establishedearly, before rumen development. However, these resultsrequire confirmation with a wider sampling range in futurestudies, to eliminate the potential effect of the host onmicrobial community composition [46].

As methanogens resident in the rumen prior to 24 h ofage are present at low numbers and could not be detected bycultivation methods in this and other studies [2, 9, 11], it isprobable that they are uncultivable using selective mediumand H2/CO2 as substrates for growth. Perhaps some of theseearly rumen methanogens have particular nutritionalrequirements that are not facilitated by these culture con-ditions, for example, utilisation of alternative substrates (e.g.methyl compounds) for heterotrophic growth [47], or theymay exhibit an interspecies dependence which preventscultivation. Methanogen have been shown to develop sym-biotic relationships with bacteria, fungi and protozoa, andtheir associations with protozoa has been particularly stud-ied (see review: [45]). These findings could also relate to thematuration state of the rumen as it has been previously

reported that hydrogenotrophic capacity of the ruminalmicrobiota increases with animal age for both conventionaland gnotobiotically reared lambs, suggesting that this func-tion is affected by the maturation of the ecosystem andpossibly also the physiology of the host [7]. The establish-ment of the inoculated methanogenic strain, Mbb. sp. 87.7,at a high density (>106 rrs copies per microgram of DNA)outcompeting the already present methanogens, indicatesthat this strain finds all that it needs to be active and develop(e.g. nutritional requirements, microbial interactions) in therumen microbiota acquired soon after birth. However, asfungi and protozoa were not observed in the rumen of ouranimal model, interactions with these microorganisms isprobably not involved. While early colonising methanogensrequire further investigation, the present findings affirm theneed for use of molecular techniques in future rumen colo-nisation studies, particularly for studies investigating‘methanogen-free’ models of the rumen.

Broad level bacterial diversity of isolated lambs inthis study was only slightly altered after inoculationwith Mbb. sp 87.7, and rrs libraries from the lambsboth before and after inoculation with methanogenswere clearly different to that from conventional animals(Table 3). At a high taxonomic level, the bacterialdiversity observed for conventional sheep in this studyis comparable to that described for hay-fed cattle [48]and from ruminal microbiomes [49].

01020304050607080910111213141516171819202122

01020304050607080910111213141516171819202122




OTU OTUMbbMbbMbbMbbuncMbbMspMbbMbbMspMbb*MbbMbbMbbMbbMbbMspMbbMbbuncuncunc

Classification

Figure 4 Heatmap analysis of deduced mcrA amino acid sequencesfrom isolated lambs and conventional sheep. Analysis performed forOTUs at ≤0.025 for sequences from: A: isolated lambs pre-inoculationwith Mbb. sp. 87.7; B: isolated lambs post-inoculation with Mbb. sp.87.7; and C: conventional sheep. The scale bar indicates relative

abundance of each OTU within a sample group. OTUs have beenarbitrarily numbered. Classification of sequences based on phyloge-netic placement (refer to Online Resource 3) are as follows: Mbb:Methanobrevibacter spp.; Mbb*: Mbb. sp 87.7; Msp: Methanosphaeraspp.; Unc: deep branching uncultured Archaea


Potential acetogens identified in isolated lambs were E.limosum, R. obeum and novel species that affiliated with theBlautia group and the Lachnospiraceae. The novel acsBsequences that affiliated between the Lachnospiraceae andClostridiaceae in isolated lambs pre-inoculation with Mbb.sp. 87.7 were not detected in the lambs post-inoculation withMbb. sp. 87.7. However, this observation was not reflected infhs analyses and could indicate incomplete sequencing cover-age of the acsB library from isolated lambs post-inoculationwith Mbb. sp. 87.7. Almost all detected acetogen functionalgene sequences (acsB and fhs) were common to isolatedlambs pre- and post-inoculation withMbb. sp. 87.7 indicatingthat the diversity of potential acetogen species present in therumen of young lambs are unaffected by colonisation of amethanogen at a high level (qPCR and mcrA diversity analy-ses indicated successful establishment of Mbb. sp 87.7 and a1,000-fold increase in methanogen numbers post-inoculationwith Mbb. sp 87.7). However, data calculated from SCFAssuggested that reductive acetogenesis accounted for only 26%of hydrogen capture before inoculation with Mbb. sp 87.7inoculation (Table 2). These data are similar to those foundpreviously for meroxenic lambs lacking methanogens [7] or inyoung lambs with a weak methane-producing microbiota[24]. They show that acetogenesis is much less efficient thanmethanogenesis as a hydrogen sink in this animal model.After Mbb. sp 87.7 establishment, hydrogen recovery wasclose to the expected value of 90% [24, 25] indicating afunctional optimal methanogenesis; then, reductive acetogen-esis was not significant, as found previously in conventionaland meroxenic methanogenic rumens [7, 24, 50]. BeforeMbb.sp. 87.7 establishment, hydrogen recovery was calculated atabout 20%, which is very low and much less than the 90%expected on the basis of the ‘normal’ fermentation stoichiom-etry. This indicates that the equation used for the calculationdoes not correspond to the real stoichiometry in the rumen,and suggests that H2 probably accumulates in the ecosystem,or is disposed through acetogenesis or other unknown sinks.Acetogens are metabolically versatile and can readily utilize arange of substrates [5, 51] so potentially, heterotrophic metab-olism enabled the putative acetogens present before methano-gen inoculation to persist in the rumen in the presence ofMbb.sp. 87.7. Perhaps, even before methanogen inoculation theseacetogens may have been consuming hydrogen during mixo-trophic growth. Mixotrophic growth is energetically morefavourable than autotrophic growth and more favourable thanmethanogenesis in some cases [52, 53].

A possibility that could not be explored in this study waswhether potential acetogen numbers diminished after estab-lishment of Mbb. sp. 87.7 in the rumen. An assay (fhs1) forquantifying acetogens from environmental samples has beenpublished [54] however as well as compromised specificity[37], the fhs1 assay primers include fhs sequences from non-acetogens in their target range. Because very similar

FTHFSs are found in acetogens and non-acetogens, qPCRfor all acetogens exclusively, based on fhs, is not possible[39]. An alternative quantitative assay for the acetogens isurgently needed for future investigations into interactionsbetween hydrogenotrophic populations in the rumen.

Conventional sheep hosted a different, novel potential ace-togen population to that in isolated lambs and most acetogenfunctional genes from conventional sheep affiliated broadlybetween the Lachnospiraceae and Clostridiaceae. HS scoresfor FTHFS sequences in that group were high (most >90%)and placed phylogenetically similarly to ACS sequences fromthe same samples, supporting the likelihood that they origi-nated from acetogens. Identical and similarly placed novelfunctional genes have been found in other ruminants and inthe tammar wallaby forestomach previously [37, 39, 55]which makes this potential acetogen group of even moreinterest. Obtaining and characterizing isolates from this un-known group will be important to shed light on their activityin the rumen. None of the acetogen functional genes present inisolated lambs were also found in conventional sheep whichmay indicate that early potential acetogens are adverselyaffected, either directly or indirectly, by microbial species thatcolonise the rumen later than 17 h after birth, including fungiand protozoa. Hydrogen utilization threshold is higher inmethanogens than in acetogens and methanogenesis is morethermodynamically favourable than aetogenesis [56, 57]. Inaddition, acetogens are able to grow on substrates other thanH2/CO2 such asmonosaccharides [51]. In consequence, a highabundance of methanogens might direct the metabolism ofacetogenic species towards heterotrophy. Hence, their putativeecological niches might be more autotrophic than heterotro-phic in young animals before methanogen establishment, andthe reverse in adults [1]. Both biotic and abiotic factors areinvolved in the maturation of the rumen ecosystem, includingflow rate of digesta, redox potential, pH, epithelial secretionsand diet. However, microbial interactions are probably themajor factor shaping the successive species colonisation ofthe rumen in combination with changing abiotic factors in thegut associated with the maturation of the animal. Future workto monitor a wider range of microbial populations usingmolecular techniques during the course of rumen develop-ment may shed light on the factors that affect early rumenacetogens and methanogens.

Conclusions

This study is the first molecular diversity analysis of putativeacetogen andmethanogen populations in isolated lambs raisedaseptically. It provides a new insight into the microbial diver-sity of the early developing rumen and into lambs isolatedsoon after natural birth and thereafter raised aseptically—amodel which has been used widely in rumen colonisation


studies.Methanogens were present in lambs isolated 17 h afterbirth, though were undetectable using traditional cultivationtechniques. Methanogen numbers were low in these lambs(<104 rrs copies per microgram of DNA) however mcrAdiversity was not dissimilar to that found in 2-year-old con-ventional sheep. This suggests that early colonising methano-gens may persist in the rumen. However, a wider samplingrange of animals is required to confirm this result. In contrastto the observation for methanogens, total bacterial diversityand potential acetogen diversity in isolated lambs and con-ventional sheep were significantly different and the establish-ment of the methanogenic strain Mbb. sp 87.7 in isolatedlambs (1,000-fold increase in methanogen numbers) did notsubstantially affect potential acetogen diversity. These find-ings indicate that in the early rumen, diversity of the potentialacetogen population is unaffected by methanogen numbersalone. Presumably factors relating to microorganisms thatcolonise the rumen after 17 h affect acetogens, resulting in adifferent potential acetogen population in the mature rumen.Urgent work is needed to develop a molecular, quantitativemonitoring tool for rumen acetogens to better resolve thefactors that affect the acetogen population changes duringrumen development. Continued rumen colonisation studiesusing molecular analyses and omics global approaches arenecessary to refine our understanding of the ecology of hydro-genotrophs in the developing rumen in order to identify keyfactors that lead to the prevalence of methanogens in themature rumen.

Acknowledgements We are very grateful to GérardVert andChristophede Martrin (Unit of Microbiology, INRA Clermont-Ferrand/Theix) for therearing of lambs and rumen sampling, and to Pascale Lepercq, Rémy Rouxand Gérard Andant for technical assistance. This work was partly fundedby the French and Australian governments, through the French–AustralianScience and Technology (FAST) program. EmmaGagen was a recipient ofscholarships from The University of Queensland and CSIRO LivestockIndustries.

References

1. Morvan B, Bonnemoy F, Fonty G, Gouet P (1996) Quantitativedetermination of H2-utilizing acetogenic and sulfate-reducing bac-teria and methanogenic Archaea from digestive tract of differentmammals. Curr Microbiol 32:129–133

2. Morvan B, Dore J, Rieu-Lesme F, Foucat L, Fonty G, Gouet P(1994) Establishment of hydrogen-utilizing bacteria in the rumenof the newborn lamb. FEMS Microbiol Lett 117:249–256

3. Morvan B, Rieu-Lesme F, Fonty G, Gouet P (1996) In vitrointeractions between rumen H2-producing cellulolytic microorgan-isms and H2-utilizing acetogenic and sulfate-reducing bacteria.Anaerobe 2:175–180

4. Nollet L, Demeyer D, Verstraete W (1997) Effect of 2-bromoethansulfonic acid and Peptostreptococcus productus ATCC35244 addition on stimulation of reductive acetogensis in theruminal ecosystem by selective inhibition of methanogenesis.Appl Environ Microbiol 63:194–200

5. Joblin KN (1999) Ruminal acetogens and their potential to lowerruminant methane emissions. Aust J Agric Res 50:1307–1313

6. Olsson K, Evans P, Joblin KN (2006) Ruminal acetogens in dairycows: dietary effects and quantification of E. limosum. ReprodNutr Devel 46(suppl 1):S109

7. Fonty G, Joblin KN, Chavarot M, Roux R, Naylor GE, Michallon F(2007) Methanogen-free lambs: establishment and development ofruminal hydrogenotrophs. Appl Environ Microbiol 73:6391–6403

8. Fonty G, Gouet P, Nebout JM (1989) Development of the cellulo-lytic microflora in the rumen of lambs transferred into sterileisolators a few days after birth. Can J Microbiol 35:416–422

9. Fonty G, Williams AG, Bonnemoy F, Morvan B, Withers SE,Gouet P (1997) Effect of Methanobrevibacter sp MF1 inoculationon glycoside hydrolase and polysaccharide depolymerase activi-ties, wheat straw degradation and volatile fatty acid concentrationsin the rumen of gnotobiotically-reared lambs. Anaerobe 3:383–389

10. Chaucheyras-Durand F, Fonty G (2001) Establishment of cellulo-lytic bacteria and development of fermentative activities in therumen of gnotobiotically-reared lambs receiving the microbialadditive Saccharomyces cerevisiae CNCM I-1077. Reprod NutrDev 41:57–68

11. Chaucheyras-Durand F, Masseglia S, Fonty G, Forano E (2010)Influence of the composition of the cellulolytic flora on the devel-opment of hydrogenotrophic microorganisms, hydrogen utilisationand methane production in the rumen of gnotobiotically-rearedlambs. Appl Environ Microbiol 76:7931–7937

12. Williams AG, Joblin KN, Fonty G (1994) Interactions between therumen chytrid fungi and other microorganisms. In: Mountford DO,Orpin CG (eds) Anaerobic Fungi: Biology, Ecology, and Function.Marcel Dekker, Inc, New York

13. Hobson PN, Fonty G (1997) Biological models of the rumenfunction. In: Hobson PN, Stewart CS (eds) The Rumen MicrobialEcosystem. Blackie Academic & Professional, London, pp 661–684

14. Fonty G, Gouet P, Jouany J-P, Senaud J (1987) Establishment ofthe microflora and anaerobic fungi in the rumen of lambs. J GenMicrobiol 133:1835–1843

15. Fonty G, Gouet P, Jouany JP, Senaud J (1983) Ecological factorsdetermining establishment of cellulolytic bacteria and protozoa inthe rumens of meroxenic lambs. J Gen Microbiol 129:213–223

16. Bera-Maillet C, Mosoni P, Kwasiborski A, Suau F, Ribot Y, ForanoE (2009) Development of a RT-qPCR method for the quantifica-tion of Fibrobacter succinogenes S85 glycoside hydrolase tran-scripts in the rumen content of gnotobiotic and conventional sheep.J Microbiol Methods 77:8–16

17. Raskin L, Stromley JM, Rittmann BE, Stahl DA (1994) Group-specific 16S rRNA hybridization probes to describe natural com-munities of methanogens. Appl Environ Microbiol 60:1232–1240

18. Ducluzeau R, Raibaud P (1979) Ecologie Microbienne du TubeDigestif. Masson, Paris

19. Brossard L, Martin C, Chaucheyras-Durand F, Michalet-Doreau B(2004) Protozoa involved in butyric rather than lactic fermentativepattern during latent acidosis in sheep. Reprod Nutr Dev 44:195–206

20. Halliwell G, Bryant MP (1963) The cellulolytic activity of purestrains of bacteria from the rumen of cattle. J Gen Microbiol32:441–448

21. Greening RC, Leedle JAZ (1989) Enrichment and isolation ofAcetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteriafrom the bovine rumen. Arch Microbiol 151:399–406

22. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe RS (1979)Methanogens: Reevaluation of a unique biological group. Micro-biol Rev 43:260–296

23. Clarke KR, Owens NJP (1983) A simple and versatile micro-computer program for the determination of 'most probable num-ber'. J Microbiol Meth 1:133–137


24. Faichney GJ, Graham NM, Walker DM (1999) Rumen character-istics, methane emissions and digestion in weaned lambs reared inisolation. Aust J Agric Res 50:1083–1089

25. Demeyer DI, Van Nevel CJ (1975) Methanogenesis, an integratedpart of carbohydrate fermentation and its control. In: McDonaldIW, Warner ACI (eds) Digestion and metabolism in the ruminant.The University of New England Publishing Unit, Armidale,Australia

26. Yu Z, Morrison M (2004) Improved extraction of PCR-qualitycommunity DNA from digest and fecal samples. Biotechniques36:808–812

27. Mosoni P, Martin C, Forano E, Morgavi DP (2011) Long-termdefaunation increases the abundance of cellulolytic ruminococciand methanogens but does not affect the bacterial and methanogendiversity in the rumen of sheep. J Anim Sci 89:783–791

28. Sambrook J, Russell DW (2001) Molecular cloning: A laboratorymanual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

29. Ohene-Adjei S, Teather RM, Ivan M, Forster RJ (2007) Postinoc-ulation protozoan establishment and association patterns of meth-anogenic archaea in the ovine rumen. Appl Environ Microb73:4609–4618

30. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E,Goodfellow M (eds) Nucleic Acid Techniques in Bacterial Sys-tematics. Wiley, London

31. Cole JR, Chai B, Marsh TL et al (2003) The Ribosomal DatabaseProject (RDP-II) previewing a new autoaligner that allows regularupdates and the new prokaryotic taxonomy. Nucl Acids Res31:442–443

32. DeSantis TZ, Hugenholtz P, Larsen N et al (2006) Greengenes, achimera-checked 16S rRNA gene database and workbench com-patible with ARB. Appl Environ Microbiol 72:5069–5072

33. Schloss PD, Westcott SL, Ryabin T et al (2009) Introducingmothur: open-source, platform-independent, community-supported software for describing and comparing microbial com-munities. Appl Environ Microbiol 75:7537–7541

34. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place forDNA-DNA reassociation and 16S rRNA sequence analysis in thepresent species definition in bacteriology. Int J Syst Bacteriol44:846–849

35. Leaphart AB, Lovell CR (2001) Recovery and analysis of formyl-tetrahydrofolate synthetase gene sequences from natural popula-tions of acetogenic bacteria. Appl Environ Microbiol 67:1392–1395

36. Ludwig W, Strunk O, Westram R et al (2004) ARB: a softwareenvironment for sequence data. Nucleic Acids Res 32:1363–1371

37. Gagen EJ, Denman SE, Padmanabha J, Zadbuke S, Al Jassim R,Morrison M, McSweeney CS (2010) Functional gene analysissuggests different acetogen populations in the bovine rumen andtammar wallaby forestomach. Appl Environ Microbiol 76:7785–7795

38. Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixedmodels. Bioinformatics 22:2688–2690

39. Henderson G, Naylor GE, Leahy SC, Janssen PH (2010)Presence of novel, potentially homoacetogenic bacteria in therumen as determined by analysis of formyltetrahydrofolatesynthetase sequences from ruminants. Appl Environ Microbiol76:2058–2066

40. Luton PE, Wayne JM, Sharp RJ, Riley PW (2002) The mcrA geneas an alternative to 16S rRNA in the phylogenetic analysis ofmethanogen populations in landfill. Microbiology 148:3521–3530

41. Lysons RJ, Alexander TJL, Wellstead PD (1976) Defined bacterialpopulations in the rumens of gnotobiotic lambs 94:257-269

42. Anderson KL, Nagaraja TG, Morrill JL, Avery TB, Galitzer SJ,Boyer JE (1987) Ruminal microbial development in convention-ally or early-weaned calves. J Anim Sci 64:1215–1226

43. Wright A-DG, Williams AJ, Winder B, Christophersen CT, RodgersSL, Smith KD (2004) Molecular diversity of rumen methanogensfrom sheep in Western Australia. Appl Environ Microb 70:1263–1270

44. Skillman LC, Evans PN, Naylor GE, Morvan B, Jarvis GN, JoblinKN (2004) 16S ribosomal DNA-directed PCR primers for ruminalmethanogens and identification of methanogens colonising younglambs. Anaerobe 10:277–285

45. Janssen PH, Kirs M (2008) Structure of the Archaeal communityof the rumen. Appl Environ Microbiol 74:3619–3625

46. Weimer PJ, Stevenson DM, Mantovani HC, Man SLC (2010) Hostspecificity of the ruminal bacterial comunity in the dairy cow followingnear-total exchange of ruminal contents. J Dairy Sci 93:5902–5912

47. Whitman WB, Bowen TL, Boone DR (2006) The methanogenicbacteria. In: Dworkin M, Stanley F, Rosenberg E, Schleifer K-H,Stackebrandt E (eds) The Prokaryotes: A handbook on the biologyof bacteria: Archaea. Bacteria: Firmicutes, Actinomycetes. Springer,New York

48. Pitta DW, Pinchak WE, Dowd SE et al (2010) Rumen bacterialdiversity dynamics associated with changing from bermudagrasshay to grazed winter wheat diets. Microb Ecol 59:511–522

49. Kim M, Morrison M, Zhongtang Y (2011) Status of the phyloge-netic diversity census of ruminal microbiomes. FEMS MicrobiolEcol 76:49–63

50. Le Van TD, Robinson JA, Ralph J, Greening RC, Smolenski WJ,Leedle JAZ, Schaefer DM (1998) Assessment of reductive aceto-genesis with indigenous ruminal bacterium populations and Aceti-tomaculum ruminis. Appl Environ Microbiol 64:3429–3436

51. Drake HL, Gößner AS, Daniel SL (2008) Old acetogens, newlight. Ann N YAcad Sci 1125:100–128

52. Breznak JA (1994) Acetogenesis from carbon dioxide in termiteguts. In: Drake HL (ed) Acetogenesis. Chapman & Hall, NewYork, pp 303–330

53. Breznak JA, Blum JS (1991) Mixotrophy in the termite gut ace-togen, Sporomusa termitida. Arch Microbiol 156:105–110

54. Xu K, Liu H, Du G, Chen J (2009) Real-time PCR assays targetingformyltetrahydrofolate synthetase gene to enumerate acetogens innatural and engineered environments. Anaerobe 15:204–213

55. Matsui H, Kojima N, Tajima K (2008) Diversity of the formylte-trahydrofolate synthetase gene (fhs) a key enzyme for reductiveacetogenesis, in the bovine rumen. Biosci Biotechnol Biochem72:3273–3276

56. Cord-Ruwisch R, Seitz H, Conrad R (1988) The capacity of hydro-genotrophic anaerobic bacteria to compete for traces of hydrogendepends on the redox potential of the terminal electron acceptor.Arch Microbiol 149:350–357

57. Ellis JL, Dijkstra J, Kebreab E, Bannink A, Odongo NE, McBrideBW, France J (2008) Aspects of rumen microbiology central tomechanistic modelling of methane production in cattle. J Agri Sci146:213–233


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Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17 h After Birth and Raised Aseptically