Académique Documents
Professionnel Documents
Culture Documents
Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe
Centre for Plant Genetics and Breeding, School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
School of Animal Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
d
Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
e
Department of Agriculture and Food Western Australia, Livestock Industries, 3 Baron-Hay Court, South Perth, WA 6151, Australia
f
CSIRO Livestock Industries, Queensland Bioscience Precinct, St Lucia, Qld, Australia
g
School of Chemistry and Biochemistry, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 November 2015
Received in revised form
4 April 2016
Accepted 5 April 2016
Available online 7 April 2016
Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In
ruminants, they are responsible for producing abundant amounts of methane during digestion of food,
but selected bioactive plants and compounds may inhibit this activity. Recently, we have identied that,
Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specic antimethanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro
fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against ve species of methanogens grown in pure culture. Fraction bioactivity
was assessed by measuring methane production and amplication of the methanogen mcrA gene.
Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to
distinguish between static and cidal effects. All four fractions were active against pure cultures, but the
F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four
species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions
had selective activity affecting only some methanogens, or reducing either methane production or
methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to
compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen
methanogens. Further study is required to identify the specic plant compounds in biserrula that are
responsible for the anti-methanogenic activity. These ndings will help devise novel strategies to control
methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse
gas emissions from ruminants.
Crown Copyright 2016 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Methanogenic archaea (methanogens) are phylogenetically
distinct group of very strict anaerobes, with a metabolism that
relies on CO2, H2 and methylated compounds as sole energy source,
* Corresponding author. Centre for Plant Genetics and Breeding, School of Plant
Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA
6009, Australia.
E-mail address: bidhyut.banik@research.uwa.edu.au (B.K. Banik).
http://dx.doi.org/10.1016/j.anaerobe.2016.04.004
1075-9964/Crown Copyright 2016 Published by Elsevier Ltd. All rights reserved.
174
November 2013 during the vegetative growth stage. Leaf and stem
material was cut 2 cm from the ground and randomly collected
from the area. Plant samples were pooled, freeze-dried and ground
to pass a 1 mm sieve. Material was stored at room temperature
(22 C) in sealed containers until analysis.
2.3. Plant extracts and fractions
A modied version of plant extraction [25e28] was used with
ve different solvents e water (100% v/v water), methanol (80% v/v
methanol/water), ethanol (70% v/v ethanol/water), acetone (100%
acetone) and methanol/chloroform (50%: 50% methanol: chloroform) to obtain the crude extracts from biserrula [29]. The extract
with the highest activity, i.e. 50%: 50% methanol/chloroform (Fig. 2)
was subjected to further fractionation using silica gel chromatography. Separation was achieved using a column (30 1.5 cm)
containing silica gel (15 g, Davisil silica, LC60A) (Grace-Davison,
Columbia, USA) equilibrated with hexanes. The column was eluted
with hexanes (100 mL) followed by solvent mixtures containing
increasing concentrations of ethyl acetate in petrol to 100% ethyl
acetate, followed by increasing amounts of methanol in ethyl acetate. This method afforded 10 fractions as shown in Table 1, which
were then tested in the IVFT. Fractions were also examined by high
performance liquid chromatography (HPLC). Briey, a sub-sample
of each fraction (ca. 10%) was taken for the bioassay and evaporated under a stream of N2 (gas) to dryness. A sample of each
fraction (~1 mg/mL in methanol) was analysed by HPLC using an
Agilent 1200 HPLC with a photodiode array detector. Separation
was achieved using an Apollo C18 reversed-phase column
(250 4.6 mm i.d., 5 mm) (Grace-Davison, Columbia, USA) with a
33 mm 7 mm guard column of the same material. The column
was eluted at 1 mL/min with 1% acetonitrile/water increasing to
100% acetonitrile/water over 30 min, and held for 5 min. Finally, the
most active fractions were analysed by nuclear magnetic resonance
(NMR) spectroscopy using Bruker ARX-600 (Bruker, Alexandria,
NSW, Australia) and assessed by using UV absorbance measured at
wavelengths of 220, 254 and 280 nm.
2.4. In vitro fermentation technique (IVFT)
Plant extracts and fractions were tested in an in vitro batch
fermentation system modied for testing of plant extracts [30].
Briey, aliquots (100 mL) of the extracts or fractions (dissolved in
70% v/v ethanol/water) were mixed with 10 mL of buffered rumen
uid and 0.1 g oaten chaff as substrate transferred to an anaerobic
chamber (Coy Vinyl Anaerobic Chamber; Coy Laboratory Products
Inc., Grace Lake, MI, USA) maintained at 39 C and supplied with
80% N2, 10% CO2 and 10% H2, to remove oxygen from the incubation.
Table 1
Yields (mg) from silica gel fractionation of biserrula crude extract methanol: chloroform (50%: 50%). EtOAc, ethyl acetate; MeOH, methanol.
Fraction
Elution solvent
Yield (mg)
Initial extract
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
Recovered
100% Petrol
20% EtOAc: Petrol
20% EtOAc: Petrol
40% EtOAc: Petrol
60% EtOAc: Petrol
80% EtOAc: Petrol
100% EtOAc
20% MeOH: EtOAc
50% MeOH: EtOAc
100% MeOH
1490
60
50
10
510
5
7
6
50
170
380
1248
175
176
3. Results
3.1. Bioassay guided fractionation and HPLC of biserrula fractions
The HPLC chromatogram of the methanol: chloroform (50%:
50%) crude extract of biserrula (Fig. 1) indicated that it contained a
complex mixture of different compounds. To simplify the mixture,
the crude extract was chromatographed on silica gel using mixtures
of solvents with increasing polarity (from hexanes to ethyl acetate
to methanol) to give 10 fractions. Out of these 10 fractions, 40%
EtOAc: Petrol (F4) and 100% MeOH (F10) yielded (510 and 380 mg,
respectively) as the highest extracted yields from HPLC chromatogram (Table 1). The three fractions found active in the subsequent
IVFT (Fig. 3) had yield of 50 mg (F2 and F8) and 170 mg (F9), but
there was no direct link between yield and activity. The HPLC of
these fractions revealed that each active fraction still contained a
mixture of compounds (Fig. 1). Fraction F8 and F9 contained similar
compounds and NMR spectroscopy suggested the presence of
avonoid glycosides in these two fractions. Fraction F2 on the other
hand was quite different and based on UV detection prole seemed
to still contain a large number of compounds.
Fig. 1. HPLC chromatograms of biserrula crude extract (50%: 50%) methanol: chloroform compared with active fractions F2, F8 and F9.
177
Fig. 3. In vitro methane concentration (mean SEM) of biserrula fractions (F), plant
(Biserrula pelecinus) and assay controls. PC e substrate only control; PC 70% EtOH e
substrate ethanol solvent control; Means that do not share common superscripts are
signicantly different (P < 0.05); the standard error bars represent SE of means (n 3).
178
Fig. 4. Methane concentration in headspace (mean SEM) in pure cultures of selected methanogens (a) M. bryantii, (b) M. gallocaecium, (c) M. gottschalkii, (d) M. ruminantium and
(e) M. stadtmanae, when exposed to different fractions of biserrula. Control, methanogen grown in presence of EtOH; * signicantly lower than control (P < 0.05, n 3).
179
Fig. 5. The fold change in mcrA gene amplication product (mean SEM, n 3) of selected methanogens (a) M. bryantii, (b) M. gallocaecium, (c) M. gottschalkii, (d) M. ruminantium
and (e) M. stadtmanae, when compared with control of methanogenic pure cultures exposed to biserrula fractions. The x axis crosses at the value for control (1.0, no change), so
negative values (below 1.0) indicate reduction compared to control and positive values (above 1.0) indicate stimulation.
180
Fig. 6. Methane concentration in headspace (mean SEM) from cultures of methanogens sub-cultured in fresh media after being exposed to active fractions F2, F3 and
F9. Line crossing at 70 mmol/10 mL distinguishes static and equivocal cidal (strongly
reduced) effect; the standard error bars represent SE of means (n 3).
Table 2
Summary of the inhibitory effects on methane production and mcrA gene amplication product and type of effect (where tested) in pure culture of methanogens exposed to
selected fractions of biserrula. e/cidal e equivocal cidal effect.
Methanogens
M. bryantii
M. gallocaecium
M. gottschalkii
M. ruminantium
M. stadtmanae
Fractions
F2
F3
F8
F9
Methane
mcrA gene amplication
e/cidal
Methane
mcrA gene amplication
e/cidal
No effect
Methane
mcrA gene amplication
Methane
No effect
e/cidal
Methane
mcrA gene amplication
static
No effect
Methane
mcrA gene amplication
Not tested
mcrA gene amplication
Methane
mcrA gene amplication
Static
mcrA gene amplication
No effect
Methane
mcrA gene amplication
Methane
No effect
Static
No effect
Methane
mcrA gene amplication
No effect
181
[11] S.E. Denman, N.W. Tomkins, C.S. McSweeney, Quantitation and diversity
analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane, FEMS Microbiol. Ecol. 62
(2007) 313e322.
[12] A.-D.G. Wright, A.F. Toovey, C.L. Pimm, Molecular identication of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured
novel archaea, Anaerobe 12 (2006) 134e139.
[13] A.D.G. Wright, A.J. Williams, B. Winder, C.T. Christophersen, S.L. Rodgers,
K.D. Smith, Molecular diversity of rumen methanogens from sheep in Western
Australia, Appl. Environ. Microbiol. 70 (2004) 1263e1270.
[14] A.K. Patra, J. Saxena, A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen, Phytochemistry 71 (2010)
1198e1222.
[15] A. Cieslak, M. Szumacher-Strabel, A. Stochmal, W. Oleszek, Plant components
with specic activities against rumen methanogens, Animal 7 (2013)
253e265.
[16] X. Li, Z. Durmic, S. Liu, C.S. McSweeney, P.E. Vercoe, Eremophila glabra reduces
methane production and methanogen populations when fermented in a
RUSITEC, Anaerobe 29 (2014) 100e107.
[17] L.P. Broudiscou, Y. Papon, A.F. Broudiscou, Effects of dry plant extracts on
fermentation and methanogenesis in continuous culture of rumen microbes,
Animal Feed Sci. Technol. 87 (2000) 263e277.
n
~ ez-Ruiz, S.M. Duval, N.R. McEwan, C.J. Newbold, Plant ex[18] K.J. Hart, D.R. Ya
tracts to manipulate rumen fermentation, Animal Feed Sci. Technol. 147
(2008) 8e35.
[19] B. Banik, Z. Durmic, W. Erskine, K. Ghamkhar, C. Revell, In vitro ruminal
fermentation characteristics and methane production differ in selected key
pasture species in Australia, Crop Pasture Sci. 64 (2013) 935e942.
[20] B.K. Banik, Z. Durmic, W. Erskine, P. Nichols, K. Ghamkhar, P. Vercoe, Variability of in vitro ruminal fermentation and methanogenic potential in the
pasture legume biserrula (Biserrula pelecinus L.), Crop Pasture Sci. 64 (2013)
409e416.
[21] L. Pistelli, Secondary metabolites of genus Astragalus: structure and biological
activity, Stud. Nat. Prod. Chem. 27 (2002) 443e545.
[22] R.J. Aerts, T.N. Barry, W.C. McNabb, Polyphenols and agriculture: benecial
effects of proanthocyanidins in forages, Agric. Ecosyst. Environ. 75 (1999)
1e12.
[23] E. Swinny, C. Revell, N. Campbell, E. Spadek, C. Russo, In search of photosensitising compounds in the annual forage legume Biserrula pelecinus L. Crop
Pasture Sci. (2015) 1161e1166.
[24] Y.Q. Guo, J.X. Liu, Y. Lu, W.Y. Zhu, S.E. Denman, C.S. McSweeney, Effect of tea
saponin on methanogenesis, microbial community structure and expression
of mcrA gene, in cultures of rumen micro-organisms, Lett. Appl. Microbiol. 47
(2008) 421e426.
[25] M. Billo, P. Cabalion, J. Waikedre, C. Fourneau, S. Bouttier, R. Hocquemiller, et
al., Screening of some new Caledonian and Vanuatu medicinal plants for
antimycobacterial activity, J. Ethnopharmacol. 96 (2005) 195e200.
[26] A. Geyid, D. Abebe, A. Debella, Z. Makonnen, F. Aberra, F. Teka, et al., Screening
of some medicinal plants of Ethiopia for their anti-microbial properties and
chemical proles, J. Ethnopharmacol. 97 (2005) 421e427.
[27] R.A. Mothana, U. Lindequist, Antimicrobial activity of some medicinal plants of
the island Soqotra, J. Ethnopharmacol. 96 (2005) 177e181.
[28] A. Neto, J. Costa, C. Belati, A. Vinholis, L. Possebom, A. Da Silva Filho, et al.,
Analgesic and anti-inammatory activity of a crude root extract of Pfafa
glomerata (Spreng) Pedersen, J. Ethnopharmacol. 96 (2005) 87e91.
[29] B.K. Banik, Genetic Diversity within the Core Collection of Biserrula Pelecinus
L. For Morphological Traits, Rumen Fermentation Proles, Bioactivity and its
Possible Links to Plant Secondary Compounds. Centre for Legumes in Mediterranean Agriculture (CLIMA), Faculty of Nature and Agricultural Science
(FNAS), The University of Western Australia, Perth, WA, Australia, 2011, p. 69.
[30] Z. Durmic, C. McSweeney, G. Kemp, P. Hutton, R. Wallace, P. Vercoe, Australian
plants with potential to inhibit bacteria and processes involved in ruminal
biohydrogenation of fatty acids, Animal Feed Sci. Technol. 145 (2008)
271e284.
[31] Z. Durmic, P. Hutton, D. Revell, J. Emms, S. Hughes, P. Vercoe, In vitro
fermentative traits of Australian woody perennial plant species that may be
considered as potential sources of feed for grazing ruminants, Animal Feed
Sci. Technol. 160 (2010) 98e109.
[32] J. Padmanabha, J. Liu, C. Kurekci, S. Denman, C. McSweeney, A methylotrophic
methanogen isolate from the thermoplasmatales afliated RCC clade may
provide insight into the role of this group in the rumen, in: Proceedings of the
5th Greenhouse Gases and Animal Agriculture Conference 2013, Cambridge
University Press, Dublin. Cambridge, 2013, p. 259.
[33] W.E. Balch, G. Fox, L. Magrum, C. Woese, R. Wolfe, Methanogens: reevaluation
of a unique biological group, Microbiol. Rev. 43 (1979) 260e296.
[34] S.E. Denman, C.S. McSweeney, Development of a real-time PCR assay for
monitoring anaerobic fungal and cellulolytic bacterial populations within the
rumen, FEMS Microbiol. Ecol. 58 (2006) 572e582.
[35] N. Tatsuoka, N. Mohammed, M. Mitsumori, K. Hara, M. Kurihara, H. Itabashi,
Phylogenetic analysis of methyl coenzyme-M reductase detected from the
bovine rumen, Lett. Appl. Microbiol. 39 (2004) 257e260.
[36] P.E. Luton, J.M. Wayne, R.J. Sharp, P.W. Riley, The mcrA gene as an alternative
to 16S rRNA in the phylogenetic analysis of methanogen populations in
landll, Microbiology 148 (2002) 3521e3530.
[37] E. Springer, M.S. Sachs, C.R. Woese, D.R. Boone, Partial gene sequences for the
182
[38]
[39]
[40]
[41]
[42]
[43]
[44]