Bioresource Technology 148 (2013) 352–360

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Effective reduction of enteric methane production by a combination

of nitrate and saponin without adverse effect on feed degradability,
fermentation, or bacterial and archaeal communities of the rumen
Amlan Kumar Patra a,b,⇑, Zhongtang Yu a,⇑
a
Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA
b
Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, 37 K.B. Sarani, Belgachia, Kolkata 700037, India

h i g h l i g h t s

 Addition of saponin and nitrate together markedly inhibited methane production.

 This combination increased feed degradability and total volatile fatty acids.
 Saponin–nitrate combination reduced abundances of protozoa and methanogens.
 Saponin alone and in combination with nitrate increased cellulolytic bacteria.
 Saponin plus nitrate additively lower methane with no adverse effect on digestion.

a r t i c l e i n f o a b s t r a c t

Article history: This study evaluated the effects of Quillaja saponin (0.6 and 1.2 g/L), propynoate (4 and 8 mM), and
Received 2 July 2013 nitrate (5 and 10 mM), alone or in combinations, on methanogenesis, fermentation, bacterial and archa-
Received in revised form 23 August 2013 eal communities, and abundances of select ruminal microbial populations. All treatment decreased meth-
Accepted 24 August 2013
ane production, but combination of all three inhibitors at high dose achieved the greatest inhibition
Available online 31 August 2013
(85%). Propynoate, alone or in combination with nitrate or saponin, decreased feed degradability and
total volatile fatty acid (TVFA) concentrations. However, saponin and nitrate alone at high dose and in
Keywords:
combination at low dose inhibited methanogenesis substantially while increasing feed degradability
Feed degradability
Methane inhibitors
and TVFA concentrations. The abundances of methanogens were lowered by all inhibitors except saponin
Microbial communities alone. Fibrobacter succinogenes and Ruminococcus flavefaciens were increased by saponin, both alone and
Rumen fermentation in combination with nitrate, but inhibited by propynoate. Combination of saponin and nitrate may have
practical application in mitigating methane emission from ruminants.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction sions from livestock are a major environmental concern because

The livestock production systems in both developing and devel-
oped economies have been facing a number of growing environ-
mental challenges including emissions of greenhouse gases into
the atmosphere and excretion of nitrogen polluting ground water
(Steinfeld et al., 2006). As livestock populations continue to grow
to meet the escalating demands for meat and milk products by
the increasing human population, these environmental problems
would be worsened unless practical and cost-effective mitigation
strategies are developed and implemented. Greenhouse gas emis-
⇑ Corresponding authors. Address: Department of Animal Nutrition, West Bengal
University of Animal and Fishery Sciences, 37 K.B. Sarani, Belgachia, Kolkata
700037, India. Tel.: +91 33 25569234; fax: +91 33 25571986 (A.K. Patra). Tel.: +1
(614) 292 3057, fax: +1 (614) 292 2929 (Z. Yu).
E-mail addresses: patra_amlan@yahoo.com (A.K. Patra), yu.226@osu.edu (Z. Yu).
they contribute substantially, about 12–18% in CO2-equivalent, to
total global greenhouse gas emissions (Steinfeld et al., 2006; West-
hoek et al., 2011). One major particular concern is over methane
emission resulted from rumen fermentation of feeds, which was
estimated to contribute about 37% of the total anthropogenic
methane emission (Steinfeld et al., 2006). Besides, methane pro-
duction in the rumen also causes a significant feed energy loss
depending upon the diets. Therefore, effective mitigation of meth-
ane emission from ruminant animals, mostly through dietary
interventions, has been ‘holy grail’ in alleviating the environmental
concern caused by the livestock industry.
Several methane inhibitors have been repeatedly evaluated, pri-
marily individually, for their efficacy to inhibit enteric methane
production in ruminants (Patra, 2012). In most studies, however,
the dilemma is that these inhibitors often exert adverse effects
on feed intake, digestion, and rumen fermentation when added

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.08.140
 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360
at concentrations high enough to achieve substantial or desirable
reduction in methane production, while they result in little inhibi-
tion to methane production when added at concentration that do
not reduce animal productivity or feed digestion (Patra, 2012). Ni-
trate, saponin, and propynoic acid are such methanogenic inhibi-
tors commonly evaluated (van Zijderveld et al., 2010; Zhou et al.,
2011, 2012; Patra and Yu, 2012). Quillaja saponin may inhibit
the growth of rumen protozoa (Patra et al., 2012), which have po-
sitive exo- and endo-symbiotic association with methanogens,
thus lowering protozoa-associated methanogens and their activi-
ties (Patra and Saxena, 2009); propynoic acid is directly toxic to
methanogens (Zhou et al., 2011); and nitrate may serve as compet-
itive electron acceptors diverting hydrogen away from methano-
genesis and directly inhibit methanogens in the form of nitrite
(Bozic et al., 2009; Zhou et al., 2012). Thus, it was hypothesized
that these inhibitors may act additively and/or synergistically in
inhibiting different groups of microbes involved in methane pro-
duction when used in combinations, thus achieving effective
reduction of methane production at low concentrations that do
not adversely affect feed digestion or fermentation. Therefore,
the objective of this study was to explore possible combinations
of quillaja saponin, propynoic acid, and nitrate in inhibiting meth-
ane production, and their effects on feed digestion, rumen fermen-
tation, diversity of bacteria and archaea, and abundances of major
cellulolytic bacterial populations using an in vitro model.
2. Methods
2.1. Experimental design
Quillaja saponin (from the bark of Quillaja saponaria Molina
plants) and propynoic acid (95% purity) were purchased from Sig-
ma–Aldrich (St. Louis, MO, USA), and sodium nitrate (also from Sig-
ma–Aldrich) was used as a source of nitrate. Quillaja saponin,
propynoic acid, and nitrate were used at two different doses indi-
vidually or in their two- and three-way combinations. There were
15 treatments in total: control (without any methanogenic inhibi-
tor), saponin at low (0.6 g/L; SL) and high (1.2 g/L; SH) doses, pro-
pynoic acid at low (4 mM; PL) and high (8 mM; PH) doses, nitrate
at low (5 mM; NL) and high (10 mM; NH) doses, and combinations
of the three inhibitors (Table 1). The doses of these compounds
were selected based on previous studies (Ungerfeld et al., 2007;
Zhou et al., 2011; Patra et al., 2012). Each of these treatments
was evaluated in three replicates. The sapogenin content of the
quillaja saponin was 24.2%, which was determined by the gravi-
metric method after acid hydrolysis of glycosidic linkages in sapo-
nin in a previous study (Patra et al., 2012).
2.2. Inoculum preparation and incubation
Fresh rumen fluid was collected from two cannulated lactating
Jersey cows at approximately 10 h post morning feeding. The cows
were fed a total mixed ration (TMR) that was composed (% dry
matter (DM) basis) of corn silage (45%), alfalfa hay (10%), Cargill
dairy protein product (20%), and a concentrate mixture (25%).
The concentrate mixture consisted of 46.8% ground shelled corn,
12.2% soybean meal, 30.3% AminoPlusÒ (Ag Processing Inc., Omaha,
NE), 1.8% tallow, 2.2% salt, 4.3% limestone, 0.11% magnesium oxide,
and vitamin and trace minerals. The cows had free access to the
TMR that was delivered twice a day at 5:30 am and 4:00 pm. The
rumen fluid collected from each of the cows was mixed equally,
and then filtered through three layers of sterile cheesecloth for
use as the inoculum in the in vitro batch rumen fermentation.
The in vitro batch fermentation was carried out in 120-mL ser-
um bottles as described in previous studies (Patra and Yu, 2012,
353
Table 1
Combinations of methanogenic inhibitors used in this study.
Treatment Quillaja saponin Sodium nitrate Propynoic acid
(g/L) (mM) (mM)
SL + NL 0.6 5 0
SL + PL 0.6 0 4
PL + NL 0 5 4
SH + NH 1.2 10 0
SH + PH 1.2 0 8
PH + NH 0 10 8
SL + NL + PL 0.6 5 4
SH + NH + PH 1.2 10 8
2013a). The feed substrate for the in vitro fermentation was a mix-
ture of alfalfa hay and a dairy concentrate feed at the ratio of 50:50.
The concentrate feed consisted mainly of ground corn (33.2%), soy-
bean meal (14.2%), AminoPlusÒ (15.5%), distillers grains (19.8%),
and wheat middlings (11.3%). The buffered medium for the
in vitro rumen fermentation was prepared anaerobically according
to the procedure of Menke and Steingass (1988). The anaerobic
buffered medium (30 mL) and the rumen inoculum (10 mL) were
dispensed into each serum bottle containing 400 mg of ground
feed substrate in an anaerobic chamber. The headspace of these
bottles contained carbon dioxide only. These serum bottles were
each sealed with a butyl rubber stoppers and then incubated at
39 °C for 24 h in a water bath with intermittent shaking.
2.3. Sampling and measurements
After 24 h incubation, gas pressure in each of the bottles was
measured using a manometer (TraceableÒ; Fisher Scientific, USA)
to determine total gas production. Then 10 mL of each headspace
gas was collected into a glass tube pre-filled with distilled water
by displacement. The culture samples (1 mL) were collected in
microcentrifuge tubes for microbial analysis. The pH values of
the fermentation media were recorded using a pH meter (Fisher
Scientific, USA), and then the cultures were filtered through filter
bags (ANKOM Technology, USA) to determine degradability of
feeds. The filtrates were sampled into 2 mL microfuge tubes for
VFA (1 mL) and ammonia analyses (2 mL). All the samples were
stored at 20 °C until further processing. The samples for VFA
analysis were added with equal volume of 33% metaphosphoric
acid and were then centrifuged (16,100g for 10 min at 4 °C).
The supernatants (0.5 mL) were pipetted into 2-mL clear crimp
glass vials (Suppelco, Bellefonte, USA), which were sealed with
caps (Fisher Scientific, USA) after adding an internal standard
(50 lL of 0.2% 2-ethylbutyric acid) and stored at 4 °C until gas
chromatography analysis.
The concentrations of methane in the gas samples were deter-
mined using a gas chromatograph (HP 5890 Series, Agilent Tech-
nologies, USA) equipped with a thermal conductivity detector
and a HP-PLOT Q capillary column coated with porous polymer
particles made of divinylbenzene and ethylvinylbenzene (Agilent
Technologies Inc., USA). The VFA concentrations in fermentation
media were analyzed using a gas chromatograph (HP 5890 series,
Agilent Technologies, USA) fitted with a flame ionization detector.
The concentrations of ammonia in the fermentation media were
measured by calorimetric method (Chaney and Marbach, 1962).
The DM of the feed substrates and undegraded residues in filter
bags was determined after drying at 105 °C in a hot air oven (AOAC,
2007). The concentration of neutral detergent fiber (NDF) in the
feed and residues was analyzed by treatment with a neutral deter-
gent solution without a-amylase and addition of sodium sulfite
(Van Soest et al., 1991). Apparent DM and NDF degradabilities of
the feed substrate were calculated by the difference in amounts
354 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360
between the added feed substrate and the undegraded residues
(Patra et al., 2012). True DM degradability of the feed substrate
was calculated by the difference in weights between the DM of
feed substrate and the NDF content in the residues.
2.4. DNA extraction
Metagenomic DNA was extracted from 0.5 mL of homogenized
sample of each in vitro culture using the repeated bead beating
(on a Mini-Beadbeater™, BioSpec Products, Bartlesville, OK, USA)
and column purification (using QIAamp DNA Stool Mini Kit, Qia-
gen, Valencia, CA, USA) method (Yu and Morrison, 2004a). The
DNA quality was evaluated using 1% agarose gel electrophoresis,
and DNA yield was quantified using the Quant-iT dsDNA Broad
Range Assay kit (Invitrogen Corporation, Carlsbad, CA, USA) in a
Stratagene M3000p machine (La Jolla, CA, USA). The DNA samples
were stored at 20 °C until analysis.
2.5. Quantitative real-time PCR (qPCR) analyses
The population sizes of total bacteria were quantified using a
TaqMan assay, while those of archaea, protozoa, and major cul-
tured cellulolytic bacterial species (i.e., Fibrobacter succinogenes,
Ruminococcus albus, and Ruminococcus flavefaciens) were quantified
using SYBR-based qPCR using respective specific primer sets and a
Stratagene Mx3000p machine (La Jolla, CA, USA) as reported previ-
ously (Patra and Yu, 2013b). The Prevotella populations were quan-
tified with two sets of genus Prevotella-specific primers using
SYBR-based qPCR. To minimize potential bias, instead of a qPCR
standard prepared from a single strain, one sample-derived qPCR
standard was prepared for each target group using the respective
specific PCR primer set and a composite metagenomic DNA sample
that were prepared by pooling equal amounts of all the metage-
nomic DNA samples (Yu et al., 2005). The standards were purified
using a PCR purification kit (Qiagen, USA) and quantified using a
Quant-iT dsDNA Broad Range Assay kit (Invitrogen). For each of
the standards, 16S (18S in the case of protozoa) rRNA (rrs) gene
copy number concentrations were calculated based on the length
of each PCR product and the mass concentrations. Tenfold serial
dilutions were prepared in Tris–EDTA (TE) buffer prior to qPCR as-
says. To eliminate the effect from potential primer dimers in the
SYBR-based qPCR assays, the fluorescence signal was acquired at
86 °C, at which primer dimers were completely denatured, and
used in quantifying populations of the microbial groups or species
(Yu et al., 2005). The qPCR assay for each species or group was per-
formed in technical triplicates for both the standards and the
metagenomic DNA samples using the same master mix and the
same qPCR plate. The absolute abundances of microbial popula-
tions were expressed as rrs gene copies/mL of culture samples.
2.6. Denaturing gradient gel electrophoresis (DGGE)
The bacterial and the archaeal communities in each of the cul-
tures were examined separately using DGGE (Yu and Morrison,
2004b; Yu et al., 2008). Briefly, the V3 region of the 16S rRNA gene
of bacteria and archaea was amplified using domain (Bacteria and
Archaea) specific primers in a PTC-100 thermal cycler (MJ Research
Inc., Watertown, MA, USA) with a 40 bp GC clamp attached at the
50 end of the forward primers. To eliminate artifactual double
DGGE bands, a final elongation step at 72 °C for 30 min was in-
cluded at the end the of the PCR (Janse et al., 2004). After confirma-
tion on 1.2% agarose gels, all the PCR products were resolved using
a polyacrylamide gels (8%) with a denaturant gradient of 40% and
60% (Yu and Morrison, 2004b; Yu et al., 2008). Following staining
with SYBR green I (Molecular Probe, Oregan, USA), the gel images
were captured using a FlourChem Imaging System (Alpha Innotech
Corporation, San Leandro, CA, USA) and analyzed with the BioNum-
erics software (Applied Maths, Inc., Texas, USA). Diversity indices
were calculated for each of the cultures as described previously
(Patra and Yu, 2012): (i) the Shannon–Wiener diversity index,
P
H0 =  (ni/N) ln(ni/N); (ii) the Simpson dominance index,
P
k = (ni/N)2, and (iii) the evenness index, e = H0 /ln S; where S = to-
tal number of bands, ni = the peak height of ith band and N = sum of
the peak heights of all bands of each sample.
2.7. Statistical analysis
The data on rumen fermentation characteristics, microbial
abundances, and diversity indices were analyzed using the mixed
model procedure of SAS (SAS Institute Inc., version 8, Cary, NC)
in a completely randomized design. All pairwise significant differ-
ences (P < 0.05) between treatment means were separated by Fish-
er’s protected least significant difference using SAS. Principal
component analyses (PCA) of DGGE profiles of bacteria and archaea
were performed using SAS based on the peak heights and migra-
tion of the bands (Patra and Yu, 2012; Patra et al., 2012) after log-
arithmic transformation to account for normal distribution of the
data. Graphically, the PCA visualized the relative similarity of com-
munity composition as indicated by the distance among treat-
ments. The PCA scores on the first three components were
further analyzed by multivariate analysis of variance (MANOVA)
to test for differences in community composition among the
treatments using SAS.
3. Results and discussions
3.1. Gas and methane production, and substrate degradability
Saponin alone did not affect total gas production, while nitrate
and propynoic acid individually and in combinations (including
combination with saponin) lowered total gas production signifi-
cantly at both doses compared with control (Table 2). All the com-
pounds, either alone or in combinations, decreased methane
production substantially. Propynoic acid resulted in the lowest
methane production (67% and 78% at low and high doses, respec-
tively), followed by nitrate (23% and 43%) and saponin (12% and
25%). The combinations of propynoic acid with either saponin or
nitrate did not show additive inhibition to methane production.
However, the combinations of saponin and nitrate demonstrated
an additive effect in lowering methane production. Most of the
methanogens present in the rumen belong to the family Methano-
bacteriaceae (89% and 99% of the methanogens present in rumen
fluid and protozoa fractions, respectively), and washout of
protozoa resulted in a 27% decrease in methanogens population
in fermenters (Sharp et al., 1998). Thus, the decrease in methano-
genesis by saponin might be attributed to decreases in abundance
of archaea due to inhibition of protozoa, as proposed previously
(Patra and Saxena, 2009, 2010). The magnitude of methane reduc-
tion by saponin observed in the present study was similar to that
observed in vitro by Hu et al. (2005).
Propynoic acid is toxic to methanogens and might have directly
inhibited methanogens (Zhou et al., 2011). Methane inhibition by
nitrate has been proposed to be mediated through two mecha-
nisms: direct competition with carbon dioxide for hydrogen and
direct toxicity of nitrite, an intermediate of nitrate reduction, to
methanogens (van Zijderveld et al., 2010; Zhou et al., 2012). Stoi-
chiometrically, reduction of one mole of nitrate to ammonia would
consume four moles of hydrogen gas and subsequently result in a
decrease of one mole of methane production (van Zijderveld et al.,
2010). Thus, theoretically 5 and 10 mM nitrate in 40 mL of the cul-
ture media could decrease methane production by a maximum of
 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360 355

Table 2
Effects of quillaja saponins, propynoic acid, nitrate and their combinations on total gas and methane production, digestibility, ammonia concentrations and pH in in vitro
fermentation cultures.

Treatment Total gas (mL) Methane (mL) DM (%) NDFD (%) TDMD (%) NH3 (mM) pH
Control 85.6a 30.9a 69.5bcde 37.4b 77.7b 25.4bc 5.72e
SL 85.0ab 27.3b 73.3a 39.9ab 78.6ab 27.1bc 5.59g
SH 85.0ab 23.3c 71.3abc 37.4b 77.7b 25.8bc 5.59g
PL 71.9ef 10.1g 69.1cde 31.1cd 75.5cd 17.7ef 5.85d
PH 70.5fg 6.75hi 67.8cde 27.8cde 74.3cd 12.7f 5.64f
NL 80.4bc 23.7c 74.4a 40.4ab 78.8a 28.8ab 5.92bc
NH 74.3d 17.7e 72.7ab 37.4b 77.7b 29.7ab 6.03a
SL + PL 69.6gh 10.7fg 68.5cde 32.1c 75.9c 20.1de 5.83d
SH + PH 69.0hi 6.49i 66.4e 27.0de 74.0d 16.3ef 5.59g
SL + NL 79.2c 21.0d 73.6a 42.6a 79.6a 32.3a 5.85d
SH + NH 73.1de 13.0f 70.0bcd 31.8cd 75.6c 26.6bc 6.02a
PL + NL 68.7hi 10.7fg 68.5cde 29.2cde 74.8cd 28.8ab 6.02a
PH + NH 61.6k 5.58i 66.5e 25.7e 73.6d 27.9abc 6.01a
SL + PL + NL 67.5i 9.31gh 66.8de 25.0e 73.3d 27.3bc 5.95b
SH + PH + NH 63.5j 4.69i 66.4e 27.1cde 74.0d 23.3cd 5.89c
SEM 0.55 0.81 1.17 1.77 0.890 1.63 0.0076
P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Means followed by different superscript letters (a–j) in column differ significantly (P < 0.05).
DMD, dry matter digestibility; NDFD, neutral detergent fiber digestibility; TDMD, true dry matter digestibility.
Control, without any compounds; L and H indicate the dose levels of 0.6 and 1.2 g/L for saponins, 4 and 8 mM for propynoic acid, and 5 and 10 mM for nitrate, respectively.
SEM, standard error of means.

4.5 and 9 mL, respectively. However, methane production was re-
duced by 7.2 and 13.2 mL by the low and the high dose of nitrate,
respectively. It is likely that the ‘‘extra’’ reduction in methane pro-
duction by nitrate might have stemmed from direct inhibition of
methanogens or their activities by nitrite. Unfortunately, nitrite
concentrations were not determined in this study. Even the con-
centrations of nitrite were determined, it would not be possible
to determine the actual magnitudes of the two inhibitory modes
of action. Although, methanogen numbers were not lowered by ni-
trate during the short period of incubation (24 h) in this study, they
were decreased during longer period of in vitro incubation (van Zij-
derveld et al., 2010; Zhou et al., 2011). Nonetheless, it appeared
that some methanogens are resistant to nitrate/nitrite. These
methanogens can be enriched and/or isolated and they can be used
to screen for other methanogen inhibitors. The addition of these
inhibitors to the nitrate–saponin combination may further reduce
methane production.
Most of the treatments did not affect DM degradability, and
DM digestion of the feed was even increased by saponin, nitrate,
and their combination at the low dose compared with control.
Propynoic acid alone and its combinations with saponin or nitrate
significantly reduced NDF degradability at both low and high
doses. This was also reflected by the lower abundances of the cel-
lulolytic bacterial populations including F. succinogenes and R.
flavefaciens, as also noted in other studies (Zhou et al., 2011).
However, saponin and nitrate did not decrease NDF degradability
when added alone irrespective of the dose, while their combina-
tion improved NDF degradability at the low dose but decreased
NDF degradability at the high dose. The increased degradability
of the feed by saponin and nitrate at the low doses could be
attributable to the increased populations of cellulolytic bacteria
(including the three quantified species and other species, see Sec-
tion 3.3). Most of the methane inhibitors evaluated in previous
studies substantially reduced feed digestion and rumen fermenta-
tion when used at effective doses (Patra and Saxena, 2010; Patra,
2012; Patra and Yu, 2012). As hypothesized in the present study,
the combinations of saponin and nitrate resulted in substantial
reduction in methane production, by 32% and 58% at the low
and the high doses, respectively, without significantly affecting
feed digestion or fermentation. The results also substantiated that
propynoic acid has a broad-spectrum toxicity to both methano-
gens and bacteria, whereas saponin and nitrate specifically inhibit
microbes (methanogens and protozoa) that are involved in
methanogenesis.
3.2. Volatile fatty acid and ammonia concentrations
The concentrations of total VFA were significantly greater in the
cultures that received saponin or nitrate alone and both together at
both dose levels compared with the control (Table 3). However,
VFA concentrations were lowered by propynoic acid alone at both
doses and its combination with saponin at the high dose. Other
treatments had no statistically significant effect on VFA produc-
tion. The high dose of nitrate alone, the combinations of nitrate
and propynoic acid, propynoic acid alone, and the combination of
nitrate and saponin increased the molar percentage of acetate. In
contrast, propynoic acid alone, saponin plus propynoic acid at both
doses, propynoic acid plus nitrate, and the combination of the
three inhibitors at their low dose reduced molar percentage of ace-
tate. Saponin alone and its combination with nitrate had no influ-
ence on acetate percentage compared with the control. All the
treatments increased the molar percentage of propionate except
nitrate that decreased it at the low dose and did not affect it at
the high dose. Saponin or nitrate individually decreased molar per-
centage of butyrate, while propynoic acid increased it. This re-
sulted in mixed results for the combination treatments.
Combinations of propynoic acid and saponin at both doses in-
creased molar percentage of butyrate, whereas saponin plus nitrate
at both doses, propynoic acid plus nitrate, and the high-dose com-
bination of the three inhibitors lowered it. Other treatments had no
influence on this VFA pattern. Molar percentages of iso-VFAs were
lower for all treatments than for the control. Propynoic acid alone
and its combination with other compounds resulted in decreased
molar percentage of valerate, whereas saponin and nitrate alone,
and their combination did not affect valerate percentage in the
cultures.
Corroborating the results of the present study, increased con-
centrations of total VFA concentrations by saponin (Wang et al.,
2009) and nitrate (Nolan et al., 2010; van Zijderveld et al., 2010)
have been reported by other researchers. Increased percentage of
acetate but decreased percentage of butyrate by nitrate is well doc-
umented (Bozic et al., 2009; Zhou et al., 2012); however, the
underlying mechanisms are unclear. One mechanism proposes that
nitrate reduction rapidly consumes electrons leading to decreased
356 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360

Table 3
Effects of quillaja saponins, propynoic acid, nitrate and their combinations on volatile fatty acids (VFA) profiles in in vitro fermentation cultures.

Treatment TVFA (mM) Molar percentage (mol/100 mol)

Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate A/P
Control 93.9de 63.2d 18.2j 0.97a 13.6d 1.92a 2.06ab 3.47b
SL 108.2a 62.5de 21.7g 0.82cd 11.1fg 1.58c 2.36a 2.88d
SH 109.8a 62.3de 22.1f 0.85c 10.9fg 1.65c 2.27a 2.82de
PL 82.0fg 54.5h 23.7d 0.72fg 18.6a 1.27e 1.14cd 2.30g
PH 73.2h 57.4g 21.1h 0.68g 19.0a 1.00f 1.06cd 2.71e
NL 100.5bc 65.7b 17.8k 0.87bc 11.9ef 1.61c 2.10a 3.68a
NH 100.3bc 67.6a 18.0j 0.88bc 10.1gh 1.68bc 1.77b 3.76a
SL + PL 87.8ef 54.5h 26.6a 0.75ef 15.7c 1.36de 1.06cd 2.05h
SH + PH 78.8gh 54.8h 25.8b 0.52i 17.1b 0.92f 1.17c 2.12h
SL + NL 102.1b 62.8de 22.1f 0.84cd 10.4gh 1.61c 2.25a 2.85d
SH + NH 101.8b 63.2de 22.6e 0.90b 9.55h 1.76b 2.04ab 2.79de
PL + NL 94.4cd 60.6e 21.8g 0.80de 14.0d 1.43d 1.33c 2.78de
PH + NH 88.6def 65.0bc 20.5i 0.60h 12.1ef 1.00f 0.72e 3.17c
SL + PL + NL 93.8de 59.4f 24.5c 0.74f 13.1de 1.29e 0.91de 2.42f
SH + PH + NH 90.6de 64.5c 22.5ef 0.45j 11.1fg 0.79g 0.71e 2.87d
SEM 2.25 0.40 0.175 0.018 0.456 0.034 0.117 0.041
P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Means followed by different superscript letters (a–j) in column differ significantly (P < 0.05).
TVFA, total volatile fatty acids; A/P, acetate to propionate ratio.
Control, without any compounds; L and H indicate the dose levels of 0.6 and 1.2 g/L for saponins, 4 and 8 mM for propynoic acid, and 5 and 10 mM for nitrate, respectively.
SEM, standard error of means.

concentrations of NADH and increased concentrations of NAD+,
which in turn favor production of acetate over production of more
reduced VFA such as butyrate (Nolan et al., 2010). In the present
study, however, hydrogen concentration in the headspace gas
was higher (2.19% in culture headspace) at the low-dose nitrate
but lower (0.19% in culture headspace) at the high-dose nitrate
compared with the control (0.61%) (data not shown). Thus, it is
likely that nitrate may also promote acetate production by other
pathways.
The ammonia concentrations were lowered by propynoic acid
alone and its combination with saponin at both doses, which was
probably due to the toxicity of propynoic acid to protein-degrading
and ammonia-producing rumen bacteria. In contrast, concentra-
tions of ammonia were increased by the combination of saponin
and nitrate at the low dose. As observed previously (Zhou et al.,
2011), nitrate and its combinations resulted in increases in ammo-
nia concentration due to assimilatory reduction of the added ni-
trate. Other treatments had no effect on ammonia concentration
compared to the control. The pH values were lower for saponin
at both doses and propynoic acid at the high dose than the control.
In contrast, pH values were higher for all other treatments com-
pared to the control. Increase in pH can benefit ruminal fermenta-
tion especially when large amounts of concentrate are fed.
3.3. Abundance of microbial populations
The abundances of the microbial groups quantified in the pres-
ent study are summarized in Table 4. Overall, the abundances of
total bacterial population were similar for all the treatments ex-
cept for the combination of the three inhibitors at their high dose
that lowered the abundance of total bacteria. It was noted that the
abundance of genus Prevotella was significantly greater in the cul-
tures receiving nitrate alone and its combination with saponin at
the low dose than in the control, but were lower in the cultures
receiving the combination of propynoic acid and saponin, and com-
bination of all the three inhibitors at the high dose. It should be
noted that, the Prevotella-specific primer set reported by Stevenson
and Weimer (2007) consistently resulted in a much higher abun-
dance of Prevotella compared with the primer set reported by Bek-
ele et al. (2010). This observation is consistent with the finding in a
previous study that showed the primer set reported by Stevenson
and Weimer (2007) matched many non-Prevotella sequences in the
RDP database (Kim and Yu, 2012). The population of F. succinogenes
increased due to addition of saponin alone and its combination
with nitrate at the low dose. In contrast, propynoic acid and its
combinations with saponin and/or nitrate had a significant inhibi-
tory effect on F. succinogenes. The high dose of saponin plus nitrate
also reduced F. succinogenes population. The population of R. flav-
efaciens had similar responses to these inhibitors as that of F. suc-
cinogenes. The population of R. albus increased in response to
addition of saponin at both doses, nitrate alone at the lose dose,
and the combination of saponin and nitrate at their low dose. How-
ever, other treatments did not affect this bacterial population ex-
cept a decreased population by the combinations of propynoic
acid with either nitrate or saponin at the high dose. The increased
abundances of F. succinogenes, R. albus, R. flavefaciens, and genus
Prevotella by the low-dose of saponin, which was also observed
in another study (Patra et al., 2012), could be due to its direct stim-
ulation of cellulolytic bacteria or defaunation (Patra and Saxena,
2009; Patra et al., 2012). Given that nitrate was found to be toxic
to rumen bacteria including cellulolytic bacteria only at high dose
(>12 mM) (Zhou et al., 2012), the reason of increasing populations
of F. succinogenes, R. albus, R. flavefaciens, and genus Prevotella by
the low dose of nitrate is not apparently known. Importantly, the
combination of saponin and nitrate at the low dose not only re-
sulted in reduced methane production but also improved feed
degradability and fermentation profile. These results were corrob-
orated by increased abundances of F. succinogenes, R. albus, R. flav-
efaciens, and genus Prevotella, but decreased numbers of
methanogens. The effect of the inhibitors on rumen fungi was
not evaluated in the present study since they only play a limited
role in fiber digestion in the rumen (i.e. reviews of Ho and Abdul-
lah, 1999). However, future studies may examine the effect on ru-
men fungi because they can make an important contribution to
fiber digestion when high-forage diets are fed (Ho and Abdullah,
1999).
Saponin alone and its combination with either nitrate or propy-
noic acid drastically decreased the abundance of protozoal popula-
tions. This can be explained by the anti-protozoal action of
saponin, which interacts with the cholesterol in the cell mem-
branes resulting in the destruction of protozoal cells (Patra and
Saxena, 2009). Protozoal population was not affected by nitrate
or propynoic acid at both doses. The archaeal population was sig-
nificantly reduced by all the treatments except saponin alone at
 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360 357

Table 4
Effects of quillaja saponins, propynoic acid, nitrate and their combinations on abundances (log 16S rDNA copy numbers/mL) of microbial population in in vitro fermentation
cultures.

Treatment TB FS RF RA AR PRS PRK PT

Control 10.91ab 6.72bc 6.24cde 7.04de 7.11a 10.23bc 9.77cdef 9.26ab
SL 10.93ab 7.32a 6.99ab 7.34ab 7.03ab 10.31ab 9.83bcd 8.44c
SH 10.83abc 7.12ab 6.50bcd 7.32ab 6.89b 10.46a 9.92abc 7.67e
PL 11.12a 6.00de 5.62fg 6.81ef 6.87bc 10.18bc 9.71def 9.14b
PH 10.93ab 5.78def 5.36g 6.82ef 6.89b 10.00d 9.66ef 8.94b
NL 11.16a 7.14ab 6.73bc 7.35ab 7.03ab 10.42a 9.89abc 9.67a
NH 11.16a 6.78b 6.47bcd 7.18bcd 6.99ab 10.48a 9.94ab 9.34ab
SL + PL 11.00ab 5.29f 6.13def 7.16bcd 6.93ab 10.06cd 9.61f 8.15cd
SH + PH 10.40bc 3.79g 4.69h 6.32g 6.58de 9.64e 9.20g 4.91f
SL + NL 11.33a 7.49a 7.54a 7.49a 6.70cd 10.42a 10.02a 7.95de
SH + NH 10.72abc 5.69ef 5.68efg 7.05cde 6.60de 10.21bc 9.80bcde 4.87f
PL + NL 10.83abc 6.25cd 6.80bc 7.08bcde 6.59de 10.32ab 9.85bcd 9.02b
PH + NH 10.70abc 5.68ef 5.47g 6.76f 6.70cd 10.08cd 9.63f 7.98cde
SL + PL + NL 11.29a 5.45f 6.73bc 7.17bcd 6.91ab 10.22bc 9.79bcde 8.11cde
SH + PH + NH 10.21c 3.82g 4.26h 6.37g 6.43e 9.28f 8.72h 4.10g
SEM 0.239 0.182 0.207 0.096 0.071 0.064 0.056 0.165
P-value 0.11 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Means followed by different superscript letters (a–h) in column differ significantly (P < 0.05).
TB, total bacteria; FS, F. succinogenes; RF, R. flavefaciens; RA, R. albus; AR, archaea; PRS, genus Prevotella using the primer set of Stevenson and Weimer (2007); PRK, genus
Prevotella using the primer set of Bekele et al. (2010); PT, total protozoa.
Control, without any compounds; L and H indicate the dose levels of 0.6 and 1.2 g/L for saponins, 4 and 8 mM for propynoic acid, and 5 and 10 mM for nitrate, respectively.
SEM, standard error of means.

the low dose, the combinations of saponin and propynoic acid, the
three inhibitors in combination, and both doses of nitrate. In gen-
eral, the abundances of methanogens were lowered to a greater ex-
tent by the combinations of the inhibitors than by each of the
inhibitors suggesting additive or synergistic inhibition of methano-
gens by these compounds, which was also reflected in methane
inhibition. Although saponin at the low dose decreased protozoal
abundances by 84%, it did not result in a decrease in the abun-
dances of methanogens. This cannot be explained by the data of
the present study, but compensatory increase in free-living meth-
anogens after decrease in protozoa-associate methanogens might
be one possible reason. This type of interactions was noted in
anaerobic digesters where one group of methanogens outcompet-
ed another group of methanogens in response to substrate avail-
ability or presence of inhibitors (Hao et al., 2013; Williams et al.,
2013).
3.4. Microbial community profiles
Community profiling by DGGE is a rapid approach to examine
overall effects of any treatments on microbial communities, and
it has been widely used in recent studies (Li et al., 2013; Bonmatí
et al., 2013). In this study, the DGGE fingerprints of rumen archaea
demonstrated that all archaea were not equally sensitive to the
compounds and their combinations tested in this study (Fig. 1).
As revealed by the increasing number of weak DGGE bands, the
three inhibitors were more effective in inhibiting methanogens
when used in combination than when used alone, even at high
doses. The PCA plot clustered the archaeal DGGE profiles of some
of the treatments (Fig. 2). All the treatments with low doses, except
for propynoic acid, clustered together, indicating a dose effect of
the treatments. In general, the treatments with individual com-
pounds formed a large cluster, while combination treatments
changed the archaeal composition in an inhibitor-specific manner,
as illustrated by the similar archaeal communities observed in the
cultures that received saponin in combination with nitrate or/and
propynoic acid at the high dose. This signified that the archaeal
communities were affected differently by the inhibitor combina-
tions than by individual inhibitors, and the different inhibitors
inhibited different populations of rumen methanogens. In order
to further compare the microbial communities, MANOVA was
performed using the PCA scores of the first three components.
The MANOVA suggested that archaeal communities were different
among most of the treatments (Table 5). Shannon index for archaea
was greater only for propynoic acid at low dose. In contrast, Shan-
non index was lower for all the combinations than the control ex-
cept for the low-dose combination of saponin and propynoic acid
(Table 6), which implied that the diversity of archaea was de-
creased by the inhibitors in an additive or synergistic manner
when used in combinations. Evenness index was higher for nitrate
at both high and low doses, but was lower for all the combination
groups except saponin plus propynoic acid at their low dose. Sim-
ilarly, dominance index was greater for the combination treat-
ments except for the low-dose combination of propynoic acid
and saponin. Increased dominance index of archaea due to addition
of inhibitors suggested that a few methanogens were not inhibited.
As revealed by the bacterial DGGE profiles, a few bacteria dom-
inated in the bacterial communities in some of the treatments
(Fig. 3). The PCA plots of bacterial DGGE profiles showed that the
bacterial communities were generally similar when the inhibitors
were added individually but became distinctly different when
the inhibitors were added in combinations (Fig. 4). It was also
noted that the DGGE profiles of the low-dose combination treat-
ments clustered together, and this cluster also contained the com-
bination treatment of saponin plus nitrate at the high dose. The
bacterial DGGE profiles for saponin at the low and high doses, ni-
trate alone, saponin plus nitrate, and propynoic acid in combina-
tion with nitrate or/and saponin at the high dose were distinctly
separated. The MANOVA on the PCA scores of the first three com-
ponents also suggested that the bacterial communities in most of
the treatments were different. The Shannon index was significantly
greater for the treatments with nitrate at the high dose and nitrate
(5 mM) plus saponin (0.6 g/L). However, this index decreased due
to inclusion of saponin (1.2 g/L), propynoic acid (4 and 8 mM)
alone, and saponin (1.2 g/L) plus propynoic acid (8 mM). The even-
ness index for bacteria decreased for the treatments with propy-
noic acid alone and saponin plus propynoic acid at their high
dose. The dominance index was significantly greater for the above
two treatments but was the lowest for the nitrate treatments, sug-
gesting that propynoic acid, but not nitrate, can be toxic to many
different bacteria. Other treatments did not change the Shannon
diversity, evenness, or dominance indices for bacteria.
358 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360

Fig. 1. DGGE profile of archaea. The letters C, S, P and N stand for the control, quillaja saponins, propynoic acid and nitrate, respectively. The letters L and H represent the low
and high dose, respectively, for S (0.6 and 1.2 g/L), P (4 and 8 mM) and N (5 and 10 mM). All the treatments were tested in triplicates.

Fig. 2. PCA plots of archaeal DGGE profile. The letters C, S, P and N stand for the control, quillaja saponins, propynoic acid and nitrate, respectively. The letters L and H
represent the low and high dose, respectively, for S (0.6 and 1.2 g/L), P (4 and 8 mM) and N (5 and 10 mM). PC1, PC2 and PC3 denote first, second and third principal
component, respectively. All the treatments were tested in triplicates.

Table 5
Pairwise comparisons of DGGE profiles of bacteria and archaea as affected by quillaja saponins, nitrate and propynoic acid, and their combinations. P-values (bold font for
archaea) were calculated from the first three principal components applying MANOVA.

Treatment C NH NL PH PL P + NH P + NL SH SL S + NH S + NL S + PH S + PL S + N + PH S + N + PL
C <0.01 0.04 0.15 0.29 0.06 <0.01 0.10 0.87 0.05 <0.01 0.02 <0.01 <0.01 0.02
NH 0.04 0.54 <0.01 <0.01 <0.01 0.02 <0.01 <0.01 0.01 <0.01 0.01 0.06 <0.01 <0.01
NL 0.08 0.58 0.02 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.01 <0.01 <0.01 0.01
PH 0.03 0.17 0.01 0.18 0.06 0.01 <0.01 <0.01 0.02 0.01 <0.01 <0.01 <0.01 0.01
PL 0.52 0.05 0.20 0.17 0.15 <0.01 0.08 0.14 <0.01 <0.01 <0.01 <0.01 <0.01 <0..01
P + NH <0.01 <0.01 0.01 0.02 0.01 <0.01 <0.01 0.02 0.22 0.01 0.09 <0.01 0.06 0.02
P + NL <0.01 <0.01 <0.01 <0.01 <0.01 0.05 <0.01 <0.01 <0.01 0.34 <0.01 <0.01 <0.01 0.25
SH <0.01 0.01 <0.01 0.06 0.05 <0.01 <0.01 <0.01 0.02 <0.01 <0.01 <0.01 <0.01 <0.01
SL <0.01 0.09 <0.01 0.30 0.27 <0.01 0.02 0.19 0.05 <0.01 0.02 <0.01 <0.01 0.01
S + NH <0.01 0.05 0.03 0.05 0.04 0.07 0.01 0.03 0.03 <0.01 0.56 <0.01 <0.01 0.03
S + NL <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.35 <0.01 <0.01 0.09 0.02 <0.01 <0.01 0.02
S + PH <0.01 <0.01 <0.01 0.01 <0.01 0.42 0.03 <0.01 <0.01 0.07 <0.01 <0.01 0.11 0.01
S + PL 0.17 0.24 0.04 0.34 0.27 0.04 0.04 0.08 0.21 0.08 0.01 0.03 <0.01 <0.01
S + N + PH 0.03 <0.01 <0.01 <0.01 <0.01 0.34 <0.01 <0.01 <0.01 0.05 <0.01 0.34 <0.01 <0.01
S + N + PL 0.04 <0.01 <0.01 <0.01 <0.01 0.05 0.49 <0.01 <0.01 0.20 0.03 0.05 0.04 0.05

The letters C, S, N and P denote control (without any compounds) quillaja saponins, nitrate and propynoic acid, respectively. The last letters L and H indicate the dose levels of
0.6 and 1.2 g/L for S, 4 and 8 mM for P, and 5 and 10 mM for N, respectively.

The prominence or disappearance of some of the DGGE bands microbial communities by nitrate, saponin, and propynoic acid
suggests that both the archaeal and the bacterial functionality alone have been reported earlier (Zhou et al., 2011; Patra et al.,
were influenced by the methane inhibitors. The changes in 2012). As demonstrated in the present study, the bacterial Shannon
 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360 359

Table 6
Effects of quillaja saponins, propynoic acid, nitrate and their combinations on bacterial and archaeal diversity.

Treatment Bacteria Archaea

Shannon Evenness Dominance Shannon Evenness Dominance
Control 3.00cd 0.917abc 0.065bcd 2.76bc 0.922bc 0.073efg
SL 2.95cde 0.917abc 0.063cde 2.77bc 0.925ab 0.072efg
SH 2.85ef 0.931a 0.070abc 2.73c 0.926ab 0.076ef
PL 2.89ef 0.909bcd 0.072ab 2.87a 0.927ab 0.066g
PH 2.82f 0.893d 0.078a 2.79bc 0.922bc 0.072efg
NL 3.03bc 0.928a 0.058def 2.82ab 0.941a 0.068fg
NH 3.14a 0.924ab 0.051f 2.82ab 0.941a 0.067g
SL + PL 2.95cde 0.916abc 0.067bc 2.80ab 0.935ab 0.069fg
SH + PH 2.87ef 0.895d 0.076a 2.43g 0.882efg 0.107b
SL + NL 3.11ab 0.928ab 0.056ef 2.49fg 0.866g 0.102bc
SH + NH 3.04abc 0.918abc 0.062cde 2.46g 0.888ef 0.103bc
PL + NL 3.04abc 0.906cd 0.062cde 2.64d 0.907c 0.085d
PH + NH 2.94cde 0.911bc 0.065bcd 2.54ef 0.874fg 0.098c
SL + PL + NL 3.00cd 0.911bcd 0.066bcd 2.61de 0.893de 0.087d
SH + PH + NH 2.90def 0.919abc 0.070abc 2.24h 0.873fg 0.127a
SEM 0.037 0.0067 0.0030 0.026 0.0062 0.0029
P-value <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Means followed by different superscript letters (a–h) in column differ significantly (P < 0.05).
Control, without any compounds; L and H indicate the dose levels of 0.6 and 1.2 g/L for saponins, 4 and 8 mM for propynoic acid, and 5 and 10 mM for nitrate, respectively.
SEM, standard error of means.

Fig. 3. PCA plots of bacterial DGGE profile. The letters C, S, P and N stand for the control, quillaja saponins, propynoic acid and nitrate, respectively. The letters L and H
represent the low and high dose, respectively, for S (0.6 and 1.2 g/L), P (4 and 8 mM) and N (5 and 10 mM). All the treatments were tested in triplicates.

Fig. 4. PCA plots of bacterial DGGE profile. The letters C, S, P and N stand for the control, quillaja saponins, propynoic acid and nitrate, respectively. The letters L and H
represent the low and high dose, respectively, for S (0.6 and 1.2 g/L), P (4 and 8 mM) and N (5 and 10 mM). PC1, PC2 and PC3 denote first, second and third principal
component, respectively. All the treatments were tested in triplicates.
360 A.K. Patra, Z. Yu / Bioresource Technology 148 (2013) 352–360
index was, however, greater or similar for nitrate and the combina-
tion of saponin and nitrate compared to the control, whereas dom-
inance index was lower or similar for these treatments. Thus, the
bacterial communities appeared to be affected to a small extent
by these two inhibitors, and the relatively small alteration of bac-
terial community by saponin and nitrate at low dose should not af-
fect the stable function of rumen microbiome. The data on feed
substrate degradation and VFA concentrations support this
premise.
4. Conclusions
Saponin and nitrate substantially decreased methane produc-
tion and/or abundances of methanogens in an additive manner.
This was reflected in the archaeal community composition and
diversity. Moreover, saponin and nitrate in combination improved
feed digestion and rumen fermentation. This study demonstrated
that combination of multiple methane inhibitors with complemen-
tary mechanisms at low doses could be more effective and practi-
cal in mitigating methane emissions from ruminants without
impairing feed digestion, and combination of saponin and nitrate
may be such a practical strategy. Future studies can further im-
prove the efficacy of the saponin–nitrate combination.
Acknowledgements
This work was supported in part by an OARDC Grant (2010-
007). A.K. Patra’s tenure at The Ohio State University was sup-
ported by a BOYSCAST fellowship from the Department of Science
and Technology, India.
References
AOAC, 2007. Official Methods of Analysis eighteenth ed.. AOAC International,
Gaithersburg, MD, USA, Revision 2.
Bekele, A.Z., Koike, S., Kobayashi, Y., 2010. Genetic diversity and diet specificity of
ruminal Prevotella revealed by 16S rRNA gene-based analysis. FEMS Microbiol.
Lett. 305, 49–57.
Bonmatí, A., Sotres, A., Mu, Y., Rozendal, R., Rabaey, K., 2013. Oxalate degradation in
a bioelectrochemical system: reactor performance and microbial community
characterization. Bioresour. Technol. 143, 147–153.
Bozic, A.K., Anderson, R.C., Carstens, G.E., Ricke, S.C., Callaway, T.R., Yokoyama, M.T.,
Wang, J.K., Nisbet, D.J., 2009. Effects of the methane-inhibitors nitrate,
nitroethane, lauric acid, lauricidin and the Hawaiian marine algae Chaetoceros
on ruminal fermentation in vitro. Bioresour. Technol. 100, 4017–4025.
Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and
ammonia. Clin. Chem. 8, 130–132.
Hao, L., Lü, F., Li, L., Wu, Q., Shao, L., He, P., 2013. Self-adaption of methane-
producing communities to pH disturbance at different acetate concentrations
by shifting pathways and population interaction. Bioresour. Technol. 140, 319–
327.
Ho, Y.W., Abdullah, N., 1999. The role of fungi in fiber digestion: review. Asian-Aust.
J. Anim. Sci. 12, 104–112.
Hu, W.L., Wu, Y.M., Liu, J.X., Guo, Y.Q., Ye, J.A., 2005. Tea saponins affect in vitro
fermentation and methanogenesis in faunated and defaunated rumen fluid. J.
Zhejiang Univ. Sci. B 6, 787–792.
Janse, I., Bok, J., Zwart, G., 2004. A simple remedy against artifactual double bands in
denaturing gradient gel electrophoresis. J. Microbiol. Method 57, 279–281.
Kim, M., Yu, Z., 2012. Quantitative comparisons of select cultured and uncultured
microbial populations in the rumen of cattle fed different diets. J. Anim. Sci.
Biotechnol. 3, 28.
Li, H., Xu, X., Chen, H., Zhang, Y., Xu, J., Wang, J., Lu, X., 2013. Molecular analyses of
the functional microbial community in composting by PCR-DGGE targeting the
genes of the b-glucosidase. Bioresour. Technol. 134, 51–58.
Menke, K.H., Steingass, H., 1988. Estimation of the energetic feed value from
chemical analysis and in vitro gas production using rumen fluid. Anim. Res.
Develop. 28, 7–55.
Nolan, J.V., Hegarty, R.S., Hegarty, J., Godwin, I.R., Woodgate, R., 2010. Effects of
dietary nitrate on fermentation, methane production and digesta kinetics in
sheep. Anim. Prod. Sci. 50, 801–806.
Patra, A.K., 2012. Enteric methane mitigation technologies for ruminant livestock: a
synthesis of current research and future directions. Environ. Monit. Assess. 184,
1929–1952.
Patra, A.K., Saxena, J., 2009. A review of the effect and mode of action of saponins on
microbial population and fermentation in the rumen and ruminant production.
Nutr. Res. Rev. 22, 204–219.
Patra, A.K., Saxena, J., 2010. A new perspective on the use of plant secondary
metabolites to inhibit methanogenesis in the rumen. Phytochemistry 71, 1198–
1222.
Patra, A.K., Yu, Z., 2012. Effects of essential oils on methane production,
fermentation, abundance and diversity of rumen microbial populations. Appl.
Environ. Microbiol. 78, 4271–4280.
Patra, A.K., Yu, Z., 2013a. Effects of coconut and fish oils on ruminal methanogenesis,
fermentation, and abundance and diversity of microbial populations in vitro. J.
Dairy Sci. 96, 1782–1792.
Patra, A.K., Yu, Z., 2013b. Effects of vanillin, quillaja saponin, and essential oils on
in vitro fermentation and protein degrading microorganisms of the rumen. Appl.
Microbiol. Biotechnol.. http://dx.doi.org/10.1007/s00253-013-4930-x, E-pub
ahead of print 30 April 2013.
Patra, A.K., Stiverson, J., Yu, Z., 2012. Effects of quillaja and yucca saponins on
communities of ruminal bacteria and archaea, select rumen microbial
populations, and fermentation. J. Appl. Microbiol. 113, 1329–1340.
Sharp, R., Ziemer, C.J., Stern, M.D., Stahl, D.A., 1998. Taxon-specific associations
between protozoal and methanogen populations in the rumen and a model
rumen system. FEMS Microbiol. Ecol. 26, 71–78.
Steinfeld, H., Costales, A., Rushton, J., Scherf, B., Bennett, T., Hall, D., 2006. Livestock
Report 2006. FAO, Rome.
Stevenson, D.M., Weimer, P.J., 2007. Dominance of Prevotella and low abundance of
classical ruminal bacterial species in the bovine rumen revealed by relative
quantification real-time PCR. Appl. Microbiol. Biotechnol. 75, 165–174.
Ungerfeld, E.M., Rust, S.R., Burnett, R., 2007. Increases in microbial nitrogen
production and efficiency in vitro with three inhibitors of ruminal
methanogenesis. Can. J. Microbiol. 53, 496–503.
Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral
detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J.
Dairy Sci. 74, 3583–3597.
van Zijderveld, S.M., Gerrits, W.J., Apajalahti, J.A., Newbold, J.R., Dijkstra, J., Leng,
R.A., Perdok, H.B., 2010. Nitrate and sulfate: effective alternative hydrogen sinks
for mitigation of ruminal methane production in sheep. J. Dairy Sci. 93, 5856–
5866.
Wang, C.J., Wang, S.P., Zhou, H., 2009. Influences of flavomycin, ropadiar, and
saponin on nutrient digestibility, rumen fermentation, and methane emission
from sheep. Anim. Feed Sci. Technol. 148, 157–166.
Westhoek, H., Rood, T., van den Berg, M., Janse, J., Nijdam, D., Reudink, M., Stehfest,
E., 2011. The Protein Puzzle. PBL Netherlands Environmental Assessment
Agency, The Hague.
Williams, J., Williams, H., Dinsdale, R., Guwy, A., Esteves, S., 2013. Monitoring
methanogenic population dynamics in a full-scale anaerobic digester to
facilitate operational management. Bioresour. Technol. 140, 234–242.
Yu, Z., Morrison, M., 2004a. Improved extraction of PCR-quality community DNA
from digesta and fecal samples. Biotechniques 36, 808–812.
Yu, Z., Morrison, M., 2004b. Comparisons of different hypervariable regions of rrs
genes for use in fingerprinting of microbial communities by PCR-denaturing
gradient gel electrophoresis. Appl. Environ. Microbiol. 70, 4800–4806.
Yu, Z., Michel Jr, F.C., Hansen, G., Wittum, T., Morrison, M., 2005. Development and
application of real-time PCR assays for quantification of genes encoding
tetracycline resistance. Appl. Environ. Microbiol. 71, 6926–6933.
Yu, Z., Garcia-Gonzalez, R., Schanbacher, F.L., Morrison, M., 2008. Evaluations of
different hypervariable regions of archaeal 16S rRNA genes in profiling of
methanogens by archaea-specific PCR and denaturing gradient gel
electrophoresis. Appl. Environ. Microbiol. 74, 889–893.
Zhou, Z., Meng, Q., Yu, Z., 2011. Effects of methanogenic inhibitors on methane
production and abundance of methanogen and cellulolytic bacteria in in-vitro
ruminal cultures. Appl. Environ. Microbiol. 77, 2634–2639.
Zhou, Z., Yu, Z., Meng, Q., 2012. Effects of nitrate on methane production,
fermentation, and microbial populations in in vitro ruminal cultures.
Bioresour. Technol. 103, 173–179.