Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Effects of garlic oil, nitrate, saponin and their combinations supplemented to different substrates on in vitro fermentation, ruminal methanogenesis, and abundance and diversity of microbial populations A.K. Patra1,2 and Z. Yu1 1 Department of Animal Sciences, The Ohio State University, Columbus, OH, USA 2 Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Kolkata, India

Keywords garlic oil-nitrate-saponin combinations, methanogenesis, microbial diversity, rumen fermentation, substrate degradability. Correspondence Zhongtang Yu and Amlan K. Patra, Department of Animal Sciences, The Ohio State University, Columbus, Ohio 43210, USA. E-mails: [email protected] and [email protected] 2015/0121: received 19 January 2015, revised 25 March 2015 and accepted 31 March 2015 doi:10.1111/jam.12819

Abstract Aims: To investigate the effect of garlic oil (G), nitrate (N), saponin (S) and their combinations supplemented to different forage to concentrate substrates on methanogenesis, fermentation, diversity and abundances of bacteria and Archaea in vitro. Methods and Results: The study was conducted in an 8 9 2 factorial design with eight treatments and two substrates using mixed ruminal batch cultures obtained. Quillaja S (0
6 g l 1), N (5 mmol l 1) and G (0
27 g l 1) were used separately or in binary and tertiary combinations. The two substrates contained grass hay and a dairy concentrate mixture at a 70 : 30 (high-forage substrate) ratio or a 30 : 70 (high-concentrate substrate) ratio. Ruminal fermentation and cellulolytic bacterial populations were affected by interaction between substrate and anti-methanogenic compounds. The inhibitor combinations decreased the methane production additively regardless of substrate. For the high-concentrate substrate, S decreased methane production to a greater extent, so did G and N individually for the high-forage substrate. Feed degradability and total volatile fatty acid (VFA) concentrations were not decreased by any of the treatments. Fibre degradability was actually improved by N+S for the high-forage substrate. VFA concentrations and profiles were affected differently by different anti-methanogenic inhibitors and their combinations. All treatments inhibited the growth of Archaea, but the effect on Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens varied. Conclusions: The results suggest that substrate influences the efficacy of these inhibitors when they are used separately, but in combinations, they can lower methanogenesis additively without much influence from the substrate. Significance and Impact of the Study: The presented research provided evidence that binary and tertiary combination of garlic oil, nitrate and saponin can lower the methane production additively without adversely impacting rumen fermentation and degradability, and forage to concentrate ratio does not change the above effects. These anti-methanogenic inhibitors in combination may have practical application to mitigate methane emission from ruminants.

Journal of Applied Microbiology 119, 127--138 © 2015 The Society for Applied Microbiology

127

Substrate-methane inhibitors interactions

A.K. Patra and Z. Yu

ria and Archaea in an in vitro rumen model using two substrates that differed in forage:concentrate ratio.

Introduction Hydrogenotrophic methanogenesis in the rumen accounts for a substantial loss of the ingested gross energy, thus reducing the efficiency of feed energy utilization. Enteric methane production from livestock also significantly contributes to greenhouse gas emission. Therefore, researchers are keen to explore various technologies and policies to lessen enteric methane emissions (Patra 2012; Hristov et al. 2013). Most of the anti-methanogenic compounds that have been tried to suppress methane production in the rumen negatively influence the fermentation and productivity when they are used at doses that can effectively and persistently decrease methane emission (Patra 2012). Combining several anti-methanogenic compounds with complementary modes of actions at low doses appeared to be promising to decrease synergistically or additively methane emissions while maintaining or even improving rumen fermentation (Patra and Yu 2013). Saponins (S) decrease methane production via inhibition of protozoa-associated methanogens (Cieslak et al. 2013; Patra and Yu 2013; Manatbay et al. 2014); garlic oil (G) lowers methane production by directly inhibiting methanogens (Calsamiglia et al. 2007; Patra and Yu 2012); and nitrate (N) decreases methanogenesis by acting as an more competitive electron acceptor as well as directly inhibiting the growth of methanogens (Nolan et al. 2010; Patra and Yu 2013). Because these inhibitors possess complementary mechanisms of action on methanogenesis, it was hypothesized that their combinations can additively lower methane production while not affecting the degradability or the rumen fermentation. Several anti-methanogenic compounds have been evaluated using in vitro and in vivo studies for their effect on methane production, which varied considerably depending upon type, dose, animal and dietary management (Patra 2012). Although different feed substrates had been used to evaluate the effects of individual anti-methanogenic inhibitors in different studies (Castro-Montoya et al. 2012), the effects of diets on the efficacy of combined anti-methanogenic inhibitors are not reported in the literature. Because populations, density of protozoa, Archaea and cellulolytic bacteria can be affected by diets (Tajima et al. 2001; Petri et al. 2013), and G, N and S affect these microbial populations differently (Patra and Yu 2013), it was hypothesized that methane production and rumen fermentation can be affected by interaction between basal diets and these anti-methanogenic compounds (both individually or in combination). Therefore, this study was undertaken to comprehensively investigate the effect of G, N, S and their combinations on fermentation, methanogenesis, diversity and abundances of bacte128

Materials and methods Experimental design The study was conducted in an 8 9 2 factorial design with eight treatments and two substrate mixtures (referred as substrate hereafter). Quillaja saponin (0
6 g l 1), nitrate (5 mmol l 1), and garlic oil (0
27 g l 1) were used separately and in binary or tertiary combinations, resulting in eight treatments: control (C, without any methanogenic inhibitor), garlic oil (G), nitrate (N), saponin (S), garlic oil plus nitrate (G+N), garlic oil plus saponin (G+S), nitrate plus saponin (N+S), and garlic oil plus nitrate plus saponin (G+N+S). Garlic oil and Quillaja saponin (prepared from the bark of Quillaja saponaria Molina plants containing 24% sapogenin) were purchased from Sigma-Aldrich (St. Louis, MO); and sodium nitrate was used as a source of nitrate. Two substrates comprised (% dry matter (DM) basis) of grass hay (67% neutral detergent fibre (NDF) and 11
1% crude protein (CP) and a dairy concentrate mixture (19
6% NDF and 26% CP) at ratios of 70 : 30 (high-forage substrate) or 30 : 70 (high-concentrate substrate). The concentrate mixture was composed mainly of ground corn (33
2%), soybean meal, 48% CP (14
2%), AminoPlusâ (15
5%; Ag Processing Inc., Omaha, NE), corn distillers grains (19
8%) and wheat middlings (11
3%). In vitro fermentation The in vitro fermentation was carried out in 120-mL serum bottles in triplicates as described earlier (Patra and Yu 2012). Fresh rumen fluid was collected from two cannulated lactating Jersey cows at approx. 10 h postmorning feeding. The cows were fed a total mixed ration (TMR) containing 37% NDF and 17% CP. The TMR was composed of corn silage (33
2%), alfalfa and grass hay (8
8%), corn wet-milling co-product (25%; Cargill, Inc. Minneapolis, MN), ground corn (11
7%), soybean hulls (9
8%), soybean meal (48%), CP (5
5%) and AminoPlusâ (2
27%), tallow (0
91%), limestone (1
16%), trace mineralized salt (0
50%), and other mineral and vitamin supplements. The cows were fed the TMR (17
3 kg) twice daily at 6:00 and 18:00. During the sample collection, the animals were handled following the protocols approved by The Ohio State University Animal Care and Use Committee. The rumen fluid samples from the two cows was mixed equally, filtered through three layers of cheesecloth in an anaerobic chamber, and used as the inoculum. The medium for the in vitro fermentation was prepared

Journal of Applied Microbiology 119, 127--138 © 2015 The Society for Applied Microbiology

Substrate-methane inhibitors interactions

A.K. Patra and Z. Yu

Effect of the inhibitors on methane production and abundance of methanogens It is well-established that saponins decrease protozoal numbers in the rumen (Patra and Saxena 2009; McMurphy et al. 2014; Manatbay et al. 2014) by interacting with the cholesterol in their cell membranes thereby destructing protozoal cells (Patra and Saxena 2009). Decreased rumen protozoa population decreases hydrogen production, leading to decreased methane production by decreasing hydrogen availability. Additionally, some rumen methanogens remains associated with protozoa either ecto- and endo-symbiotically (Sharp et al. 1998; Belanche et al. 2014), and logically, lowered methanogenesis by saponin might be attributed to decreased abundances of methanogens associated with protozoa (Cieslak et al. 2013). Furthermore, a decrease in Archaeal number by saponins was also noted earlier (Patra and Yu 2013; Manatbay et al. 2014). Nitrate diminishes the methane production by acting as an electron sink as well as directly inhibiting the growth of methanogens depending upon species (Kl€ uber and Conrad 1998; Patra and Yu 2013), whereas garlic oil directly suppresses the growth and activity of methanogens probably by inhibiting the activity of hydroxymethyglutaryl co-enzyme A reductase that catalyses the synthesis of isoprenoid unit, a components of the cell membrane of Archaea (Calsamiglia et al. 2007). Therefore, these inhibitors in combinations are expected to additively lower methane production. Among the binary combinations, methane inhibition was greater for G+N and N+S than for G+S. It is likely that garlic oil and saponin overlap in mode of methane inhibition. The Archaeal population was significantly reduced by all the treatments, but by small magnitude. As observed previously (Guo et al. 2008; Patra and Yu 2014), none of the treatments, even the binary and the tertiary combinations that substantially decreased methane production, had pronounced effect on the abundance of methanogens. As the culture was incubated for only a short period of 24 h, the methane inhibition might have been attributed to decreased activities of methanogens, rather than decreased abundance of methanogens. The short incubation time of 24 h batch fermentation may not be an actual representative of the in vivo rumen conditions. Thus, the results obtained in this study might have limitations, and should be interpreted accordingly. The extent of methane inhibition by the different methanogenic inhibitors showed dependence forage to concentrate ratio. Saponin had greater methane inhibition for the high-concentrate substrate than for the highforage substrate. Goel et al. (2008) also reported that saponins extracted from Sesbenia sesban and fenugreek seeds decreased methane production to a greater extent 134

with a high-concentrate diet compared with a high-forage diet. Because S improved degradability of the forage substrate, it might have increased the availability of hydrogen for methanogenesis, thus thwarting its inhibitory effect on methane production. However, Castro-Montoya et al. (2012) reported that Quillaja saponins suppressed methane to a greater extent with a high-silage substrate than with a high-concentrate substrate. In contrast to S, G and N were much inhibitory to methanogenesis with the high-forage substrate than with the high-concentrate substrate. The G+S and N+S combinations resulted in greater methane inhibition for the high-concentrate substrate than for the high-forage substrate, but G+N showed the opposite trend. The substrate-dependent efficacy of G, N and S and their combinations are not clearly understood. However, it might be attributed to differences in rumen microbiome that are resulted from different substrates. Comprehensive analysis of rumen microbiome may help further understand the diet-inhibitor interactions in mitigating methane emission from cattle. Effect of the inhibitors on substrate degradability and abundance of cellulolytic bacteria Degradability of DM, especially NDF was improved by S and N+S with the high-forage substrate, but not with the high-concentrate substrate. The combination of N+S also improved degradability of feed in our earlier study (Patra and Yu 2013). The increased degradability is consistent with the increased populations of cellulolytic bacteria (including R. albus and R. flavefaciens). The increased abundances of R. albus and R. flavefaciens by S, which was also observed in other studies (Narvaez et al. 2012; Patra et al. 2012), could be due to its direct stimulation of cellulolytic bacteria or anti-protozoal properties of S (Patra and Saxena 2009; Patra et al. 2012). Fungi can also be stimulated by S (Narvaez et al. 2012). Mixed observations have been noted on the effect of N on rumen cellulolytic bacteria. At high concentrations (>12 mmol l 1), nitrate is toxic to rumen bacteria including cellulolytic bacteria (Zhou et al. 2012), but at low concentration (5 mmol l 1), it increases the populations of F. succinogenes, R. albus and R. flavefaciens (Patra and Yu 2013). In the current study, populations of these bacterial species did not significantly increased, but showed an increasing trend when nitrate was added alone. Combination of S and N lowered the methane production additively and improved feed degradability of the high-forage substrate and fermentation profile. These results were further substantiated by increased abundances of R. albus and R. flavefaciens, but decreased numbers of methanogens.

Journal of Applied Microbiology 119, 127--138 © 2015 The Society for Applied Microbiology

Substrate-methane inhibitors interactions

A.K. Patra and Z. Yu

Table 1 Effects of garlic oil (G), nitrate (N), Quillaja saponin (S) and their combinations supplemented to different substrates on total gas and methane production (ml), degradability (%) of feeds and ammonia concentration (mmol l 1) in in vitro mixed culture of rumen micro-organisms Treatment (T)

Total Gas Forage Concentrate Methane Forage Concentrate DMD Forage Concentrate NDFD Forage Concentrate Ammonia Forage Concentrate Mean pH Forage Concentrate

Effects

C

G

N

S

G+N

G+S

N+S

G+N+S

SEM

T

D

T9D

93
0f 100
0f

91
8f 97
7e

89
9e 94
0d

88
8e 89
2c

81
9b 87
1b

86
2d 85
4b

84
1c 85
8b

80
1a 81
5a

0
354