Application of MootralTM Reduces Methane Production by Altering the Archaea Community in the Rumen Simulation Technique

Abstract

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Keywords: Illumina; RUSITEC; citrus; garlic; methanogens; organosulphur; polyphenols.

FIGURE 1

Production of fermentation gas at the last day of experimental period (EP) and withdrawal period (WP). The daily production rate of methane (A) and carbon dioxide (B) was calculated by multiplying the proportions of methane (C) and carbon dioxide (D) in the fermentation gas with the volume of fermentation gas corrected for standard conditions (1013 hPa, 0°C). Effects of a low dose (LD, light gray bars) and a high dose (HD, dark gray bars) of the experimental mixture were compared with a negative control (CON, black bars) and a positive control (monensin, MON, white bars). Two-way ANOVA followed by Sidak post-test was applied to detect significant effects of the factors Time, Treatment or interaction Time × Treatment. ANOVA revealed significant effects of Treatment (P = 0.001) on methane production and of Treatment (both P < 0.001), Time (both P = 0.003) and interaction of Time × Treatment (both P = 0.02) on the percentages of methane and carbon dioxide. Carbon dioxide production only tended to be affected by Time (P = 0.076). Significant differences in Sidak post-test are indicated using different capital letters, small letters and Greek letters for differences among treatment groups for both time-points, among treatment groups in experimental period and among treatment groups in withdrawal period (at least p < 0.05), respectively. Time-dependent alterations within groups are indicated by asterisks. ^∗∗∗P < 0.001. Data are presented as means ± SD. Number of replicates n = 3.

FIGURE 2

Similarities of archaeal communities analyzed by single strand conformation polymorphism. Dendrogram analysis based on UPGMA clustering was used to compare similarities of band patterns on the SSCP gel (A) and the similarity matrix was visualized as NMDS plot (B, Stress = 0.09). During the experimental period fermenters A, F, L received no addition (CON14, light blue), fermenters B,G,K received 1 g of the experimental mixture (LD14, orange), fermenters C, E, I were treated with 2 g of the experimental mixture (HD14, dark green) and 4.1 mg Monensin was added to fermenters D, J, H (MON14, dark purple). During withdrawal period no additions were made (CON18, blue green; LD18, light purple; HD18, red; MON18, brown).

FIGURE 3

Number of observed operational taxonomic units (OTUs), richness and diversity of the archaeal community under different treatment conditions during the experimental period (day 14, A) and the withdrawal period (day 18, B). Data are shown as Box and Whiskers with Min to Max range. Results were analyzed by One-way ANOVA for day 18. A significant effect of the Treatment was observed for Shannon index at day 18 (P = 0.014). Asterisks indicate significant differences in Tukey post-test with P < 0.05.

FIGURE 4

Analysis of microbial communities under different treatment conditions. (A) Detrended coordinate analysis of beta-diversity using unifrac distances. (B) Clustering of samples based on normalized abundances and phylogenetic predictions of OTUs. During the experimental period fermenters A, F, L received no addition (CON14, light blue), fermenters B, G, K were treated with 1 g of the experimental mixture (LD14, orange), fermenters C, E, I received 2 g of the experimental mixture (HD14, dark green) and 4.1 mg Monensin was added to fermenters D, J, H (MON14, dark purple). During the withdrawal period no additions were made (CON18, blue green; LD18, light purple; HD18, red; MON18, brown).

FIGURE 5

Significant changes (P < 0.05) in the normalized abundance of archaeal operational taxonomic units (OTUs). Differential OTU analysis based on normalized abundance counts contrasting the different experimental groups was performed using the software DESeq2 v1.12.4. P-values were corrected for multiple comparisons using Benjamini–Hochberg correction. In the heatmap, the log2 fold change of significantly different abundant OTUs (P < 0.05) is indicated by the red to green color scale. A threshold of ±2 was used for the log2 fold change. White areas indicate that no significant differences were observed, or the log2 fold change was between –2 and +2. Changes are depicted among treatment groups at day 14 (A, CON: control group, LD: 1 g of the experimental mixture per day, HD 2 g of the experimental mixture per day, MON: 4.1 mg Monensin per day), among treatment groups at day 18 (B, withdrawal period: no additions) and within treatment groups among days (C).