16S amplicon sequencing and untargeted metabolomics reveal changes in rumen microorganisms and metabolic pathways involved in the reduction of methane by cordycepin

doi:10.1016/j.jia.2024.04.014

Journal of Integrative Agriculture

2025, Vol. 24

Issue (4): 1310-1326 DOI: 10.1016/j.jia.2024.04.014

Section 3: Diet components and feed additives

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16S amplicon sequencing and untargeted metabolomics reveal changes in rumen microorganisms and metabolic pathways involved in the reduction of methane by cordycepin

Haokai Ma^1*, Dengke Liu²^*, Rui Liu¹, Yang Li¹^#, Modinat Tolani Lambo¹, Baisheng Dai^3#, Weizheng Shen³, Yongli Qu⁴^{, 5}, Yonggen Zhang¹

¹College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China

²Hebei Shounong Modern Agricultural Technology Co., Ltd., Baoding 073000, China

³College of Electrical Engineering and Information, Northeast Agricultural University, Harbin 150030, China

⁴College of Animal Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

⁵Key Laboratory of Low-carbon Green Agriculture in Northeastern China, Ministry of Agriculture and Rural Affairs, Daqing 163319, China

Highlights
● Cordycepin significantly reduces methane production in in vitro rumen fermentation.
● The in vitro rumen fermentation type gradually shifts from acetate formation to propionate formation with the addition of cordycepin.
● Cordycepin alters the rumen microbial composition, metabolite profiles, and KEGG pathways.

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摘要

作为农业生产中贡献较大的甲烷生产者，反刍动物需要一种合适的甲烷抑制剂来降低其甲烷的排放，去尽可能地降低甲烷对气候的影响。虫草素在此前研究中已被证明能够提高免疫和调节菌群，另外含有虫草素的蛹虫草被证明可以降低体外甲烷排放，但虫草素能否降低体外甲烷产量尚不清楚。因此本试验选用虫草素来探究其对瘤胃发酵参数、产气量、瘤胃微生物组和瘤胃代谢产物的影响。我们选择0.00, 0.08, 0.16, 0.32和0.64 g L^-1五种浓度的虫草素加入到含TMR的发酵瓶内进行体外产气试验，测量了各瓶内发酵液瘤胃发酵参数以及各瓶的产气量，并选用0.00和0.64 g L^-1浓度组进行16s rRNA测序及非靶向代谢组学分析。结果表明，虫草素的添加可以增加pH、氨态氮、总挥发酸、丙酸盐、异戊酸盐和戊酸盐的浓度，降低丁酸盐浓度、总产气量、甲烷产量、二氧化碳产量、氢气产量、甲烷浓度和二氧化碳浓度，并且和虫草素浓度呈线性关系，另外氢气浓度和虫草素浓度呈二次关系。同时，Succiniclasticum、Prevotella、Rikenellaceae_RC9_gut_group、NK4A214_group、Christensenellaceae_R_7_group、unclassified_F082、Veillonellaceae_UCG_001、Dasytricha、Ophryoscolex、Isotricha、unclassified_Eukaryota、Methanobrevibacter和Piromyces的相对丰度在添加最大剂量(0.64 g L^-1)的虫草素后显著降低，而Succinivibrio、unclassified_Succinivibrionaceae、Prevotellaceae_UCG_001、unclassified_Lachnospiraceae、Lachnospira、Succinivibrionaceae_UCG_002、Pseudobutyrivibrio、Entodinium、Polyplastron、unclassified_Methanomethylophilaceae、Methanosphaera和Candidatus_Methanomethylophilus的相对丰度显著升高。虫草素的添加还影响了biosynthesis of unsaturated fatty acids和purine metabolism等代谢通路，以及arachidonic acid、adenine和2′-deoxyguanosine等代谢产物。基于此，我们认为虫草素是一种效果较好的甲烷排放抑制剂，其通过影响瘤胃微生物区系改变了瘤胃代谢产物和发酵参数，从而调节了瘤胃甲烷产量。本试验能够为开发含有虫草素的虫草类副产物或添加剂作为反刍动物的甲烷抑制剂的“减碳”策略提供参考。

Abstract

As a major contributor to methane production in agriculture, there is a need for a suitable methane inhibitor to reduce ruminant methane emissions and minimize the impact on the climate. This work aimed to explore the influence of cordycepin on rumen fermentation, gas production, microbiome and their metabolites. A total of 0.00, 0.08, 0.16, 0.32, and 0.64 g L^–1 cordycepin were added into fermentation bottles containing 2 g total mixed ration for in vitro ruminal fermentation, and then the gas produced and fermentation parameters were measured for each bottle. Samples from the 0 and 0.64 g L^–1 cordycepin addition were selected for 16S rRNA gene sequencing and metabolome analysis. The result of this experiment indicated that the addition of cordycepin could linearly increase the concentration of total volatile fatty acid, ammonia nitrogen, the proportion of propionate, valerate, and isovalerate, and linearly reduce ruminal pH and methane, carbon dioxide, hydrogen and total gas production, as well as the methane proportion, carbon dioxide proportion and proportion of butyrate. In addition, there was a quadratic relationship between hydrogen and cordycepin addition. At the same time, the relative abundance of Succiniclasticum, Prevotella, Rikenellaceae_RC9_gut_group, NK4A214_group, Christensenellaceae_R_7_group, unclassified_F082, Veillonellaceae_UCG_001, Dasytricha, Ophryoscolex, Isotricha, unclassified_Eukaryota, Methanobrevibacter, and Piromyces decreased significantly after adding the maximum dose of cordycepin. In contrast, the relative abundance of Succinivibrio, unclassified_Succinivibrionaceae, Prevotellaceae_UCG_001, unclassified_Lachnospiraceae, Lachnospira, Succinivibrionaceae_UCG_002, Pseudobutyrivibrio, Entodinium, Polyplastron, unclassified_Methanomethylophilaceae, Methanosphaera, and Candidatus_Methanomethylophilus increased significantly. Metabolic pathways such as biosynthesis of unsaturated fatty acids and purine metabolism and metabolites such as arachidonic acid, adenine, and 2´-deoxyguanosine were also affected by the addition of cordycepin. Based on this, we conclude that cordycepin is an effective methane emission inhibitor that can change the rumen metabolites and fermentation parameters by influencing the rumen microbiome, thus regulating rumen methane production. This experiment may provide a potential theoretical reference for developing Cordyceps byproduct or additives containing cordycepin as methane inhibitors.

Keywords: cordycepin in vitro rumen fermentation rumen microbiome metabolome methane production

Received: 10 May 2023 Accepted: 19 March 2024

Fund:

This study was financially supported by the National Key Research and Development Program of China (2023YFD2000701), the Natural Science Foundation of Heilongjiang Province, China (YQ2023C011), the Key Research and Development Program of Heilongjiang Province, China (Grant no. 2022ZX01A24) and the Key Laboratory of Low-carbon Green Agriculture in Northeastern China, Ministry of Agriculture and Rural Affairs of China (LCGANE14).

About author: Haokai Ma, E-mail: m907073443@outlook.com; Dengke Liu, E-mail: 13910356698@163.com; #Correspondence Yang Li, Tel: +86-451-55190840, E-mail: liyang1405053@neau.edu.cn; Baisheng Dai, E-mail: bsdai@neau.edu.cn * These authors contributed equally to this study.

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Haokai Ma, Dengke Liu, Rui Liu, Yang Li, Modinat Tolani Lambo, Baisheng Dai, Weizheng Shen, Yongli Qu, Yonggen Zhang. 2025. 16S amplicon sequencing and untargeted metabolomics reveal changes in rumen microorganisms and metabolic pathways involved in the reduction of methane by cordycepin. Journal of Integrative Agriculture, 24(4): 1310-1326.

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