Effect of Lactiplantibacillus plantarum on Greenhouse Gas Emissions during Alfalfa Silage

doi:10.13560/j.cnki.biotech.bull.1985.2025-1442

Abstract

Abstract:

Objective This study aimed to investigate the effect of Lactiplantibacillus plantarum (LP) on greenhouse gas emissions during the ensiling of alfalfa (Medicago sativa L.). Method Two treatments were established: A control (CK, no additive) and an LP inoculation group. Gas samples were collected at day 3, 7, 15, 30, and 45 of ensiling to measure emissions of carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O). The pH, ammonia nitrogen (NH₃-N), and dry matter (DM) contents were determined. Crude protein (CP) content was analyzed on day 7 and 45. Bacterial community structure was assessed using high-throughput sequencing, and co-occurrence networks and random forest models were constructed. Result Compared with CK, LP treatment significantly reduced the emissions of CO₂, CH₄, and N₂O (P<0.05), decreased pH and NH₃-N content, and increased DM preservation, with no significant change in CP content. LP treatment also significantly reduced bacterial diversity on day 7 and 45. Principal coordinate analysis revealed clear separation between LP and CK samples along the Y-axis. Network analysis indicated that LP treatment reduced node number and connectivity, lowered vulnerability, enhanced negative cohesion, and decreased positive cohesion. The random forest model identified the relative abundance of Enterobacter, Lactiplantibacillus, and Lactococcus, pH, Shannon index, and the contribution of PCoA1 as significant contributors to CO₂ emissions; the relative abundance of Enterobacter, NH₃-N content, pH and the relative abundance of Lactococcus as key factors for CH₄ emissions; and pH, the relative abundance of Lactococcus, Shannon index, the relative abundance of Lactiplantibacillus, the contribution of PCoA1 andthe relative abundance of Enterobacter as major contributors to N₂O emissions. Correlation analysis showed that CO₂ emissions were negatively correlated with the relative abundance of Lactiplantibacillus and CP content; CH₄ emissions were positively correlated with the relative abundance of Enterobacter; and N₂O emissions were positively correlated with the relative abundance of Enterobacter and Shannon index, but negatively correlated with the relative abundance of Lactiplantibacillus, CP content, and the degree of explanation of PCoA1. Conclusion Inoculation with L. plantarum improves fermentation quality, reduces greenhouse gas emissions, and modulates the bacterial community structure in alfalfa silage, with Enterobacter, Lactiplantibacillus, and microbial diversity serving as key microbial factors influencing gas emissions.

Key words: alfalfa silage, Lactiplantibacillus plantarum, greenhouse gas, bacterial community, network analysis, random forest

WANG Yan-ping, SUN Pin-tian, GU Song-song, TANG Xiao-xue, MENG Chen-yang, HAN Xiu-ju, JIAN Yuan, WANG En-zhao. Effect of Lactiplantibacillus plantarum on Greenhouse Gas Emissions during Alfalfa Silage[J]. Biotechnology Bulletin, 2026, 42(5): 185-192.

Figures/Tables 6

Fig. 1 Greenhouse gas emissionsA: Carbon dioxide emissions at different time points. B: Methane emissions at different time points. C: Nitrous oxide emissions at different time points. CK: The treatment without bacterial addition. LP: The treatment with L. plantarum added. Different lowercase letters indicate the significant difference at the P<0.05 level. The same below

Fig. 2 Silage fermentation quality

Fig. 3 Bacterial community structure and diversity

Fig. 4 Bacterial co-occurrence networks(A, B) and topological properties(C, D, E)A: Bacterial co-occurrence network under CK treatment. B: Bacterial co-occurrence network under LP treatment. Eb: OTU node representing Enterobacter, Lb: OTU node representing Lactiplantibacillus. Node colors indicate different modules; node colors: red for positive correlations, green for negative correlations

Fig. 5 Sum of node degrees at the genus level

Fig. 6 Random forest model and correlation analysisA: Contribution of different indicators to carbon dioxide emissions. B: Contribution of different indicators to methane emissions. C: Contribution of different indicators to nitrous oxide emissions. D: Correlation between different indicators and greenhouse gas emissions

References 31

[1]	Zhang XX, Wu LY, Ma XZ, et al. Dynamic computable general equilibrium simulation of agricultural greenhouse gas emissions in China [J]. J Clean Prod, 2022, 345: 131122.
[2]	Li LD, Awada T, Shi YY, et al. Global greenhouse gas emissions from agriculture: pathways to sustainable reductions [J]. Glob Change Biol, 2025, 31: e70015.
[3]	Zhang X, Zhang HX, Wang DC, et al. From waste to value: Multi-omics reveal pomegranate peel addition improves corn silage antioxidant activity and reduces methane and nitrogen losses [J]. Bioresour Technol, 2025, 429: 132544.
[4]	Xue YL, Wu NE, Na N, et al. Dynamics of gas and greenhouse gases production during fermentation of barley silage with lactic acid bacteria [J]. Chem Biol Technol Agric, 2024, 11: 82.
[5]	Hou ZP, Zheng X, Zhang XL, et al. Effects of urea supplementation on the nutritional quality and microbial community of alfalfa (Medicago sativa L.) silage [J]. Arch Microbiol, 2022, 204(7): 414.
[6]	郑敏娜, 康佳惠, 陈燕妮, 等. 紫花苜蓿新品种同苜2号的选育及高产栽培技术 [J]. 农业科技通讯, 2025(12): 189-191.
	Zheng MN, Kang JH, Chen YN, et al. Breeding and high-yield cultivation techniques of a new alfalfa variety Tongmu No.2 [J]. Bull Agric Sci Technol, 2025(12): 189-191.
[7]	Zhao SS, Wang YP, Yang FY, et al. Screening a Lactobacillus plantarum strain for good adaption in alfalfa ensiling and demonstrating its improvement of alfalfa silage quality [J]. J Appl Microbiol, 2020, 129(2): 233-242.
[8]	高海娟, 刘泽东, 孙蕊, 等. 不同贮藏时间对苜蓿青贮品质的影响 [J]. 中国饲料, 2019(23): 95-98.
	Gao HJ, Liu ZD, Sun R, et al. The effects of different storage time on alfalfa silage quality [J]. China Feed, 2019(23): 95-98.
[9]	Zong C, Jiang WQ, Shao T, et al. Investigation of fermentation profiles, bacterial community structure and bacterial β-carotene synthesis of alfalfa silage treated with propionic acid or its combination with squalene [J]. J Agric Sci, 2024, 162(2): 118-129.
[10]	孙雨欣, 刘庭玉, 石凯. 不同青贮条件与添加剂对提升高粱青贮品质的研究进展 [J]. 中国饲料, 2025(21): 20-26.
	Sun YX, Liu TY, Shi K. Research progress on the improvement of sorghum silage quality by different ensiling conditions and additives [J]. China Feed, 2025(21): 20-26.
[11]	Bai BC, Qiu R, Sun L, et al. Effect isolated lactic acid bacteria inoculation on the quality, bacterial composition and metabolic characterization of Caragana korshinskii silage [J]. Chem Biol Technol Agric, 2024, 11: 67.
[12]	Liu YC, Du S, Sun L, et al. Volatile metabolomics and metagenomics reveal the effects of lactic acid bacteria on alfalfa silage quality, microbial communities, and volatile organic compounds [J]. Commun Biol, 2024, 7: 1565.
[13]	Yang S, Mahmood M, Baral R, et al. Forage conservation is a neglected nitrous oxide source [J]. PNAS Nexus, 2024, 3(9): 373.
[14]	Muck RE. Silage microbiology and its control through additives [J]. R Bras Zootec, 2010, 39: 183-191.
[15]	Ellis JL, Hindrichsen IK, Klop G, et al. Effects of lactic acid bacteria silage inoculation on methane emission and productivity of Holstein Friesian dairy cattle [J]. J Dairy Sci, 2016, 99(9): 7159-7174.
[16]	Zhao SS, Yang FY, Wang Y, et al. Dynamics of fermentation parameters and bacterial community in high-moisture alfalfa silage with or without lactic acid bacteria [J]. Microorganisms, 2021, 9(6): 1225.
[17]	Thiex N, Novotny L, Crawford A. Determination of ash in animal feed: AOAC official method 942.05 revisited [J]. J AOAC INTERNATIONAL, 2012, 95(5): 1392-1397.
[18]	Chen DK, Zheng MY, Guo X, et al. Altering bacterial community: a possible way of lactic acid bacteria inoculants reducing CO₂ production and nutrient loss during fermentation [J]. Bioresour Technol, 2021, 329: 124915.
[19]	Mu L, Xie Z, Hu LX, et al. Cellulase interacts with Lactobacillus plantarum to affect chemical composition, bacterial communities, and aerobic stability in mixed silage of high-moisture amaranth and rice straw [J]. Bioresour Technol, 2020, 315: 123772.
[20]	热孜姑丽·库尔班, 崔卫东, 龙宣杞, 等. 乳酸菌对青贮饲料发酵品质影响的研究进展 [J]. 饲料研究, 2023, 46(19): 155-158.
	Kurban R, Cui WD, Long XQ, et al. Research progress on effect of lactic acid bacteria on fermentation quality of silage [J]. Feed Res, 2023, 46(19): 155-158.
[21]	Yang WH, Yang FY, Feng CS, et al. Fermentation properties and bacterial community composition of mixed silage of mulberry leaves and smooth bromegrass with and without Lactobacillus plantarum inoculation [J]. Fermentation, 2023, 9(3): 279.
[22]	Wang X, An Q, Zhao B, et al. Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL [J]. Appl Microbiol Biotechnol, 2018, 102(4): 2019-2030.
[23]	张建云, 谷立坤, 王亚西, 等. 植物叶际好氧反硝化细菌的筛选及其脱氮性能研究 [J]. 微生物学报, 2024, 64(5): 1521-1537.
	Zhang JY, Gu LK, Wang YX, et al. Aerobic denitrifying bacteria in the phyllosphere: screening and characterizing of nitrogen removal performance [J]. Acta Microbiol Sin, 2024, 64(5): 1521-1537.
[24]	Kumari K, Sharma PK, Singh RP. The transcriptome response of Enterobacter sp. S-33 is modulated by low pH-stress [J]. Genes Genom, 2024, 46(6): 671-687.
[25]	Chen JW, Li YD, Zhong C, et al. Genomic insights into niche partitioning across sediment depth among anaerobic methane-oxidizing Archaea in global methane seeps [J]. mSystems, 2023, 8(2): e01179-e01122.
[26]	Mand TD, Metcalf WW. Energy conservation and hydrogenase function in methanogenic Archaea, in particular the genus Methanosarcina [J]. Microbiol Mol Biol Rev, 2019, 83(4): e00020-e00019.
[27]	Herr C, Mannheim T, Müller T, et al. Effect of nitrification inhibitors on N₂O emissions after cattle slurry application [J]. Agronomy, 2020, 10(8): 1174.
[28]	Khajooie S, Gaus G, Dohrmann AB, et al. Methanogenic conversion of hydrogen to methane in reservoir rocks: an experimental study of microbial activity in water-filled pore space [J]. Int J Hydrog Energy, 2024, 50: 272-290.
[29]	Zolkefli N, Shui X, Ma KD, et al. Unveiling the impact of indole derivatives on methanogenic Archaea and microbial functions in anaerobic digestion of waste sewage sludge [J]. Appl Biochem Biotechnol, 2026, 198(1): 378-399.
[30]	Luo WS, Wu WL, Du XY, et al. Regulation of the nitrite, biogenic amine and flavor quality of Cantonese pickle by selected lactic acid bacteria [J]. Food Biosci, 2023, 53: 102554.
[31]	Huang YY, Jia XZ, Yu JJ, et al. Effect of different lactic acid bacteria on nitrite degradation, volatile profiles, and sensory quality in Chinese traditional Paocai [J]. LWT, 2021, 147: 111597.