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American Journal Of Agriculture And Horticulture Innovations

Peer Reviewed | Open Access | E-ISSN: 2771-2559
Published Article

Elucidating Microbial Community Dynamics, Co-Occurrence Networks, And Functional Potential In Corn Stover Silage: Implications For Fermentation Efficacy And Pathogenic Risk

Elucidating Microbial Community Dynamics, Co-Occurrence Networks, And Functional Potential In Corn Stover Silage: Implications For Fermentation Efficacy And Pathogenic Risk

Authors

  • Dr. Anja Schmidt
    Department of Animal Nutrition, Wageningen University & Research, Wageningen, The Netherlands

Keywords:

Corn stover Silage Microbial community 16S rRNA sequencing

Abstract

Background: Corn stover is a globally abundant agricultural byproduct with significant potential as livestock feed. Ensiling is a crucial method for its preservation, relying on microbial fermentation. However, a deep understanding of the complex bacterial community structure, their interactions, functional roles, and associated pathogenic risks during corn stover ensiling remains limited. This study aimed to elucidate these microbial dynamics to improve fermentation efficiency and ensure feed safety.

Methods: Corn stover was ensiled in laboratory-scale silos for up to 60 days. Fermentation quality was assessed by measuring pH, organic acids, and ammonia nitrogen. The bacterial community dynamics were profiled using high-throughput 16S rRNA gene sequencing. Bioinformatic analyses were employed to determine microbial diversity and succession, construct co-occurrence networks to identify keystone taxa, and predict the functional potential of the microbiome using PICRUSt2 based on the KEGG database.

Results: The ensiling process was successful, characterized by a rapid pH drop to below 4.0 and a high concentration of lactic acid. Microbial community analysis revealed a distinct temporal succession, with initial epiphytic Proteobacteria being replaced by a dominant Firmicutes population. Specifically, Lactiplantibacillus (formerly Lactobacillus) emerged as the most abundant genus in mature silage. Co-occurrence network analysis identified several keystone taxa that likely drive community stability. Functional predictions showed a significant enrichment of pathways related to carbohydrate metabolism, consistent with vigorous lactic acid fermentation. Notably, genera containing opportunistic pathogens, such as Pseudomonas and Clostridium, were detected, though their relative abundance decreased significantly in the well-fermented silage.

Conclusion: This study provides a comprehensive ecological perspective on the corn stover silage microbiome. Integrating community structure, network interactions, and functional predictions offers a deeper understanding of the fermentation process. The findings highlight the importance of promoting LAB dominance to not only ensure preservation but also to mitigate the risks associated with potential pathogens, thereby enhancing the overall safety and quality of silage. 

References

Cai, Y.; Du, Z.; Yamasaki, S.; Nguluve, D.; Tinga, B.; Macome, F.; Oya, T. Community of natural lactic acid bacteria and silage fermentation of corn stover and sugarcane tops in Africa. Asian-Australas. J. Anim. Sci. 2020, 33, 1252–1264.

Cai, Y.; Du, Z.; Yamasaki, S.; Nguluve, D.; Tinga, B.; Macome, F.; Oya, T. Influence of microbial additive on microbial populations, ensiling characteristics, and spoilage loss of delayed sealing silage of Napier grass. Asian-Australas. J. Anim. Sci. 2020, 33, 1103–1112.

Tamboli, P.; Chaurasiya, A.K.; Upadhyay, D.; Kumar, A. Climate Change Impact on Forage Characteristics: An Appraisal for Livestock Production. In Molecular Interventions for Developing Climate-Smart Crops: A Forage Perspective; Singhal, R.K., Ahmed, S., Pandey, S., Chand, S., Eds.; Springer: Singapore, 2023.

Wiedmeier, R.D.; Provenzat, F.D.; Burritt, E.A. Exposure to ammoniated wheat straw as suckling calves improves performance of mature beef cows wintered on ammoniated wheat straw. J. Anim. Sci. 2002, 80, 2340–2348.

Khota, W.; Pholsen, S.; Higgs, D.; Cai, Y. Natural lactic acid bacteria population of tropical grasses and their fermentation factor analysis of silage prepared with cellulase and inoculant. J. Dairy Sci. 2016, 99, 9768–9781.

Erenstein, O.; Jaleta, M.; Sonder, K.; Mottaleb, K.; Prasanna, B.M. Global maize production, consumption and trade: Trends and R&D implications. Food Secur. 2022, 14, 1295–1319.

Zabed, H.M.; Akter, S.; Yun, J.; Zhang, G.; Zhao, M.; Mofijur, M.; Awasthi, M.K.; Kalam, M.A.; Ragauskas, A.; Qi, X. Towards the sustainable conversion of corn stover into bioenergy and bioproducts through biochemical route: Technical, economic and strategic perspectives. J. Clean. Prod. 2023, 400 (Suppl. C), 136699.

He, Y.; Dijkstra, J.; Sonnenberg, A.S.M.; Mouthier, T.M.B.; Kabel, M.A.; Hendriks, W.H.; Cone, J.W. The nutritional value of the lower maize stem cannot be improved by ensiling nor by a fungal treatment. Anim. Feed Sci. Technol. 2019, 247, 92–102.

Yang, W.; Li, X.; Zhang, Y. Research progress and the development trend of the utilization of crop straw biomass resources in China. Front. Chem. 2022, 10, 904660.

Phiri, R.; Mavinkere Rangappa, S.; Siengchin, S. Agro-waste for renewable and sustainable green production: A review. J. Clean. Prod. 2024, 434, 139989.

Kazemi, M.; Valizadeh, R.; Ibrahimi Khoram Abadi, E. Yogurt and molasses can alter microbial-digestive and nutritional characteristics of pomegranate leaves silage. AMB Expr. 2022, 12, 111.

Kazemi, M.; Mokhtarpour, A.; Saleh, H. Toward making a high-quality silage from common reed (Phragmites australis). J. Anim. Physiol. Anim. Nutr. 2024, 108, 338–345.

Du, Z.; Nakagawa, A.; Fang, J.; Ridwan, R.; Astuti, W.D.; Sarwono, K.A.; Sofyan, A.; Widyastuti, Y.; Cai, Y. Cleaner anaerobic fermentation and greenhouse gas reduction of crop straw. Microbiol. Spectr. 2024, 12, e0052024.

Du, Z.; Lin, Y.; Sun, L.; Yang, F.; Cai, Y. Microbial community structure, co-occurrence network and fermentation characteristics of woody plant silage. J. Sci. Food Agric. 2022, 102, 1193–1204.

Wensel, C.R.; Pluznick, J.L.; Salzberg, S.L.; Sears, C.L. Next-generation sequencing: Insights to advance clinical investigations of the microbiome. J. Clin. Investig. 2022, 132, e154944.

Yang, J.X.; Cao, J.L.; Xu, H.Y.; Hou, Q.C.; Yu, Z.J.; Zhang, H.P.; Sun, Z.H. Bacterial diversity and community structure in Chongqing radish paocai brines revealed using PacBio single-molecule real-time sequencing technology. J. Sci. Food Agric. 2018, 98, 3234–3245.

Association of Official Agricultural Chemists (AOAC). Official Methods of Analysis, 17th ed; Association of Official Agricultural Chemists: Arlington, VA, USA, 2000.

Murphy, R.P. A method for the extraction of plant sample and the determination of total soluble carbohydrates. J. Sci. Food Agric. 1958, 9, 714–717.

Cai, Y. Analysis Method for Silage. In Japanese Society of Grassland Science, Field and Laboratory Methods for Grassland Science; Tosho Printing Co., Ltd.: Tokyo, Japan, 2004; pp. 279–282.

Wang, S.; Wang, Y.; Zhao, J.; Dong, Z.; Li, J.; Nazar, M.; Kaka, N.A.; Shao, T. Influences of growth stage and ensiling time on fermentation profile, bacterial community compositions and their predicted functionality during ensiling of Italian ryegrass. Anim. Feed Sci. Technol. 2023, 298, 115606.

Broderick, G.A.; Kang, J.H. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 1980, 63, 64–75.

Cai, Y.; Benno, Y.; Ogawa, M.; Kumai, S. Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics and aerobic deterioration of silage. J. Dairy Sci. 1999, 82, 520–526.

Du, Z.; Sun, L.; Chen, C.; Lin, J.; Yang, F.; Cai, Y. Exploring microbial community structure and metabolic gene clusters during silage fermentation of paper mulberry, a high-protein woody plant. Anim. Feed Sci. Technol. 2021, 275, 114766.

Du, Z.; Sun, L.; Lin, Y.; Yang, F.; Cai, Y. The use of PacBio SMRT technology to explore the microbial network and fermentation characteristics of woody silage prepared with exogenous carbohydrate additives. J. Appl. Microbiol. 2021, 131, 2193–2211.

Lozupone, C.; Knight, R. UniFrac: A new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 2005, 71, 8228–8235.

Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glöckner, F.O. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596.

Price, M.N.; Dehal, P.S.; Arkin, A.P. FastTree: Computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 2009, 26, 1641–1650.

Amato, K.R.; Yeoman, C.J.; Kent, A.; Righini, N.; Carbonero, F.; Estrada, A.; Gaskins, H.R.; Stumpf, R.M.; Yildirim, S.; Torralba, M.; et al. Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J. 2013, 7, 1344–1353.

Su, M.; Hao, Z.; Shi, H.; Li, T.; Wang, H.; Li, Q.; Zhang, Y.; Ma, Y. Metagenomic analysis revealed differences in composition and function between liquid-associated and solid-associated microorganisms of sheep rumen. Front. Microbiol. 2022, 13, 851567.

Wang, J.; Wang, J.; Wu, S.; Zhang, Z.; Li, Y. Global geographic diversity and distribution of the myxobacteria. Microbiol. Spectr. 2021, 9, e00012-21.

Xiao, C.C.; Ran, S.J.; Hunag, Z.W.; Liang, J.P. Bacterial diversity and community structure of supragingival plaques in adults with dental health or caries revealed by 16S pyrosequencing. Front. Microbiol. 2016, 7, 1145.

Yang, Q.; Cahn, J.K.B.; Piel, J.; Song, Y.; Zhang, W.; Lin, H. Marine sponge endosymbionts: Structural and functional specificity of the microbiome within euryspongia arenaria cells. Microbiol. Spectr. 2022, 10, e0229621.

Wang, L.; You, L.X.; Zhang, J.M.; Yang, T.; Zhang, W.; Zhang, Z.X.; Liu, P.X.; Wu, S.; Zhao, F.; Ma, J. Biodegradation of sulfadiazine in microbial fuel cells: Reaction mechanism, biotoxicity removal and the correlation with reactor microbes. J. Hazard. Mater. 2018, 360, 402–411.

Li, X.X.; Shi, S.; Rong, L.; Feng, M.Q.; Zhong, L. The impact of liposomal linolenic acid on gastrointestinal microbiota in mice. Int. J. Nanomed. 2018, 13, 1399–1409.

Gao, P.F.; Ma, C.; Sun, Z.; Wang, L.F.; Huang, S.; Su, X.Q.; Xu, J.; Zhang, H.P. Feed-additive probiotics accelerate yet antibiotics delay intestinal microbiota maturation in broiler chicken. Microbiome 2017, 5, 91.

Bai, S.; Hou, G. Microbial communities on fish eggs from Acanthopagrus schlegelii and Halichoeres nigrescens at the XuWen coral reef in the Gulf of Tonkin. Peer J. 2020, 8, e8517.

Duan, T.; Li, Z.; Han, X.; Hong, Q.; Yang, Y.; Yan, J.; Xing, C. Changes functional prediction of ear canal flora in chronic bacterial otitis externa. Front. Cell. Infect. Microbiol. 2024, 14, 1434754.

Steel, R.G.; Torrie, J.H. Principles and Procedures of Statistics: A Biometrical Approach; Mc Graw Hill Company: New York, NY, USA, 1980.

Ingersent, K.A. World agriculture: Towards 2015/2030-an FAO perspective. J. Agric. Econ. 2003, 54, 513–515.

Yan, Y.H.; Li, X.M.; Guan, H.; Huang, L.K.; Ma, X.; Peng, Y.; Li, Z.; Nie, G.; Zhou, J.Q.; Yang, W.Y. Microbial community and fermentation characteristic of Italian ryegrass silage prepared with corn stover and lactic acid bacteria. Bioresour. Technol. 2019, 279, 166–173.

McDonald, P. The Biochemistry of Silage; John Wiley and Sons Inc.: Hoboken, NJ, USA, 1981.

Echegaray, N.; Yilmaz, B.; Sharma, H.; Kumar, M.; Pateiro, M.; Ozogul, F.; Lorenzo, J.M. A novel approach to Lactiplantibacillus plantarum: From probiotic properties to the omics insights. Microbiol. Res. 2023, 268, 127289.

Muck, R.E.; Nadeau, E.M.G.; McAllister, T.A.; Contreras-Govea, F.E.; Santos, M.C.; Kung, L. Silage review: Recent advances and future uses of silage additives. J. Dairy Sci. 2018, 101, 3980–4000.

Willis, A.D. Rarefaction, alpha diversity, and statistics. Front. Microbiol. 2019, 10, 2407.

Pang, H.; Tan, Z.; Wang, Y. Dominant Factors Affecting Silage Fermentation. In Cultural History and Modern Production Technology of Silage; Cai, Y., Ataku, K., Eds.; Springer: Singapore, 2025.

Drouin, P.; Tremblay, J.; Chaucheyras-Durand, F. Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination. Microorganisms 2019, 7, 595.

Wang, Y.; Wang, R.; Kou, F.; He, L.; Sheng, X. Cadmium-tolerant facultative endophytic Rhizobium larrymoorei S28 reduces cadmium availability and accumulation in rice in cadmium-polluted soil. Environ. Technol. Innov. 2022, 26, 102294.

Cui, Y.; Wang, M.; Zheng, Y.; Miao, K.; Qu, X. The carbohydrate metabolism of Lactiplantibacillus plantarum. Int. J. Mol. Sci. 2021, 22, 13452.

Ogunade, I.M.; Jiang, Y.; Pech Cervantes, A.A.; Kim, D.H.; Oliveira, A.S.; Vyas, D.; Weinberg, Z.G.; Jeong, K.C.; Adesogan, A.T. Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives. J. Dairy Sci. 2018, 101, 2048–2059.

Liu, X.; Wang, A.; Zhu, L.; Guo, W.; Guo, X.; Zhu, B.; Yang, M. Effect of additive cellulase on fermentation quality of whole-plant corn silage ensiling by a Bacillus inoculant and dynamic microbial community analysis. Front. Microbiol. 2024, 14, 1330538.

Du, Z.; Yamasaki, S.; Oya, T.; Nguluve, D.; Euridse, D.; Tinga, B.; Macome, F.; Cai, Y. Microbial network and fermentation modulation of Napier grass and sugarcane top silage in southern Africa. Microbiol. Spectr. 2024, 12, e0303223.

Juhász, E.; Iván, M.; Pongrácz, J.; Kristóf, K. Uncommon non-fermenting Gram-negative rods as pathogens of lower respiratory tract infection. Orvosi Hetil. 2018, 159, 23–30.

Qin, S.; Xiao, W.; Zhou, C.; Pu, Q.; Deng, X.; Lan, L.; Liang, H.; Song, X.; Wu, M. Pseudomonas aeruginosa: Pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct. Target. Ther. 2022, 7, 199.

Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27–30.

Ganzle, M.G. Lactic metabolism revisited: Metabolism of lactic acid bacteria in food fermentations and food spoilage. Curr. Opin. Food Sci. 2015, 2, 106–117.

Wang, S.; Wang, Y.; Liu, H.; Li, X.; Zhao, J.; Dong, Z.; Li, J.; Kaka, N.; Nazar, M.; Shao, T. Using PICRUSt2 to explore the functional potential of bacterial community in alfalfa silage harvested at different growth stages. Chem. Biol. Technol. Agric. 2022, 9, 98.

Wang, S.; Ding, C.; Tian, J.; Cheng, Y.; Xu, N.; Zhang, W.; Wang, X.; Nazar, M.; Liu, B. Fermentation profile, bacterial community structure, co-occurrence networks, and their predicted functionality and pathogenic risk in high-moisture Italian ryegrass silage. Agriculture 2024, 14, 1921.

Zong, C.; Wang, L.; Zhao, J.; Dong, Z.; Li, J.; Yuan, X.; Xu, C.; Shao, T. Evaluation of high-moisture oat silage inoculated with synthetic lactic acid bacteria consortia in mini-silos: Fermentation, microbial, metabolic and safety profiles. Ital. J. Anim. Sci. 2025, 24, 1353–1369.