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Bacillus coagulans inoculation generates an alternative microbial succession trajectory associated with nitrogen preservation and aerobic stability in alfalfa silage

This study demonstrates that inoculating alfalfa silage with *Bacillus coagulans* establishes a unique, staged microbial succession trajectory that outperforms traditional *Lactiplantibacillus plantarum* inoculation by significantly enhancing nitrogen preservation and extending aerobic stability through distinct functional shifts in acetate production and nutrient retention.

Original authors: Junming Lu, Jinlong Xu, Wenjing Wei, Qingshan Fan, Ting Jiao, Shengguo Zhao, Jie Bai

Published 2026-08-26

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CC BY 4.0

Original authors: Junming Lu, Jinlong Xu, Wenjing Wei, Qingshan Fan, Ting Jiao, Shengguo Zhao, Jie Bai

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine a field of alfalfa, a lush green crop prized by farmers for its high protein content, which feeds dairy cows and other livestock. To keep this valuable feed fresh through the winter, farmers often chop it up and pack it tightly into airtight bags or towers, a process called ensiling. Inside this sealed environment, the plant material undergoes a natural fermentation, where microscopic organisms break down sugars and produce acids that preserve the food. However, alfalfa is notoriously difficult to preserve. Because it is rich in protein but low in the sugars needed for fermentation, it often fails to acidify quickly enough. This allows unwanted bacteria to break down the precious protein into waste products, reducing the feed's nutritional value. Furthermore, once the storage bag is opened and air rushes in, the feed can spoil rapidly, heating up and rotting due to the activity of yeasts and molds. The challenge for scientists has long been to find a way to guide this microscopic world so that the protein stays intact and the feed remains stable even after the seal is broken.

Researchers at Gansu Agricultural University in China set out to solve this puzzle by testing how different microscopic helpers, known as inoculants, change the course of fermentation in alfalfa. They compared a standard treatment using a common lactic acid bacterium against a newer approach using a different type of bacteria called Bacillus coagulans, as well as a combination of both. The goal was to see which method best preserved the protein and kept the feed from spoiling when exposed to air. The study revealed that the two types of bacteria create very different paths for the microbial community to follow. The traditional lactic acid bacterium acts quickly, rapidly lowering the acidity of the feed and taking over the environment almost immediately. In contrast, the Bacillus coagulans treatment creates a more gradual, staged process where different types of bacteria take turns, resulting in a higher concentration of acetic acid, a different type of preservative acid.

The results showed that while the fast-acting lactic acid bacterium successfully lowered the pH early on, it did not protect the protein as well as the Bacillus coagulans treatment. After sixty days of storage, the silage treated with Bacillus coagulans retained the highest amount of crude protein and had the lowest levels of nitrogen waste. More importantly, when the researchers opened the bags and exposed the feed to air to test its stability, the difference became even clearer. The control group, which received no treatment, began to heat up and spoil after just forty hours. The silage treated with the fast-acting lactic acid bacterium lasted longer, surviving for about one hundred and five hours before showing signs of spoilage. However, the silage treated with Bacillus coagulans, and the mix containing it, remained stable for one hundred and thirty-three hours. This suggests that the presence of acetic acid, which is more effective at fighting off the yeasts and molds that cause spoilage in air, was the key factor in keeping the feed fresh.

To understand why this happened, the scientists looked deep inside the microbial communities using advanced genetic sequencing. They found that the fast-acting lactic acid bacterium created a very simple community dominated by a single type of microbe. While this was good for making acid quickly, it left the system vulnerable once the air returned. The Bacillus coagulans treatment, however, supported a more diverse and shifting community. This diversity allowed the feed to maintain a better balance of acids and preserved specific genetic functions related to nitrogen and sulfur metabolism. When air was introduced, the community in the Bacillus coagulans silage adjusted locally without falling apart, whereas the untreated silage suffered a broad collapse in its microbial structure. The study concludes that preserving high-protein feed is not just about making it acidic as fast as possible. Instead, success depends on guiding the microbial community through a specific sequence of changes that builds a resilient environment, one that can protect the protein during storage and withstand the shock of air exposure when the feed is finally ready to be eaten.

Technical Summary: Bacillus coagulans Inoculation in Alfalfa Silage

Problem Statement
Alfalfa (Medicago sativa L.) is a critical high-protein forage crop but presents significant challenges for ensiling due to its high buffering capacity, low water-soluble carbohydrate (WSC) concentration, and active plant proteases. These characteristics often lead to slow acidification, extensive protein degradation (resulting in nitrogen loss), and poor aerobic stability upon silo opening. While lactic acid bacteria (LAB) inoculants like Lactiplantibacillus plantarum are widely used to accelerate acidification, rapid early pH decline does not always guarantee optimal nitrogen preservation or resistance to aerobic spoilage in high-protein, low-WSC substrates. Furthermore, the functional mechanisms by which alternative inoculants, such as Bacillus coagulans, influence microbial succession, carbon flow, and nitrogen retention across both anaerobic and aerobic phases remain insufficiently resolved, particularly regarding the link between community structure and metagenomic functional potential.

Methodology
The study employed a laboratory-scale bag silo experiment using alfalfa harvested at the early flowering stage and wilted to approximately 350 g/kg dry matter (DM). Four treatments were compared:

CK: Uninoculated control.
LP: Inoculated with Lactiplantibacillus plantarum MTD/1 ( $1 \times 10^6$ CFU/g FW).
BC: Inoculated with Bacillus coagulans ( $1 \times 10^6$ CFU/g FW).
BL: Combined inoculation of LP and BC ( $5 \times 10^5$ CFU/g FW each).

Samples were collected at 3, 15, and 60 days of anaerobic ensiling, with additional sampling after 7 days of aerobic exposure. The experimental design included:

Chemical Analysis: Measurement of pH, organic acids (lactic, acetic, propionic, butyric), water-soluble carbohydrates (WSC), crude protein (CP), non-protein nitrogen (NPN), and ammonia nitrogen (NH $_3$ -N).
Aerobic Stability Testing: Temperature monitoring over 7 days to determine the time required for silage temperature to rise >2°C above ambient.
Microbial Analysis: 16S rRNA gene amplicon sequencing (V3-V4 region) to assess bacterial community structure and succession.
Metagenomic Analysis: Shotgun metagenomic sequencing to evaluate functional gene potential (KEGG modules) related to carbon, nitrogen, phosphorus, and sulfur metabolism.
Statistical Analysis: Two-way and one-way ANOVA, Spearman correlation analysis, and integrated association network construction linking phenotypic traits, taxa, and functional modules.

Key Results

Fermentation Trajectories: LP induced rapid early acidification, establishing Lactiplantibacillus dominance and high lactic acid concentrations by day 3. In contrast, BC generated a "staged" succession involving Bacillus, Pediococcus, and Lactiplantibacillus taxa, resulting in slower initial pH decline but higher acetic acid accumulation throughout the ensiling period.
Nitrogen Preservation: After 60 days, BC-treated silage exhibited the highest crude protein (CP) content and the lowest concentrations of NPN and NH $_3$ -N, outperforming both the control and the LP treatment. This suggests that BC was more effective at limiting proteolysis under the specific high-protein, low-WSC conditions.
Aerobic Stability: BC and BL treatments significantly extended aerobic stability to 133.5 hours, compared to 105 hours for LP and 40.5 hours for the control. Notably, LP, despite achieving the lowest early pH, was less stable than BC upon exposure to air.
Metagenomic Insights:
- BC Trajectory: Associated with sustained enrichment of acetate-related functional modules (e.g., acetate kinase/phosphotransacetylase pathway) and the retention of nitrogen- and sulfur-assimilation modules during anaerobic fermentation.
- Aerobic Response: Upon aerobic exposure, the control (CK) showed broad functional disturbance and community drift. LP and BL maintained a relatively stable Lactiplantibacillus-dominated structure. BC showed localized functional adjustments, enriching regulatory and transport-related modules (e.g., two-component systems, phosphotransferase systems) rather than experiencing broad disruption.
Network Analysis: An integrated association network revealed that Lactiplantibacillus dominance correlated with rapid acidification, whereas acetate-associated functional potential and nitrogen/sulfur assimilation modules were linked to the BC-associated phenotype of nitrogen preservation and aerobic stability.

Key Contributions

Differentiation of Inoculant Mechanisms: The study distinguishes between the "rapid dominance" strategy of homofermentative LAB (LP) and a "staged community succession" strategy driven by B. coagulans (BC). It demonstrates that these strategies lead to divergent fermentation profiles and preservation outcomes.
Linking Succession to Function: By integrating shotgun metagenomics with phenotypic data, the paper connects specific microbial succession patterns (e.g., the transient presence of Pediococcus and retention of Bacillus) to functional potentials (acetate production, nitrogen/sulfur retention) that drive nitrogen preservation and aerobic stability.
Acid Profile vs. pH Depth: The results challenge the assumption that the lowest early pH is the sole determinant of silage quality. Instead, the study highlights the importance of the terminal organic acid profile (specifically acetic acid accumulation) and the continuity of microbial function across anaerobic and aerobic phases.
Mechanistic Insight into Aerobic Stability: The findings suggest that aerobic stability is not merely a function of early acidification but relies on the "anaerobic legacy"—specifically, the acid profile and functional reservoir established during fermentation that allows for localized community adjustment rather than broad collapse upon oxygen exposure.

Significance and Claims
The authors claim that microbial succession patterns, rather than just the presence of a specific inoculant strain, serve as a design principle for improving high-protein biomass fermentation. The study posits that B. coagulans acts as a stability-oriented inoculant that facilitates a staged community assembly, leading to an acetate-favoring fermentation phenotype. This phenotype is associated with superior nitrogen preservation and extended aerobic stability compared to traditional homofermentative LAB inoculants.

The paper concludes that for high-protein, low-WSC forages like alfalfa, effective preservation requires balancing rapid early acidification with late-stage acid-profile maintenance and functional continuity. The combination of LP and BC (BL) is presented as a practical strategy that leverages the early acidification of LP and the late-stage stability mechanisms of BC. The authors modestly note that while metagenomic data reveals functional potential, further transcriptomic and flux-based analyses are required to confirm gene expression and enzymatic activity levels. The study emphasizes that the observed benefits are likely mediated by the complex interplay between the inoculated strain and the indigenous microbiome, rather than the activity of the inoculant alone.

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