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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.46 No.2 pp.76-85
DOI : https://doi.org/10.5333/KGFS.2026.46.2.76

Effect of Moisture Content and Microbial Inoculant on Fermentation Characteristics of Alfalfa Silage during Long-term Storage

Seung Min Jeong, Ki Won Lee, Hyung Soo Park*
Forages Production Systems Division, National Institute of Animal Science, Cheonan 31000, Republic of Korea
*Corresponding author: Hyung Soo Park, Forages Production Systems Division, National Institute of Animal Science, Cheonan 31000, Republic of Korea. Tel: +82-41-580-6751, Fax: +82-41-580-6779, E-mail: anpark69@korea.kr
May 29, 2026 June 22, 2026 June 22, 2026

Abstract


Alfalfa silage production is challenged by high moisture, strong buffering capacity, and low water-soluble carbohydrate concentrations, yet long-term storage is common under Korean production conditions. This study evaluated how target moisture content at ensiling and a lactic acid bacterial inoculant affect the chemical composition, fermentation profile, and bacterial community of alfalfa silage stored for 12, 18, or 24 months. Alfalfa harvested at National Institute of Animal Science (NIAS, Cheonan) was ensiled at target moisture levels of 30, 40, 50, 60, or 70% with either no additive (CON) or a mixed inoculant containing Lactococcus lactis and Pediococcus pentosaceus (INO). Moisture content exerted the dominant and most consistent influence on silage quality. Crude protein generally decreased and acid detergent fiber (ADF) generally increased as moisture content increased, whereas neutral detergent fiber (NDF) responses were not consistently quadratic across all storage periods. The 40–50% moisture treatments showed the most favorable lactic fermentation, with lower pH and higher lactic acid concentrations than the drier 30% and wetter 60–70% treatments. Inoculation effects were limited and depended on moisture level and storage duration; significant inoculation-related effects were observed for pH and propionic acid at 12 months and lactic acid at 24 months, but inoculation did not consistently improve lactic acid accumulation. The 60–70% moisture treatments showed elevated butyric acid and Clostridium relative abundance, indicating increased risk of undesirable fermentation. At 70% moisture, Clostridium relative abundance was numerically lower in INO than in CON; however, this pattern was not consistently supported by butyric acid responses. Overall, controlling ensiling moisture to approximately 40–50% appears more reliable than relying on inoculation alone for maintaining long-term alfalfa silage quality.


Silage , Fermentation characteristics , Microbial community

초록

    Ⅰ. INTRODUCTION

    Alfalfa (Medicago sativa L.) is a leguminous forage widely recognized for its high crude protein and mineral concentrations and is valued as a feed resource in many countries. Because of these nutritional advantages, alfalfa is strategically cultivated to support livestock production. However, due to its high moisture content, strong buffering capacity, and low concentrations of water-soluble carbohydrates (WSC), alfalfa is not ideally suited to ensiling and is therefore used predominantly as hay rather than silage (Muck et al., 2018;McDonald et al., 1991). Nevertheless, in humid subtropical climates or when fresh, succulent forage is desired to preserve nutrients there has been sustained interest in optimizing alfalfa silage production (Borreani et al., 2018).

    The ensiling process involves microbial and biochemical interactions that are strongly influenced by forage moisture, epiphytic and inoculated microflora, and storage duration (McDonald et al., 1991). Silage fermentation quality is driven primarily by lactic acid bacteria (LAB), which acidify the mass, lower pH, and suppress undesirable microbes (Pahlow et al., 2003). Given alfalfa’s high buffering capacity, the use of inoculants, organic acids, or fermentable substrates (sugars) is often required to improve fermentation efficiency and storage stability (Kung et al., 2018;Muck et al., 2018). Among LAB, Lactococcus lactis and Pediococcus pentosaceus are commonly applied homofermentative strains in silage manufacture; numerous studies report that P. pentosaceus can improve the fermentation quality of alfalfa silage and related systems (Jiang et al., 2020;Nascimento Agarussi et al., 2019).

    In Korea, where haymaking is frequently constrained by weather, conserved forages are often prepared as silage or low-moisture silage for long-term storage (Park et al., 2015). In practice, some forages are harvested late in the previous year and fed the following year, thereby exposing stored silage to winter and summer temperature regimes that may affect fermentation dynamics and microbial growth (Borreani et al., 2018;Muck et al., 2018). However, systematic evaluations of the long-term storage stability and fermentation characteristics of alfalfa silage under domestic conditions remain limited. Accordingly, this study aimed to evaluate the effects of moisture content at ensiling and microbial inoculant treatment on the storage stability and fermentation quality of alfalfa silage under long-term storage conditions, with the overarching goal of informing practical conservation strategies under Korean production conditions.

    Ⅱ. MATERIAL AND METHODS

    1. Plant material and site

    This study was conducted at the Forage Production Systems Division field of the National Institute of Animal Science (NIAS), Seonghwan-eup, Cheonan, Republic of Korea. Alfalfa (Medicago sativa L.) was harvested at early bloom (~10%) in July 2022 from NIAS experimental fields in Cheonan and used for silage manufacture.

    2. Treatments, sampling, and silage preparation

    After mowing, the forage was field-cured outdoors with periodic swath inversion until the material reached target moisture contents of 30, 40, 50, 60, and 70% on a wet basis, respectively. The treatment labels indicate target moisture categories, and the actual pre-ensiling dry matter values are presented in Table 1. At each moisture target, 3,000 g of forage was collected. Two additive treatments were prepared: CON (no additive), with 10 mL of distilled water per portion; and INO (microbial inoculant), a mixed culture of Lactococcus lactis and Pediococcus pentosaceus applied at a stated rate of 1.5 × 105 cfu g−1 FM after dilution in distilled water (0.1 g / 10 mL) and sprayed onto the forage. For each moisture × additive combination, the treated alfalfa was vacuum-sealed in polyethylene bags after expelling air, and three replicate bags were prepared at 500 g FM per bag (n = 3). Bags were stored under non-temperature-controlled conditions, allowing temperature to fluctuate with ambient conditions. The 12- and 24-month silages were opened in July, and the 18-month was opened in January. The outside temperatures during the opening period were 25.4, 0.4, and 26.0 °C, respectively.

    3. Sample processing and chemical analyses

    Upon opening, samples were taken for analysis. For dry matter (DM) determination, subsamples were dried at 65 °C for 72 h in a forced-air oven. The dried material was ground and passed through a 1-mm sieve using a Wiley mill for subsequent analysis. All nutrient value analyses were performed by the Association of Official Analytical Chemists (AOAC, 1990a). The crude protein (CP) content was measured using an elemental analyzer (Vario MAX cube; Elementar, Langenselbold, Germany) according to Dumas' method (AOAC, 1990b). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed by Goering and Van Soest (1970) using an Ankom200 fiber analyzer (Ankom Technology, Macedon, NY, USA).

    4. Fermentation characteristics

    For fermentation profiling, 10 g of fresh matter (FM) was extracted with 90 mL of distilled water using an orbital shaker. The pH of the filtrate was measured with an Inolab pH meter (Thomas Scientific, NJ, USA). Extracts were pre-filtered through filter paper, followed by 0.2 µm membrane filtration, and stored frozen until analysis. Lactic acid was quantified using HPLC (HP1100; Agilent, USA), and acetic, propionic, and butyric acids were determined (Filipek and Dvorak, 2009) by gas chromatography (GC-450; Varian, USA).

    5. DNA extraction and 16S rRNA gene sequencing

    Fresh subsamples were frozen at −70 °C before DNA extraction. Genomic DNA was extracted using a Qiagen kit (Qiagen, Hilden, Germany) and quantified with the Quant-iT PicoGreen Kit (Invitrogen, USA). Libraries were prepared with the Illumina 16S Metagenomic Sequencing Library Kit according to the manufacturer’s protocol and sequenced on an Illumina MiSeq™ platform (San Diego, USA) by Macrogen (Seoul, Korea).

    6. Statistical analysis

    Groupwise comparisons within each storage period were evaluated by ANOVA with Tukey’s HSD test (p < 0.05) using SAS 9.4 (SAS Institute, 2013). To evaluate the overall effects of microbial inoculation, moisture content, and storage period, the full dataset was analyzed using PROC GLM. The model included microbial inoculation (CON vs. INO), moisture content at ensiling (30, 40, 50, 60, and 70%), storage period (12, 18, and 24 months), and their two- and three-way interactions as fixed effects. Least-squares means were compared using Tukey adjustment when significant effects were detected.

    The main objective was to evaluate the effects of moisture content and microbial inoculation within each long-term storage period. Period-specific slice analyses were also conducted to test the effects of microbial inoculation, moisture content, and their interaction within each storage period. The p-values presented in Tables 2 and 3 for MI, MO, and MI × MO correspond to these period-specific analyses, whereas storage-period effects and interactions involving storage period were summarized separately from the overall model (Supplementary Table 1). Because storage period was partially confounded with opening season and ambient temperature, storage-period effects were interpreted cautiously as period-related variation rather than as pure storage-duration effects.

    Ⅲ. RESULTS AND DISCUSSION

    1. Chemical composition

    The pre-ensiling nutrient composition is summarized in Table 1, and the post-ensiling composition is shown in Table 2. Averaged across post-ensiling treatments and storage durations, CP, NDF, and ADF were approximately 17.4, 51.4, and 40.1% of DM, respectively. Relative to the pre-ensiling values, CP generally decreased after long-term storage, whereas NDF did not show a consistent overall decrease. Across storage durations, moisture content at ensiling (MO) had a significant main effect on CP, NDF, and ADF (p < 0.05). CP showed a strong negative linear response to increasing moisture at 12, 18, and 24 months (MOL; p < 0.001), indicating lower CP at higher moisture levels. ADF generally increased with increasing moisture; however, the response included both linear and quadratic components at some storage periods rather than a purely linear pattern. In contrast, the NDF response was not consistently quadratic across all storage periods: at 12 months, the linear effect was significant and the quadratic effect showed only a tendency; at 18 months, the quadratic effect was significant; and at 24 months, the linear effect was significant while the quadratic effect again showed only a tendency. The inoculant main effect and inoculant × moisture interaction were not significant for chemical composition variables (p > 0.05). These patterns suggest that higher moisture conditions may have promoted greater loss or solubilization of more degradable non-fiber fractions, resulting in a proportional increase in ADF (Kung et al., 2018).

    In the overall factorial analysis using the full dataset, storage period affected several chemical and fermentation variables, whereas interactions involving storage period were variable among traits. However, because the 12- and 24-month silages were opened in July and the 18-month silages were opened in January, these storage-period effects should be interpreted as period-related variation potentially confounded with opening season and ambient temperature rather than as pure storage-duration effects. Therefore, the period-specific results were used mainly to describe the effects of moisture content and microbial inoculation within each long-term storage period.

    2. Fermentation characteristics

    Table 3 summarizes fermentation profiles by storage duration.

    Across all period points, pH, lactic acid (LA), acetic acid (AC), propionic acid (PR), and butyric acid (BU) exhibited a significant moisture-at-ensiling main effect (MO; p < 0.05). The 40 and 50% treatments showed lower pH than the other moisture groups (MOQ, 12 month; p = 0.023, 18 month; p < 0.001), which is attributable to their relatively higher LA concentrations. This U-shaped response is consistent with ensiling theory for alfalfa at 12 and 18 month. At intermediate moisture (40–50%), adequate water activity and packing density allow LAB to dominate and depress pH (Kung et al., 2018), whereas at 30% desiccation limits LAB growth and at ≥60% dilution of fermentable substrates and stronger buffering impede rapid acidification. Numerically, the INO groups tended to have lower pH and higher LA than the control groups; however, these differences were generally not significant (MI; p > 0.05), except for PR at 12 months (MI; p = 0.037) and LA at 24 months (MI; p < 0.01). At 12 months, AC (MOL; p < 0.001), PR (MOL; p = 0.002), and BU (MOL; p < 0.001) increased linearly with higher initial moisture, whereas pH (MOQ; p = 0.023) and LA (MOQ; p = 0.001) exhibited quadratic responses. A similar pattern was observed at 18 months AC (MOL; p < 0.001), PR (MOL; p < 0.001), and BU (MOL; p < 0.001) rose linearly with moisture, while pH (MOQ; p < 0.001) and LA (MOQ; p < 0.001) followed quadratic trends. By contrast, at 24 months, only BU showed a positive linear response to initial moisture (MOL; p < 0.001). The persistence of a moisture-driven butyric signal at 24 months suggests that late-stage clostridial pathways and/or proteolysis account for a disproportionate share of residual variation; tracking NH3–N and biogenic amines alongside BU would strengthen causal attribution to clostridial activity in long-stored silage. At 30% moisture, the elevated pH together with non-detectable lactic acid indicates that microbial fermentation did not proceed, consistent with forage being too dry to support an active lactic fermentation. Notably, the 30% treatments failed to approach pH values (4.2–4.5) typical of well-preserved legume silages (Kung et al., 2018), and low moisture also compromises compaction, leaving oxygen entrapped and predisposing the silage to aerobic losses at opening. Acetate and propionate should not be regarded as inherently negative fermentation products because both acids can inhibit yeasts and molds and may improve aerobic stability after silo opening (Danner et al., 2003;Muck et al., 2018). However, their contribution to preservation depends on the overall fermentation context, particularly sufficient acidification and limited undesirable fermentation. In the present study, the 60% and 70% moisture treatments showed high pH and non-detectable LA together with elevated AC, PR, and BU. Thus, the increased AC and PR in these treatments were unlikely to reflect a desirable antifungal fermentation pattern; rather, when considered alongside BU accumulation and poor acidification, they indicate abnormal fermentation, most plausibly clostridial or butyric activity (Kung et al., 2018). A contribution from heterofermentative LAB is also plausible (shift from LA to AC and 1,2-propanediol, with secondary conversion to PR by associated microbes), in line with reports on heterofermentative pathways in silage (Li et al., 2020;Schmidt and Kung, 2010). Mechanistically, Lentilactobacillus buchneri converts lactate to acetate and 1,2-propanediol during storage, and associated microbes can further oxidize 1,2-propanediol to propionate—providing a biochemical rationale for the linear rise in PR with moisture (Muck et al., 2018). Regarding the opening month, the 12 and 24-month silages were opened in July (summer), exposure to higher ambient temperatures during late storage and sampling could have favored secondary fermentations and proteolysis, helping to explain the higher BU observed relative to 18 months. Elevated storage-to-opening temperatures are a recognized risk factor for aerobic deterioration and secondary fermentations, and thermal load coupled with oxygen ingress accelerates DM and quality losses; thus, the summer-opening context likely amplified the butyric signal at high moisture.

    The dominant effect of moisture content suggests that the ensiling environment, rather than inoculation alone, primarily determined the fermentation pathway of alfalfa silage. The 40–50% moisture treatments likely provided a favorable balance between sufficient water activity for LAB growth and adequate restriction of undesirable microorganisms, resulting in greater lactic acid accumulation and lower pH. In contrast, the 30% treatment appeared too dry to support active fermentation, whereas the 60–70% treatments likely delayed acidification because of substrate dilution, high buffering capacity, and conditions favorable for anaerobic spoilage microorganisms. This interpretation is supported by the microbial community results, in which LAB-associated genera were more closely aligned with lactic acid production at intermediate moisture levels, while Clostridium detection at 60–70% moisture corresponded with higher butyric acid concentrations. Similar moisture-dependent responses have been reported in alfalfa silage, where appropriate wilting improved lactic fermentation, whereas high-moisture conditions increased the risk of clostridial fermentation and undesirable metabolites (Li et al., 2020;Yang et al., 2022).

    3. Microbial community

    Fig. 1 presents the genus-level 16S rRNA gene relative abundances of the microbiota in alfalfa silages as affected by storage duration. Overall, Lacticaseibacillus, Lactiplantibacillus, and Levilactobacillus predominated across treatments, whereas the inoculant genus Lactococcus remained at trace or low relative abundance. By contrast, Pediococcus was clearly detectable in the INO silages at 18 months. This pattern should be interpreted cautiously because the 18-month silages were opened in January, whereas the 12- and 24-month silages were opened in July. Temperature can influence LAB succession and the overall silage microbiota; for example, moderate temperatures may favor rapid development of Lactiplantibacillus- and Pediococcus-related populations during early fermentation, whereas low-temperature conditions can alter LAB succession and select for different lactic acid bacterial communities (Zhou et al., 2016). Nevertheless, Pediococcus should not be assumed to be suppressed under winter conditions because cold-tolerant P. pentosaceus strains have been reported to grow at low temperature and improve silage fermentation under cool conditions (Xu et al., 2019). Therefore, the higher detection of Pediococcus in the INO 18-month silages may reflect a combined effect of inoculation, storage duration, and lower ambient temperature during the final storage/opening period, which may have reduced the activity of competing aerobic spoilage microorganisms and preserved a LAB-dominated profile.

    Seasonal effects may also influence the broader microbial community. Warmer summer conditions during opening and feed-out can favor aerobic spoilage organisms, including yeasts, molds, acetic acid bacteria, and other aerobic or facultative anaerobic bacteria, thereby altering relative abundances after oxygen exposure. In contrast, cooler winter conditions may slow aerobic deterioration and allow LAB-associated DNA signatures to remain relatively more prominent. Farm-scale studies have similarly shown that season-dependent factors affect the chemical profile and microbiota of corn and grass-legume silages, including seasonal shifts in Lactobacillaceae, Acetobacteraceae, and metabolites associated with secondary fermentation (Huffman et al., 2023). Therefore, the microbial differences among storage periods in the present study should be interpreted not only as an effect of storage duration but also as potentially influenced by the season at silo opening.

    At 30% moisture, most LAB failed to proliferate; however, Lacticaseibacillusshowed limited detection in the CON 12-month silage. In all treatments at moisture levels of 60% and 70%, Clostridiumwas detected, indicating an elevated risk of clostridial or butyric fermentation under wetter ensiling conditions. Notably, at 70% moisture, Clostridium relative abundance was lower in INO (5.15, 14.2, and 12.3%) than in CON (8.01, 21.5, and 30.2%) at each storage period, suggesting a partial suppressive effect of inoculation on clostridia under high-moisture conditions; however, this pattern was not consistently supported by butyric acid responses.

    Ⅳ. CONCLUSION

    This study evaluated the effects of target moisture content and microbial inoculation on the chemical composition, fermentation characteristics, and bacterial community of alfalfa silage during long-term storage. Moisture content at ensiling had the strongest and most consistent effect on silage quality. CP generally decreased and ADF generally increased as moisture increased, whereas NDF responses were not consistently quadratic across all storage periods. Fermentation characteristics also showed clear moisture-dependent responses: the 40–50% moisture treatments produced the most favorable lactic fermentation, while the 60–70% moisture treatments showed higher BU and greater Clostridium relative abundance, indicating increased risk of undesirable fermentation. The effect of microbial inoculation was limited and condition-dependent. Although some significant inoculant effects and inoculant × moisture interactions were detected, inoculation did not consistently increase LA or reduce BU across storage periods. At 70% moisture, INO showed lower Clostridium relative abundance than CON, but this pattern was not consistently supported by fermentation acid profiles. Therefore, maintaining ensiling moisture around 40–50% appears to be the most reliable practical strategy for long-term alfalfa silage quality.

    Ⅴ. ACKNOWLEDGEMENT

    This work was carried out with the support of "The Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01593902)", 2026 collaborative research program between university and Rural Development Administration, Republic of Korea.

    Figure

    KGFS-46-2-76_F1.jpg

    Effects of microbial inoculant and moisture contents on alfalfa silage genus-level microbial community during the storage period for 24 months. CON 12, control treatment stored 12 month; INO 12, microbial inoculant treatment stored 12 month; CON 18, control treatment stored 18 month; INO 18, microbial inoculant treatment stored 18 month; CON 24, control treatment stored 24 month; INO 24, microbial inoculant treatment stored 24 month.

    Table

    Chemical composition of alfalfa before ensiling

    Effects of microbial inoculant and moisture content on alfalfa silage chemical composition during long-term storage for 24 months (% of DM)

    CON, control; INO, inoculant; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; MI, microbial inoculant effect; MO, moisture content effect; MI×MO, interaction effect of microbial inoculant and moisture content; MOL, moisture linear effect; MOQ, moisture quadratic effect. a~e means significant differences in the same row.
    Effects of microbial inoculant and moisture contents on alfalfa silage fermentation characteristics during the storage period for 24 months (% of DM)
    CON, control treatment; INO, inoculant treatment; LA, lactic acid; AC, acetic acid; PR, propionic acid; BU, butyric acid; MI, microbial inoculant effect; MO, moisture content effect; MI×MO, interaction effect of microbial inoculant and moisture content; MOL, moisture linear effect; MOQ, moisture quadratic effect. a~d means significant differences in the same row.
    P-values from the overall factorial analysis for chemical composition and fermentation characteristics of alfalfa silage during long-term storage
    CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; LA, lactic acid; AC, acetic acid; PR, propionic acid; BU, butyric acid; MI, microbial inoculant effect; MO, moisture content effect; SP, storage period effect; MO×SP, interaction effect of moisture content and storage period; SP×MI, interaction effect of storage period and microbial inoculant; MO×MI, interaction effect of microbial inoculant and moisture content; MO×SP×MI, interaction effect of moisture content, storage period, and microbial inoculant.

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