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Cited by 30Ruminant Nutrition and Digestive PhysiologyProbiotics and Fermented FoodsAnimal Nutrition and Physiology

Succession of Microbial Communities of Corn Silage Inoculated with Heterofermentative Lactic Acid Bacteria from Ensiling to Aerobic Exposure

Open access
Guan, Hao, Ran, Qifan, Li, Haiping, Zhang, Xinquan·2021·Fermentation
DOI 10.3390/fermentation7040258
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Abstract

To further explore the effects of heterofermentative lactic acid bacteria (LAB) on silage fermentation and aerobic stability, whole-plant corn at around the 1/2 milk-line stage was freshly chopped and ensiled in laboratory silos with deionized water (control), Lactobacillus buchneri (LB), or L. rhamnosus (LR). Each treatment was prepared in triplicate for 3, 14, and 60 d of fermentation, followed by 3 and 7 days of aerobic exposure. The dynamic changes in microbial community were studied by single molecule real-time (SMRT) sequencing. The results showed that the two LAB inoculants altered the microbial communities in different ways. Succession from L. plantarum to L. buchneri and L. rhamnosus was observed in LB- and LR-treated silage, respectively. Both silages improved aerobic stability (82 and 78 h vs. 44 h) by occupying the microbial niche to produce higher levels of acetic acid at terminal fermentation. Because Acetobacter fabarum dominated in the silages after aerobic exposure, beta diversity dramatically decreased. In this study, a. fabarum was reported for the first time in silage and was related to aerobic spoilage. The two heterofermentative LAB produced acetic acid and improved the aerobic stability of the corn silage by occupying the microbial niche at terminal fermentation. Inoculated L. rhamnosus had a greater pH for a longer period of time after opening and less DM loss at day 7.

AI snapshot

Written from the paper’s full text.

TL;DR

In whole-plant corn silage, inoculation with heterofermentative *Lactobacillus buchneri* or *L. rhamnosus* altered bacterial succession and improved aerobic stability compared with control (82 and 78 h vs. 44 h). SMRT sequencing showed early *L. plantarum* dominance, terminal-fermentation dominance by the inoculated LAB, and *Acetobacter fabarum* dominance after aerobic exposure, with *A. fabarum* reported for the first time in silage.

Key findings

  • Both inoculated silages had greater aerobic stability than the control: LB 82 h and LR 78 h vs. control 44 h (p < 0.05). At 3 d aerobic exposure, DM loss was lower in LB (3.07%) and LR (3.76%) than control (6.64%); at 7 d, LR had lower DM loss (23.55%) than control (27.46%) and LB (26.53%) (p < 0.05).
  • At 60 d ensiling and 3 d aerobic exposure, LB and LR did not differ significantly in acetic acid, both in the range of 2.5–3.3% DM; at 7 d aerobic exposure, LR had significantly higher acetic acid than LB (p < 0.05). The control had no acetic acid except 0.78% DM at 60 d.
  • Fresh corn was dominated by *Leuconostoc* spp. (77%), *Lactobacillus* spp. (20%), and *Klebsiella* spp. (1.7%). After 3 d ensiling, *Lactobacillus* spp. dominated control (96%), LB (99%), and LR (100%); *L. plantarum* dominated early, *L. buchneri* dominated LB from 14–60 d, and *L. rhamnosus* was highest in LR after 60 d. After 7 d aerobic exposure, *Acetobacter* spp. exceeded 95% in all silages, and *A. fabarum* was first reported in silage and related to aerobic spoilage.
  • Control silage at 3 d had higher alpha diversity than other treatments (observed species 21, Shannon 0.747, Simpson 0.202, Chao1 23.117). After aerobic exposure, beta diversity dramatically decreased because *A. fabarum* dominated.

Methods

  • Whole-plant corn (*Zea mays* L. cultivar Yayu No.8) was harvested at the 1/2 milk-line stage with DM around 28%, chopped to 15 mm, and ensiled in polyethylene bags at about 300 kg/m³ at 30–35°C. Treatments were control, *L. buchneri* 40788, and *L. rhamnosus* CGMCC 18233, each applied at around 1 × 10⁶ cfu/g fresh forage, with three biological replicates; silos were opened at 3, 14, and 60 d, followed by aerobic exposure for 3 and 7 d.
  • Fermentation profiles included pH, volatile fatty acids by HPLC, NH₃-N, microbial counts (LAB, yeast, mold, enterobacteria), chemical composition (DM, CP, NDF, ADF, WSC), and DM loss.
  • Bacterial communities were analyzed by full-length 16S rRNA PCR (27F/1541R) and PacBio RS II SMRT sequencing; QIIME was used for alpha diversity, UCLUST for OTUs at 98.6% similarity, and RDP II for taxonomy. Two-way ANOVA was used for fermentation and microbial counts, one-way ANOVA for chemical components, and Tukey’s HSD with p < 0.05.

Limitations

  • Stated in the paper: the true role of *A. fabarum* during ensiling requires future study; the aerobic stability improvement lasted only 3.5 days; and a small amount of lactic acid was detected after 7 d aerobic exposure “due to an unknown reason.”
  • Evident from the text: the study used laboratory-scale polyethylene bag silos, one corn cultivar at one location and harvest, and a high storage temperature of 30–35°C, which may limit generalization to farm-scale or other conditions.
  • Evident from the text: the community analysis focused on bacteria via SMRT sequencing, while yeasts and molds were assessed only by plate counts, so fungal community dynamics during aerobic spoilage were not characterized at the same resolution.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/fermentation7040258

Cites

53 of 53 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 8 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    DM, DM loss, CP, NDF, ADF, and WSC showed no significant differences between different treatments at 60 d of ensiling (p > 0.05).

    “DM, DM loss, CP, NDF, ADF, and WSC showed no significant differences between different treatments at 60 d of ensiling (p > 0.05).”
    Quote in the paperSubstantial claim
  • Findingown data

    There was no difference in lactic acid between treatments at 14 and 60 d of ensiling (p > 0.05).

    “There was no difference between treatments at 14 and 60 d of ensiling (p > 0.05).”
    Quote in the paperNot a substantial claim
  • Findingown data

    The abundance of Acetobacter spp. eventually exceeded 95% in all silages after aerobic exposure for 7 d.

    “The abundance of Acetobacter spp.eventually exceeded 95% in all silages after aerobic exposure for 7 d.”
    Quote in the paperSubstantial claim
  • Findingown data

    There was no significant difference in pH between different treatments at the same time point during ensiling (p > 0.05).

    “During the silage process, there was no significant difference in pH between different treatments at the same time point (p > 0.05).”
    Quote in the paperNot a substantial claim
  • Findingreasoning

    Beta diversity dramatically decreased after aerobic exposure.

    “Because Acetobacter fabarum dominated in the silages after aerobic exposure, beta diversity dramatically decreased.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 15
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Findingown data

    The abundance of Acetobacter spp. in the control silage increased dramatically at 60 d, which accounted for 30%.

    “The abundance of Acetobacter spp. in the control silage increased dramatically at 60 d, which accounted for 30%.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was no significant difference in the acetic acid concentrations between the LB and LR treatments at 60 d of ensiling and 3 d of aerobic exposure (p > 0.05); both were in the range of 2.5-3.3%DM.

    “There was no significant difference in the acetic acid concentrations between the LB and LR treatments at 60 d of ensiling and 3 d of aerobic exposure (p > 0.05); both were in the range of 2.5-3.3%DM”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Lactobacillus spp. began to dominate after 3 d of ensiling; the control, LB, and LR silage accounted for 96%, 99% and 100%, respectively.

    “Lactobacillus spp. began to dominate after 3 d of ensiling; the control, LB, and LR silage accounted for 96%, 99% and 100%, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After 3 d of aerobic degeneration, the abundance of Acetobacter spp. exceeded 95% and 28% in the control and LB groups, respectively.

    “After 3 d of aerobic degeneration, the abundance of Acetobacter spp.exceeded 95% and 28% in the control and LB groups, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DM loss increased dramatically after 7 days of aerobic exposure, exceeding 20% for all treatments.

    “DM loss increased dramatically after 7 days of aerobic exposure, exceeding 20% for all treatments”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Acetic acid in the LB- and LR-treated groups increased during ensiling and then decreased after aerobic exposure.

    “In this study, acetic acid in the LB-and LR-treated groups increased during ensiling and then decreased after aerobic exposure.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The pH values of all treatments of corn silage decreased below 4 at 3 d of fermentation and remained at that level until 60 d.

    “In this study, the pH values of all treatments of corn silage decreased below 4 at 3 d of fermentation and remained at that level until 60 d.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Lactic acid in the control was higher than that in other groups (p < 0.05), reaching 5.68% DM at 3 d of fermentation.

    “Lactic acid in the control was higher than that in other groups (p < 0.05), reaching 5.68% DM at 3 d of fermentation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The number of LAB in each treatment showed a downward trend, with none detectable after 3 d of aerobic exposure.

    “In current study, the number of LAB in each treatment showed a downward trend, with none detectable after 3 d of aerobic exposure (Figure 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Firmicutes, Cyanobacteria, and Proteobacteria were determined to be different bacteria at the phylum level.

    “As shown in Figure 4, Firmicutes, Cyanobacteria, and Proteobacteria were determined to be different bacteria at the phylum level.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    L. buchneri often works after 30 d of ensiling.

    “This result is consistent with reports that L. buchneri often works after 30 d of ensiling [1].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Andrae, 2001, Effect of hybrid, maturity, and mechanical processing of corn silage on intake and digestibility by beef cattle, J. Anim. Sci. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    LAB can quickly occupy a dominant position and simplify the complex microbial community in silage.

    “LAB can quickly occupy a dominant position and simplify the complex microbial community in silage”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [48] Ranjit, 2000, The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage, J. Dairy Sci. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The two additional heterofermentative LAB were effective but mainly in the middle and late stages of ensiling.

    “The two additional heterofermentative LAB were effective but mainly in the middle and late stages of ensiling.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetobacter fabarum was first reported to be isolated from Ghanaian cocoa bean heap fermentation.

    “Acetobacter fabarum was first reported to be isolated from Ghanaian cocoa bean heap fermentation [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Villa, 2010, Study of microbiological and nutritional quality of corn silage in two Colombian ecosystems, Rev. Colomb. Cienc. Pecu. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    This result was consistent with the results observed for alpha diversity.

    “This result was consistent with the results observed for alpha diversity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    SMRT technology can be used to thoroughly explore microorganism communities at the species level.

    “SMRT technology can be used to thoroughly explore microorganism communities at the species level”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Inoculated silages dominated the microbial community, making the silage more unified.

    “while inoculated silages dominated the microbial community, making the silage more unified.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    SMRT technology provides more accurate information for the development and utilization of LAB inoculants.

    “thus providing more accurate information for the development and utilization of LAB inoculants.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Unweighted and weighted unifrac can be used to compare the differences between two different microbial communities from an evolutionary perspective.

    “Unweighted and weighted unifrac can be used to compare the differences between two different microbial communities from an evolutionary perspective [55].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] Weatherburn, 1967, Phenol-hypochlorite reaction for determination of ammonia, Anal. Chem. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The diversity of silage bacteria in each treatment became smaller after aerobic exposure because Acetobacter fabarum had the greatest advantage among the bacterial community.

    “the diversity of silage bacteria in each treatment in the present study became smaller after aerobic exposure because Acetobacter fabarum had the greatest advantage among the bacterial community.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Inhibiting the consumption of raw materials by microorganisms is considered one of the signs of successful silage.

    “inhibiting the consumption of raw materials by microorganisms is considered one of the signs of successful silage [48].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Ranjit, 2000, The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage, J. Dairy Sci. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Two heterofermentative LAB improved aerobic stability by producing higher levels of acetic acid.

    “Two heterofermentative LAB improved aerobic stability by producing higher levels of acetic acid”
    Quote in the paperSubstantial claim
22 of 22

  • The paper relies on this without support.

    There is presently no report on Acetobacter fabarum in silage.

    “However, there is presently no report on Acetobacter fabarum in silage; thus, its true role during ensiling requires future study.”

  • The paper relies on this without support.

    No report on Acetobacter fabarum in silage exists.

    “there is presently no report on Acetobacter fabarum in silage”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conclusion says LR maintained a greater pH, but it had the lowest pH at day 7

    Major

    The conclusion says the LR-treated silage had a greater pH for longer after opening. Table 2 shows that its pH at 7 days of aerobic exposure was 5.04, below both the control (6.02) and LB (6.54); it was also below the control at 3 days.

  • Citation [24] names a different study than reference 24

    Minor

    The text attributes a small-grain silage result to Duniere and colleagues as reference [24], but reference 24 lists Haas and colleagues and concerns chimeric sequencing reads.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Muck, 2006, Microbial inoculants for silage, Focus Forage, 8, 1
  2. Mcdonald, P., Henderson, A.R., and Heron, S.J.E. (1991). The Biochemistry of Silage, John Wiley & Sons.

Cited by

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Related

Authors
Guan, H., Ran, Q., Li, H., & Zhang, X.
Affiliations
  • Sichuan Agricultural University (CN)
  • Chongqing Academy of Animal Science (CN)
  • Qinghai Normal University (CN)
Corresponding author
Zhang, Xinquan
ORCID
Venue
Fermentation
Publisher
MDPI AG
Published
2021-11-04
Volume
7
Issue
4
Pages
258
ISSN
2311-5637 (ISSN-L)
DOI
10.3390/fermentation7040258
OpenAlex
W3208345373
Semantic Scholar
d973b01dea03f74a484ca2eea121cb42b312af37
Cited by
30
References
53
Citations by year
2026: 3 · 2025: 7 · 2024: 6 · 2023: 7 · 2022: 7
FWCI
5.71
Type
journal article
Language
en
Open access
Licence
Locations
  • Fermentation (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • China Agriculture Research System of MOF and MARA (CARS-34)
  • Fundamental Research Funds for the Central Universities,” Southwest Minzu University (2021PTJS30)
Topics
  • Life Sciences › Agricultural and Biological Sciences › Agronomy and Crop Science › Ruminant Nutrition and Digestive Physiology
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Nutrition and Physiology
Keywords
corn silage, heterofermentative lactic acid bacteria, Lactobacillus buchneri, Lactobacillus rhamnosus, silage fermentation, aerobic stability, microbial succession, aerobic spoilage, acetic acid production, silage inoculation, silage pH, dry matter loss

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