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Article

Effect of Inoculation of Lactic Acid Bacteria and Fibrolytic Enzymes on Microbiota in the Terminal and Aerobically Exposed Short-Growing Season Whole-Plant Corn Silage

by
Chunli Li
1,
Jayakrishnan Nair
2,
Eric Chevaux
3,
Tim A. McAllister
1 and
Yuxi Wang
1,*
1
Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Canada
2
School of Agricultural Sciences, Southern Illinois University, Carbondale, IL 62901, USA
3
Lallemand SAS, 31702 Blagnac, France
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(9), 530; https://doi.org/10.3390/fermentation11090530
Submission received: 23 July 2025 / Revised: 27 August 2025 / Accepted: 4 September 2025 / Published: 10 September 2025
(This article belongs to the Special Issue The Use of Lactobacillus in Forage Storage and Processing, 2nd Edition)
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Abstract

An experiment was conducted to evaluate the effects of mixed lactic acid bacteria (LAB) plus fibrolytic enzymes (xylanase + β-glucanase) on bacterial and fungal communities in terminal and aerobically exposed whole-plant corn silage ensiled in a temperate zone. Short-season corn forage was either uninoculated (C) or inoculated (I) with a mixture of LAB containing 1.5 × 105 colony-forming units (cfu)/g Lentilactobacillus hilgardii, 1.5 × 105 cfu/g of Lentilactobacillus buchneri, and 1.0 × 105 cfu/g Pediococcus pentosaceus plus a combination of xylanase + β-glucanase. Silage samples were taken after ensiling in bag silos for 418 days (terminal silage; TS), with subsamples of TS subsequently exposed to air for 14 days (aerobically exposed silage; AS). Regardless of treatment, Firmicutes, Proteobacteria, Cyanobacteria, and Actinobacteria were the predominant phyla in the bacterial microbiome, whilst Ascomycota and Basidiomycota were the predominant phyla in the fungal microbiome in both TS and AS. Lactobacillus, Acetobacter, and Bacillus were the most abundant bacterial genera, whilst Candida, Aspergillus, Vishniacozyma, Pichia, and Issatchenkia were the most abundant fungal genera. Use of silage additive did not change bacterial or fungal alpha or beta diversity during ensiling or aerobic exposure, but decreased (p < 0.01) the relative abundance (RA) of Proteobacteria in both TS and AS, increased (p < 0.01) RA of Firmicutes in AS, but did not affect the RA of fungal phyla in either TS or AS. At the genus level, the additive significantly decreased (p < 0.01) RA of Acetobacter in both TS and AS. The silage additive used in this study significantly affected the composition of multiple microbial genera during ensiling and aerobic exposure by shifting bacterial communities towards enhanced aerobic stability.

1. Introduction

The success of ensiling depends largely on the dynamics of the microbial community, particularly the activity of lactic acid bacteria (LAB) that produce lactic and acetic acids that inhibit microbial activity during ensiling and spoilage microorganisms during aerobic exposure, respectively. A variety of LAB inoculants, including homo- and heterofermentative strains, has been widely adopted to improve silage quality [1]. Whilst homofermentative LAB (e.g., Lactiplantibacillus plantarum (previously Lactobacillus plantarum), Pediococcus pentosaceus) produce primarily lactic acid that accelerates post-ensiling pH decline, heterofermentative LAB (e.g., Lentilactobacillus buchneri (previously Lactobacillus buchneri) enhance the aerobic stability via increasing both lactic and acetic acid production [1]. However, the reported efficacy of the LAB inoculants on ensiling and aerobic stability has been inconsistent depending on the species/strains used in inoculants, inoculation dose, type of forage, and the ensiling conditions that define the competitive interaction between inoculated strains and epiphytic microbes that reside on the forage at the time of ensiling and persist during aerobic exposure [2,3,4,5,6,7].
Inoculants containing both homo- and heterofermentative LAB strains are currently used to promote a rapid decline in silage pH and to produce sufficient acetic acid to promote aerobic stability [8,9,10,11,12]. L. buchneri is the main heterofermentative LAB strain used, but the production of acetic acid by this species typically occurs after ~60 d of ensiling [1,8]. In contrast, L. hilgardii, another heterofermentative LAB, produces acetic acid as quickly as within two weeks after ensiling [9,10,13,14,15]. Research has shown that a combination of L. buchneri and L. hilgardii, as opposed to either singly, has a greater capacity to enhance the aerobic stability of corn [9,16,17,18] and triticale silages [19]. Arriola et al. [20], however, showed that a combination of L. buchneri and L. hilgardii enhanced aerobic stability of sorghum silage and normal-harvested (34% DM), but not late-harvested (43.8% DM) corn silages. Most of these studies were conducted in warm-zone regions, and there is a lack of information regarding their impact on the ensiling fermentation of short-season corn typical of Canadian prairies.
Corn silage is an important, high-yielding, high-quality, energy-dense, and economical forage for the cattle industry worldwide, promoting increased milk production and faster body weight gain. Corn silage production has increased significantly in the Canadian prairies over the last decade due to the progress made in the availability of short-season corn hybrids and warmer growing season temperatures [21,22]. However, there is little information about the effects of mixed LAB inoculants on the ensiling, aerobic stability, and microbiome dynamics during ensiling and aerobic exposure of short-season corn silages. Nair et al. [15] reported that a combination of P. pentosaceus, L. buchneri, and L. hilgardii prolonged the aerobic stability of short-season corn silages, increased their net energy value, and improved the feed efficiency of growing beef cattle. Most of the bacterial strains used as silage inoculants do not possess the fibrolytic enzymes required to liberate carbohydrates from plant cell walls. Therefore, the addition of fibrolytic enzymes to microbial inoculants would be a feasible strategy to increase fiber digestibility and improve utilization of the silage. It has been shown that a combination of P. pentosaceus, L. buchneri, and L. hilgardii with fibrolytic enzymes increased acetic acid concentration and lowered yeast population in short-season corn silages ensiled in silo bags and markedly prolonged the stability of aerobically exposed silages [10]. However, the effects of this inoculant on the broader bacterial and fungal microbiome during ensiling and subsequent aerobic exposure of temperate short-season corn silages are lacking. It was hypothesized that inoculation of LAB strains in combination with fibrolytic enzymes would positively alter the microbiome, leading to enhanced aerobic stability of corn silage after long-term storage in silo bags. The objective of this study was to assess the effect of a mixture of homo- and heterofermentative LAB together with fibrolytic enzymes on the microbiomes associated with ensiled and aerobically exposed short-growing season whole-plant corn silages.

2. Materials and Methods

2.1. Preparation of Corn Silage

Details of corn forage production, harvesting, and ensiling in silo bags were as described by Nair et al. [10]. Briefly, whole-plant corn grown under irrigation near Lethbridge, AB, Canada, was harvested after the first killing frost at two-thirds milk line maturity (43.1% DM). Corn was chopped to a 9.5 mm theoretical length and ensiled in Silobolsa Plastar premium silo bags (2.7 m × 60.0 m; Plaster, Argentina) using an Ag-Bag bagger (Ag-Bag, St. Nazianz, WI, USA). Two silo bags were prepared for each treatment. The treatments were untreated whole-plant corn (Control silage; C) and corn inoculated with a mixture of LAB strains plus fibrolytic enzymes (Inoculated silage; I). The inoculant (Magniva Platinum; Lallemand Specialities, Inc., Milwaukee, WI, USA) was applied at the bagger with ATV sprayers at a rate of 40 mL/t, providing 4.0 × 105 colony-forming units (cfu)/g fresh forage of LAB consisting of a mixture of 1.5 × 105 cfu/g L. hilgardii (CNCM I-4785), 1.5 × 105 cfu/g of L. buchneri (NCIMB 40788), 1.0 × 105 cfu/g Pediococcus pentosaceus, and xylanase (5750 IU/g) and β-glucanase (30,000 IU/g). Control corn was sprayed with deionized water at a rate of 40 mL/t. To reduce cross-contamination between bags, spray systems were flushed with 20 L of deionized water each time treatments were alternated. Both C and I forages were ensiled in two silo bags, with each bag containing both the treatments that were separated by 3 m of untreated corn silage between them, with these regions clearly delineated and marked with spray paint on the outside of the bags. Upon delivery to the bagger, forage was sampled from each truckload with ~200 t of each forage compressed into the two silo bags. The forage was proposed to be ensiled for 120 d prior to opening. However, bags remained ensiled due to COVID-19 restrictions and were not opened until after 418 d of ensiling.

2.2. Determination of Microbial Composition in Terminal Silage

Four batches of samples from C and I silage were collected at a 3 wk interval (batches 1 and 2 from silo bag one and batches 3 and 4 from silo bag two) after opening the bags to feed cattle. For each batch of sampling, ~1.2 kg was collected from five locations across a freshly exposed silo face at a depth of 0.5 m and at least 0.5 m away from the outer perimeter of the bags. Subsamples were combined, divided into two parts, with one part processed for DNA extraction of terminal silage (TS) and the other used to assess bacterial and fungal microbiota after aerobic exposure. Approximately 1 m of silage was removed from the face of each silo and discarded when the bags were first opened.

2.3. Determination of Microbial Compositions in Aerobically Exposed Silages

Composed TS sample in each batch as described above was placed into four 4 L insulated containers (13.5 cm in diameter × 30.9 cm in height) per treatment. The containers were covered with two layers of cheesecloth and placed in a room at 20 °C for 14 d. Two Dallas Thermochron iButton temperature sensors (Embedded Data Systems, Lawrenceburg, KY, USA) were embedded in the silage at ~9.0 and ~18.0 cm within each container and set to record the temperature every 15 min. Two sensors were also placed in the room to measure ambient temperature. The content of each container was subsampled after 14 d to determine the microbial compositions in the aerobically exposed silages (AS).

2.4. DNA Extraction, Quantification, and Quality Assessment

The TS and AS samples were immediately stored at −80 °C and then freeze-dried, ball-ground, and metagenomic DNA extracted using DNeasy PowerSoil® Kit (QIAGEN GmbH, QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. Quality and concentration of the extracted DNA were evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequent to DNA isolation, the quality and quantity of the isolated DNA were evaluated. Concentration of DNA was measured by fluorescence using the Quant-iT™ PicoGreen (Thermo Fisher Scientific, Mississauga, ON, Canada). Purity of the DNA was determined by measuring the ratios of absorbance at 260/280 and 260/230 using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA preparations with a 260/280 ratio between 1.7 and 2.0 and a 260/230 ratio between 2.0 and 2.2 were regarded as suitable for further analysis. The extracted DNA was stored at −80 ˚C until sequenced.

2.5. Analysis of the Composition of the Bacteria and Fungi Communities by Next-Generation Sequencing

The 16S rRNA gene sequence libraries were generated using a two-step PCR protocol. The primers 341-F (CCTACGGGNGGCWGCAG) and 785-R (GACTACHVGGGTATCTAATCC) [23] were used to amplify the V3-V4 hypervariable region of the 16S rRNA gene of bacteria, and the primers ITS1F (5′-TCC GTA GGT GAA CCT TGC GG-3′) and ITS2R (5′-GCT GCG TTC TTC ATC GAT GC-3′) to amplify the ITS region 1 of fungi [24]. Sequencing was carried out using an Illumina MiSeq (Illumina, Inc, San Diego, CA, USA) and the MiSeq Reagent Kit v2 (500 cycles; Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. All PCR amplification and sequencing steps were carried out at Genome Quebec (Montreal, QC, Canada).
Sequences were processed using QIIME2 ver. 2018.2 [25] and the R-package DADA2 (Version 1.40) denoise method. Briefly, the forward and reverse reads were each truncated at a length of 280 bp, and quality control was performed for the reads using QIIME2, with chimeric sequences identified and removed. The reads were merged and taxonomy assigned to generate operational taxonomic units (OTUs) at 99% similarity, using the naïve Bayesian RDP classifier 2.14 [26] and the SILVA SSU database release 138.2 [27] and UNITE database v9 [28]. The number of OTUs per sample and the Shannon diversity index were calculated in R using Phyloseq 1.4.4 [29], and Vegan 2.4.4 [30] was used to determine the Bray–Curtis dissimilarities. Differential bacterial and fungal taxonomy of each type of silage in the treatment of I as compared to C was identified using the R-package Deseq2 v4.3 [31]. Sequences have been submitted to NCBI under PRJNA1288518.

2.6. Statistical Analysis

The number of OTUs and Shannon diversity index were analyzed in R v.4.3.2 by treatment using a linear mixed model implemented with the lmer function in lme4 v 1.1.35 package [32]. The linear mixed model included the fixed effects of treatment (C and I). Post hoc comparison between treatments (C vs. I) was performed within each type of silage (TS or AS) using Tukey’s honestly significant difference test using the postHoc package v.0.1.3. The same comparison was also conducted between the types of silages (TS vs. AS) within each treatment (C or I). Microbial community structure was analyzed with vegan using β-dispersion and permutational multivariate analysis of variance (PERMANOVA, Adonis function; 10,000 permutations) to determine the difference between C vs. I as well as TS vs. AS. To calculate beta diversity, the filtered ASV counts were normalized with size factors calculated with GMPR. Sample–sample distances were determined by the Bray–Curtis metric using phyloseq ordinate and visualized with Non-metric Multidimensional Scaling (NMDS). The genera with the highest abundance in each treatment were identified with phyloseq and visualized. Significant (p < 0.01) differentially abundant genera were identified using DESeq2 and Log fold changes by fitting a negative binomial model to C vs. I. Data from fresh forages were not included in all analyses but were presented for reference to show the microbiota difference after ensiling and aerobic exposure.

3. Results

A total of 5,881,303 (median = 163,450, minimum = 104,573, and maximum = 232,974) and 5,387,262 (median = 147,172, minimum = 7,480, and maximum = 297,704) reads were used to identify 998 unique bacterial and 771 fungal OTUs, respectively.

3.1. Bacterial Community in TS and AS

Alpha diversity was similar (p > 0.05) between C and I in TS and AS, as identified by both the number of OTUs per sample (richness) and the Shannon diversity index (Figure 1). However, alpha diversity was higher (p < 0.05) in AS than in TS for I, but this difference was not observed for C. Permutational multivariate analysis of variance indicated that bacterial community structure differed between C and I in AS (R2 = 0.238, p = 0.006), but not in TS.
Among the phyla identified in the forage, only Firmicutes (51.9%), Proteobacteria (32.6%), Cyanobacteria (11.8%), and Actinobacteria (2.8%) had a relative abundance greater than 1.0% (Figure 2), representing >99.0% of the total bacterial community in all samples. Proteobacteria (53.4%) were the predominant phylum in fresh forage, whereas Firmicutes and Proteobacteria were the predominant phyla in TS (86.3 and 13.0%, respectively) and AS (66.4 and 31.5%, respectively), regardless of treatment. Only the relative abundance (RA) of Firmicutes and Proteobacteria was affected by the silage additive during ensiling (TS) and aerobic exposure (AS). Regardless of treatment, TS had a higher (p < 0.01) RA of Firmicutes than AS, but no difference in the RA of Proteobacteria was observed between TS and AS. The I silage had a higher RA of Firmicutes than C in AS but not in TS, whilst the RA of Proteobacteria was lower (p < 0.01) for I than that for C in both TS and AS.
Among the identified genera, Lactobacillus was the most abundant (44.8%), followed by Acetobacter (19.5%), Bacillus (11.6%), Paenibacillus (2.6%), Pseudomonas (1.3%), Serratia (1.9%), and Enterobacter (1.3%) in all samples irrespective of silage additive treatment and type of silage (Figure 3a,b,c). Acetobacter, Bacillus, and Paenibacillus were detected in TS and AS but not in fresh forage. In TS, I had a numerically higher RA of Lactobacillus (88.7 vs. 56.3%) but numerically lower RA of Bacillus (2.9 vs. 7.4%) and lower (p < 0.01) RA of Acetobacter (2.9 vs. 22.9%) as compared to C. Similarly, in AS, I had numerically higher RA of Lactobacillus (45.9 vs. 25.8%) and Bacillus (32.7 vs. 8.9%) but lower (p < 0.01) RA of Acetobacter (9.2 vs. 53.8%) than C. Comparison between TS and AS showed that within C, TS had higher (p < 0.01) RA of Lactobacillus (56.3% vs. 25.8%) than AS, but within treatment I, TS had higher (p < 0.01) RA of Lactobacillus (88.7% vs. 45.9%) and lower (p < 0.01) RA of Bacillus (2.9% vs. 32.7%) than AS.
Differential taxonomic comparisons showed that in TS, the RA of L. buchneri and B. nealsonii were higher (p < 0.01), while the RA of L. brevis (new name: Levilactobacillus brevis), B. smithii, and B. thermolactis were lower (p < 0.01) for I than C. In AS, however, the RA of L. buchneri, L. rhammnosus (new name: Lacticaseibacillus rhammnosus), L. zeae (new name: Lacticaseibacillus zeae), B. huizhouensis, and B. aryabhattai were higher (p < 0.01) whilst the RA of L. acidipiscis (new name: Ligilactobacillus acidipiscis), L. sakei (new name: Lactilactobacillus sakei), L. plantarum, B. smithii, and B. coagulans were lower (p < 0.01) for I than C. Within the C, the RA of L. buchneri, L. brevis, B. smithii, B. thermolactis were higher (p < 0.01) while the RA of L. plantarum, L. acidipiscis, B. nealsonii, B. thermoamylovorans and B. coagulans were lower (p < 0.01) for TS than for AS. Within treatment I, however, L. plantarum, and B. coagulans were higher (p < 0.01), whilst the RA of L. buchneri, L. zeae, B. huizhouensis, B. nealsonii, and B. clausii were lower for TS than for AS.

3.2. Fungal Community in TS and AS

Alpha diversity was similar (p > 0.05) between the C and I in both TS and AS, as identified by the similar number of OTUs per sample (richness) as well as similar Shannon diversity index (Figure 4). However, alpha diversity was lower (p < 0.05) in AS than in TS for I, but this difference was not observed for C. Permutational multivariate analysis of variance indicated that fungal community structure did not differ (p > 0.05) either between treatments (C vs. I) or between different types of silages (TS vs. AS).
Four fungal phyla, namely Ascomycota, Basidiomycota, Chytridiomycota, and Rozellomycota, were observed, and among these, only Ascomycota and Basidiomycota had an RA greater than 1.0% (Figure 5). These two phyla represented 99.8% of the total fungal community in all samples (fresh forage, TS, and AS) with a RA of 61.4 and 38.4%, respectively. Basidiomycota (93.7%) was the predominant phylum in fresh forage, whilst Ascomycota was the predominant phylum in TS (78.3%) and AS (99.6%), regardless of treatment. Fresh forage had a numerically higher RA of Basidiomycota (93.8%) but a numerically lower RA of Ascomycota (6.21%) as compared to TS (21.6 and 78.3%, respectively) and AS (0.07 and 99.6%, respectively). Additive did not affect the RA of Ascomycota and Basidiomycota in either TS or AS. Within C, the RA of Ascomycota (99.61 vs. 90.77%) and Basidiomycota (0.13 vs. 9.09%) were similar (p > 0.01) between TS and AS. However, the RA of Ascomycota (99.67 vs. 65.8%) was higher (p < 0.01) and that of Basidiomycota (0.004 vs. 33.48%) was lower (p < 0.01) in TS than AS within treatment I.
Among identified genera, Candida was the most abundant (27.3%), followed by Aspergillus (26.7%), Vishniacozyma (14.7%), Pichia (8.5%), Issatchenkia (6.7%), Naumovozyma (3.6%), Cystofilobasidium (2.9%), Monascus (1.2%), and Cryptococcus (1.2%), irrespective of treatment or type of silage (Figure 6a, b, c). Differential taxonomic comparisons indicated that I did not alter the RA of Candida, Aspergillus, Vishniacozyma, Pichia, Issatchenkia, Naumovozyma, Cystofilobasidium, Monascus, and Cryptococcus in either TS or AS, but decreased (p < 0.01) RA of Issatchenkia in TS. A comparison between TS and AS showed that TS had similar RA of Candida, Aspergillus, Vishniacozyma, Pichia, Issatchenkia, Naumovozyma, Cystofilobasidium, Monascus, and Cryptococcus to that of AS for both C and I, with the exception that RA of Candida and Aspergillus for I treatment were higher (p < 0.01) in AS than in TS.
In TS, the RA of C. ethanolica, A. ruber, and V. victoriae were lower (p < 0.01), but that of V. tephrensis and V. carnescens were higher (p < 0.01) for I than for C. In contrast, the RA of C. ethanolica and P. mandshuria were higher (p < 0.01), and I. orientalis was lower for I than for C in AS (Figure 6b,c). For C, the RA of Vishniacozyma spp. and Candida spp. were similar between TS and AS. For I, the RA of Candida spp. was similar, whilst that of V. tephrensis, V. victoriae, and V. carnescens was higher (p < 0.05) in TS than in AS.

4. Discussion

A previous study in our laboratory found that this silage additive increased acetate concentration and reduced yeast populations in whole-plant corn silages ensiled in silo bags and markedly prolonged the stability of silages upon aerobic exposure [10]. However, the effects of this additive on the microbiome of long-term stored short-season corn silage have not been explored. The results reported in this manuscript demonstrated that this additive affected both bacterial and fungal communities during both ensiling and aerobic exposure.

4.1. Effects of the Additive on the Bacterial Community in TS and AS

The similar bacterial alpha diversity between C and I in both TS and AS indicated that the additive used in this study had no effect on bacterial diversity during both ensiling and aerobic exposure. Similar results have also been reported by Drouin et al. [4], who found that bacterial alpha diversity in corn silage was not affected by inoculation with a combination of L. buchneri and L. hilgardii after 159 d of ensiling. However, Drouin et al. [16] observed that bacterial alpha diversity was decreased by this combination after 64 d of ensiling. Furthermore, da Silva et al. [9] reported that the same LAB combination did not affect bacterial diversity in corn silage ensiled for 34 d but decreased it after 99 d of ensiling. On the contrary, Bai et al. [33] showed increased bacterial diversity in corn silage treated with L. buchneri compared to untreated silage ensiled for 60 d. The discrepancy among studies in the effects of inoculants on silage microbiome during ensiling might reflect differences in ensiling length, ensiling conditions, as well as additive composition that affect microbiome dynamics during ensiling. Corn forage was ensiled in bag silos under commercial conditions in this study. Furthermore, in addition to L. hilgardii and L. buchneri, the silage additive used in this study also contained P. pentosaceus and a mixture of xylanase and β-glucanase. Drouin et al. [34] showed that the same silage additive as that used in this study differentially enriched bacteria in mini silos vs. a bunker silo.
The similar bacterial alpha diversity between C and I in AS is likely due to the similar bacterial alpha diversity between the treatments in TS, as well as the fact that both C and I silage deteriorated to a similar extent after 14 d exposure to air. It is well-documented that ensiling decreases microbial diversity due to the accumulation of organic acids over the course of ensiling. The markedly lower Shannon index of inoculated TS as compared to fresh forage and AS is likely attributable to the dominance of LAB over other bacteria and the increased organic acid production resulting from microbial fermentation during ensiling, which is supported by our previous observation [10].
This study revealed that Firmicutes and Proteobacteria were the most abundant phyla in corn silage. The same observation was also reported for grass [35], barley [6], alfalfa [36], and corn silages [17]. The higher abundance of Firmicutes in TS than in AS, regardless of treatment, corresponded with the numerically lower abundance of Proteobacteria in TS than in AS, indicating the deterioration of silage quality due to aerobic exposure. Proteobacteria consist mainly of bacteria such as Enterobacteriaceae, Pseudomonas, Flavobacterium, and Pantoea that are often linked to spoilage and the accumulation of undesirable fermentation products in silages [2]. Therefore, the observation that I had a higher abundance of Firmicutes in AS but a lower abundance of Proteobacteria in both TS and AS compared to C suggests that it shifted the bacterial composition in a manner that led to the improved aerobic stability of the corn silages [10]. Liu et al. [6] and McGarvey et al. [36] also reported that LAB inoculants reduced the abundance of Proteobacteria and increased the abundance of Firmicutes in barley and alfalfa silages.
The finding that Lactobacillus was the most abundant genus in TS is consistent with other studies [4,37,38]. The numerically higher Lactobacillus, but lower abundance of Acetobacter for I than for C in both TS and AS, suggests that the additive increased the abundance of Lactobacillus but decreased the abundance of Acetobacter during both ensiling and aerobic exposure. It is well known that LAB inoculants increase the growth of Lactobacillus in silage, leading to faster acid production and a more rapid decline in pH [10,39,40].
Acetobacter are acid-tolerant bacteria that can oxidize acetate and lactate, leading to aerobic spoilage [41]. In this study, Acetobacter was not detected in the fresh forage but increased to 23.9 and 53.8% in TS and AS for C, compared to 2.9 and 9.2% for I, respectively. This suggests that the additive used in this study inhibited the growth of Acetobacter during ensiling and aerobic exposure, likely due to the rapid fermentation that quickly depleted oxygen during the initial phase of ensiling, as it requires oxygen and is inhibited by the production of acetic acid. It has been shown that the growth of Acetobacter is inhibited by high concentrations but can be promoted by low concentrations of acetic acid [42]. The concentration of acetic acid for I was significantly higher than that for C in both TS and AS [10]. Increases in the RA of Acetobacter during ensiling and aerobic exposure, as well as inhibition by the addition of a LAB inoculant, have also been reported by others [11,43].
The other genera of bacteria, such as Pseudomonas, Enterobacter, Serratia, etc., are mostly epiphytic bacteria and markedly decreased during ensiling, and therefore their abundances were greatly reduced in TS as compared to fresh forage. The similar abundances of these bacteria between C and I in both TS and AS in this study suggest that they had limited effects on the terminal silage or its aerobic stability.

4.2. Effects of the Additive on the Fungal Community in TS and AS

It has been proposed that greater fungal diversity could help improve the aerobic stability of silage if it prevents the growth of the dominant fungi that promote spoilage [19]. Several studies have shown that LAB inoculants increase fungal richness and diversity during ensiling and aerobic exposure and that their efficacy is associated with bacterial species/strains, type of silage, ensiling conditions, etc. For instance, da Silva et al. [17] reported that a combination of L. hilgardii and L. buchneri increased fungal diversity of corn silage after 34 and 99 d of ensiling but had no effect when they were applied alone. Drouin et al. [34] showed that a combination of L. hilgardii, L. buchneri, P. pentosaceus, and a mixture of xylanase and β-glucanase increased richness in mini-silo silage and increased both richness and Shannon index in bunker silage. The greater fungal diversity in terminal and aerobically exposed silages was also reported for barley silage [6] with the addition of LAB inoculants containing L. plantarum, L. casei, and L. buchneri, and for corn silage by Yin et al. [11] with LAB inoculants containing L. buchneri and B. licheniformis. However, the similar fungal alpha diversity between I and C in either TS or AS observed in this study suggests that the silage additive in our study did not affect the richness of the fungal population during the ensiling or aerobic exposure. Nevertheless, the observation that fungal diversity of I was lower in AS than in TS, whilst that of C was similar between TS and AS, suggests that aerobic exposure decreased fungal diversity in I but not C silage. Decreased fungal diversity during aerobic exposure of corn silage treated with or without LAB was also reported in other studies [11,44]. In addition, Liu et al. [45] observed that the extent of a decrease in fungal diversity due to aerobic exposure of barley silage treated with LAB inoculants (L. plantarum, L. casei, and L. buchneri) was greater than that for control barley silage.
Ascomycota and Basidiomycota were the predominant fungal phyla in this study. Others have also reported the dominance of Ascomycota and Basidiomycota in barley [6] and corn silages [11,46,47]. In the current study, Basidiomycota was the dominant phylum before ensiling, accounting for over 93% of the population. However, Ascomycota subsequently became the dominant phylum post-ensiling (90.8 and 65.8% for C and I, respectively) and further increased to over 99% after 14 d aerobic exposure for both C and I. A similar trend was also reported for corn and barley silages [4,6,11]. The observation that Ascomycota abundance in the I terminal silage was lower compared to that of C terminal silage indicated that the additive used in this study decreased the abundance of Ascomycota during ensiling. Most of the yeasts found in corn silage are members of the Ascomycota [48]. Hence, the lowered Ascomycota abundance by the silage additive in this study is partially attributed to the anti-yeast activity of the acetic acid produced by this LAB additive, which is supported by our previous observation that the same silage additive significantly increased acetic acid production and decreased yeast populations in terminal silage and improved aerobic stability [10]. On the contrary, the abundance of Basidiomycota was higher in the I terminal silage than the C terminal silage (33.5 vs. 9.1%). Higher Basidiomycota in hetero-fermentative LAB-treated compared to untreated control was also observed for barley [45], wheat [49], and corn silages [11]. The biological function of Basidiomycota fungi in forage preservation is not well understood. Given its dominance in the epiphytic fungal microbiome and the dynamic changes over the ensiling and aerobic exposure, its role deserves further research.
The predominant fungal genera in TS and AS observed in this study differed from those in other reports. There is variation in the literature regarding dominant fungal genera in both terminal and aerobically exposed corn silages [16,34,44,50,51]. Kazachstania was frequently reported to be the most prominent genus in different silages [52,53,54], but it was not observed in either TS or AS regardless of the treatment in this study. The absence of Kazachstania from the main fungal genera in terminal and aerobically exposed corn silage was also reported by Wang et al. [55]. The discrepancy among the studies may reflect different methodologies used and different geographical locations of the studies. It is generally regarded that fungal microbiota in silages are contaminants from the environment and cropping operation [56] and therefore represent the specific geographical niche of the microbial populations. Because most of the fungi found in silage are related to spoilage, strategies to control these microorganisms in silage may need to consider geographical locations so that the main fungal population can be targeted.
The lower abundances of Candida (16.4 vs. 44.4%), Issatchenkia (3.4 vs. 25.4%), and Naumovozyma (0.01 vs. 5.7%) but higher abundances of Pichia (7.0 vs. 0.3%), Vishniacozyma (19.3 vs. 7.9%), and Aspergillus (27.0 vs. 13.0%) for I as compared to that of C in TS indicated that fungal communities responded differently to the silage additive. A similar trend was also observed for Issatchenkia (0.01 vs. 2.6%), Naumovozyma (0 vs. 10.9%), and Pichia (32.2 vs.0.3%) in AS. Candida and Aspergillus were the most dominant genera in TS and AS. Liu et al. [45] and Wang et al. [55] also reported that a LAB inoculant decreased the abundance of Candida in corn silages. Likewise, increased Vishniacozyma abundance by LAB inoculant was observed for corn [34] and alfalfa silages [48], but its role in silage preservation is unclear. The high abundance of Candida in both TS and AS is consistent with the fact that Candida is a facultative organism that can survive and grow in either aerobic or anaerobic conditions. However, because Aspergillus is an obligate aerobe, its high abundance in TS in this study is difficult to explain. Aspergillus can tolerate low-oxygen and high-acid conditions. The higher-than-normal DM concentration of the corn silage (43%) used in this study may have prolonged the oxygen-depletion time during the initial stage of the ensiling fermentation, leading to Aspergillus propagation. It also could not rule out the contribution of the prolonged ensiling period that increased the chance of oxygen filtration. Further research on long-term storage with periodic samplings would shed light on the mechanism. Aspergillus is a major mycotoxin producer in silages [56]. Therefore, the reduced Aspergillus abundance in I AS may indicate that the additive could reduce the risk of fungal mycotoxins in silages. It has been reported that various LAB inoculants decrease mycotoxins due to their capacity to inhibit fungal growth during ensiling and aerobic exposure [2,12,48,57].
Issatchenkia is a common yeast that contributes to silage aerobic deterioration, and it has been shown that LAB inoculants inhibited the growth of Issatchenkia [45], a result consistent with our study. It needs to be pointed out that the silage samples were aerobically stable for 79 h for C and 251 h for I [10], and therefore, after 14 d of aerobic exposure, all silages had deteriorated. This may explain why the effects of the additive on the fungal microbiome in AS were not as significant as those observed in TS. Ferrero et al. [2] also reported that the effects of LAB inoculants on fungal community were less in silage exposed to air for 14 d as compared to that exposed to 7 d.
Well-fermented silage is usually stable and can be long-term stored under proper management. However, certain microbes that are resistant to ensiling conditions may still be viable, and their metabolic processes continue, affecting the characteristics of the stored silage [34,58,59]. It has been shown that extended storage can increase the digestibility of crude protein, NDF, and starch due to continued acidic breakdown of plant cell walls and continued metabolic processes [58,59]. We have observed that the pH of this silage was similar to that of silage stored for 20-60 d, and that longer periods of ensiling did not negatively impact the nutrient composition, populations of total bacteria, LAB, yeast, and mold or AS of whole-plant corn silage [10]. This study found that Lactobacillus and Acetobacter were the top two bacterial genera for corn silage ensiled for 418 d, which is comparable to a previous study for corn silage ensiled for 220 in the bunker [34]. All together, this information suggests that longer than normal storage time of corn silage could be a viable option in the event of extreme conditions, such as during the COVID-19 pandemic. However, the extended storage time may make the efficacy of the inoculant less marked than within the regular storage time of a silo.
It is proposed that adding fibrolytic enzymes to LAB inoculants during ensiling could enhance ensiling fermentation by increasing carbohydrate availability via enzymatic action for LAB, leading to faster pH decline, thereby improving the silage quality. Irawan et al. [60], however, concluded, through a meta-analysis of 97 published results involving LAB inoculants and fibrolytic enzymes, that all types of inoculants are effective in improving silage quality, but the response of plant materials to inoculants containing fibrolytic enzymes varied. Factors including the strains of LAB used, nature of the enzyme cocktail, type of forage ensiled, ensiling process, and the duration of ensiling can have significant impacts on the fermentation profile, AS, and feed value of inoculated silages [1]. Extended ensiling periods have been reported to increase starch digestibility while having minimal impact on NDF digestibility [61]. Enzymes were part of the silage additive formulation because the LAB strains used in the present study lack fibrolytic activity. Therefore, the results reported here were the net outcomes of the LAB and enzyme combination. Due to the limitations of the experiment arrangement, it was impossible to separate the effects of enzymes from LAB strains in this study. The limited replication number also impacted the statistical power to detect the real experimental effects in this study. Enzymes alone as silage additives are practically not economically viable because an efficient amount of enzymes to really trigger a strong effect on forage digestibility (at ensiling) is cost-prohibitive. Another study using the same silage additive as that used in this study also showed improved silage aerobic stability of corn silage via altering the silage microbiome during different ensiling times [34]. Further metabolism studies evaluating the ruminal and total tract nutrient digestibility would provide detailed information on the impact of the addition of fibrolytic enzymes in silage inoculants on forage cell wall digestibility and potential improvement in feed value.

5. Conclusions

The addition of homo- and hetero-fermentative LAB mixed strains, along with exogenous enzymes, did not affect bacterial and fungal diversity, but lowered abundances of Proteobacteria and Acetobacter at phylum and genus levels, respectively, during both ensiling and aerobic exposure of short-season grown corn silage in the temperate zone. Similarly, the additive also decreased the abundance of Ascomycota during ensiling. These results demonstrate that the additive used in this study affected the microbial composition at the phylum and genus levels during ensiling and aerobic exposure by shifting bacterial and fungal communities towards enhancing aerobic stability. The longer-than-normal duration of the storage may also explain the mild differences observed compared to already reported studies.

Author Contributions

Conceptualization, Y.W., E.C. and T.A.M.; methodology, Y.W., J.N. and C.L.; software, C.L. and Y.W.; validation, C.L. and T.A.M.; formal analysis, C.L.; investigation, C.L., J.N. and Y.W.; resources, T.A.M. and E.C.; data curation, C.L. and J.N.; writing—original draft preparation, C.L.; writing—review and editing, Y.W., T.A.M., J.N. and E.C.; supervision, Y.W. and T.A.M.; project administration, Y.W. and J.N.; funding acquisition, Y.W., T.A.M. and E.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Lallemand Animal Nutrition, Canada, grant number J-002039.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request from the corresponding author.

Acknowledgments

We gratefully acknowledge Lallemand Animal Nutrition, Canada, for its financial support for this study. The authors also thank C. Barkley, H-E. Yang, A. Redman, and B. Baker, for their technical support on this project.

Conflicts of Interest

Author Eric Chevaux was employed by the company Lallemand SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu-script; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
ASAerobically exposed silage
ASVAmplicon sequence variant
CUninoculated silage
cfuColony-forming unit
DMDry matter
IInoculated silage
LABLactic acid bacteria
LFCLog fold change
NMDSNon-metric Multidimensional Scaling
OTUsOperational taxonomic units
PERMANOVAPermutational multivariate analysis of variance
RARelative abundance
TSTerminal silage

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Figure 1. Bacterial alpha-diversity (observed and Shannon) of OTUs in uninoculated (Control, C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS), and at the end of 14 d air-exposure of TS (AS) (n = 4). Different uppercase (sampling time) letters indicate significant differences, p < 0.05.
Figure 1. Bacterial alpha-diversity (observed and Shannon) of OTUs in uninoculated (Control, C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS), and at the end of 14 d air-exposure of TS (AS) (n = 4). Different uppercase (sampling time) letters indicate significant differences, p < 0.05.
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Figure 2. Relative abundance of bacterial 16S rRNA gene sequences at the phylum level observed. OTUs in uninoculated (Control, C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS), and at the end of 14 d air-exposure of TS (AS) (n = 4). All other classified OTUs comprising less than 1% of the total abundance are represented as others/unassigned taxa.
Figure 2. Relative abundance of bacterial 16S rRNA gene sequences at the phylum level observed. OTUs in uninoculated (Control, C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS), and at the end of 14 d air-exposure of TS (AS) (n = 4). All other classified OTUs comprising less than 1% of the total abundance are represented as others/unassigned taxa.
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Figure 3. Mean relative abundance of bacterial species identified in corn silage samples in uninoculated (Control, C; brown color) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I, blue color) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh, (a)), at the end of 418 d ensiling (terminal silage, TS, (b)) and at the end of 14 d air-exposure of TS (AS, (c)) (n = 4). ASV, Amplicon sequence variant; LFC, Log fold change; Node: A branching point representing a common ancestor from which descendant lineages diverge.
Figure 3. Mean relative abundance of bacterial species identified in corn silage samples in uninoculated (Control, C; brown color) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I, blue color) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh, (a)), at the end of 418 d ensiling (terminal silage, TS, (b)) and at the end of 14 d air-exposure of TS (AS, (c)) (n = 4). ASV, Amplicon sequence variant; LFC, Log fold change; Node: A branching point representing a common ancestor from which descendant lineages diverge.
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Figure 4. Fungal alpha-diversity (Observed and Shannon) of OTUs in uninoculated (Control; C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS) and at the end of 14 d air exposure of TS (AS) (n = 4). Different lowercase letters (treatment) and uppercase letters (sampling time) differ at p < 0.05.
Figure 4. Fungal alpha-diversity (Observed and Shannon) of OTUs in uninoculated (Control; C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS) and at the end of 14 d air exposure of TS (AS) (n = 4). Different lowercase letters (treatment) and uppercase letters (sampling time) differ at p < 0.05.
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Figure 5. Relative abundance of fungal ITS gene sequences at the phylum level observed OTUs in uninoculated (Control; C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS) and at the end of 14 d air-exposure of TS (AS) (n = 4). All other classified OTUs comprising less than 1% of the total abundance are represented as others/unassigned taxa.
Figure 5. Relative abundance of fungal ITS gene sequences at the phylum level observed OTUs in uninoculated (Control; C) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh), at the end of 418 d ensiling (terminal silage, TS) and at the end of 14 d air-exposure of TS (AS) (n = 4). All other classified OTUs comprising less than 1% of the total abundance are represented as others/unassigned taxa.
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Figure 6. Mean relative abundance of fungal species identified in corn silage samples in uninoculated (Control; C; brown color) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I, blue color) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh, (a)), at the end of 418 d ensiling (terminal silage, TS, (b)) and at the end of 14 d air-exposure of TS (AS, (c)) (n = 4). ASV, Amplicon sequence variant; LFC, Log fold change; Node: A branching point representing a common ancestor from which descendant lineages diverge.
Figure 6. Mean relative abundance of fungal species identified in corn silage samples in uninoculated (Control; C; brown color) or inoculated corn silage with lactic acid bacterial (LAB) inoculant (I, blue color) containing (cfu/g fresh forage) 1.5 × 105 L. hilgardii, 1.5 × 105 L. buchneri and 1.0 × 105 P. pentosaceus for a total of 4.0 × 105 LAB plus fibrolytic enzymes (xylanase + β-glucanase) whole-plant corn at the beginning of the ensiling (Fresh, (a)), at the end of 418 d ensiling (terminal silage, TS, (b)) and at the end of 14 d air-exposure of TS (AS, (c)) (n = 4). ASV, Amplicon sequence variant; LFC, Log fold change; Node: A branching point representing a common ancestor from which descendant lineages diverge.
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Li, C.; Nair, J.; Chevaux, E.; McAllister, T.A.; Wang, Y. Effect of Inoculation of Lactic Acid Bacteria and Fibrolytic Enzymes on Microbiota in the Terminal and Aerobically Exposed Short-Growing Season Whole-Plant Corn Silage. Fermentation 2025, 11, 530. https://doi.org/10.3390/fermentation11090530

AMA Style

Li C, Nair J, Chevaux E, McAllister TA, Wang Y. Effect of Inoculation of Lactic Acid Bacteria and Fibrolytic Enzymes on Microbiota in the Terminal and Aerobically Exposed Short-Growing Season Whole-Plant Corn Silage. Fermentation. 2025; 11(9):530. https://doi.org/10.3390/fermentation11090530

Chicago/Turabian Style

Li, Chunli, Jayakrishnan Nair, Eric Chevaux, Tim A. McAllister, and Yuxi Wang. 2025. "Effect of Inoculation of Lactic Acid Bacteria and Fibrolytic Enzymes on Microbiota in the Terminal and Aerobically Exposed Short-Growing Season Whole-Plant Corn Silage" Fermentation 11, no. 9: 530. https://doi.org/10.3390/fermentation11090530

APA Style

Li, C., Nair, J., Chevaux, E., McAllister, T. A., & Wang, Y. (2025). Effect of Inoculation of Lactic Acid Bacteria and Fibrolytic Enzymes on Microbiota in the Terminal and Aerobically Exposed Short-Growing Season Whole-Plant Corn Silage. Fermentation, 11(9), 530. https://doi.org/10.3390/fermentation11090530

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