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Article

Exploring the Fermentation Profile, Bacterial Community, and Co-Occurrence Network of Big-Bale Leymus chinensis Silage Treated with/Without Lacticaseibacillus rhamnosus and Molasses

by
Baiyila Wu
1,2,†,
Xue Cao
1,2,†,
Mingshan Fu
3,
Yuxin Bao
3,
Tiemei Wu
1,2,
Kai Liu
1,2,
Shubo Wen
1,2,
Fenglin Gao
4,
Haifeng Wang
3,*,
Hua Mei
1,2,* and
Yang Song
1,2,*
1
College of Animal Science and Technology, Inner Mongolia Minzu University, Tongliao 028000, China
2
Inner Mongolia Engineering Technology Research Center for Prevention and Control of Beef Cattle Diseases, Tongliao 028000, China
3
Tongliao Academy of Agriculture and Animal Husbandry Sciences, Tongliao 028015, China
4
Naiman Banner Animal Disease Prevention and Control Center, Tongliao 028000, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(8), 1888; https://doi.org/10.3390/agronomy15081888
Submission received: 26 June 2025 / Revised: 1 August 2025 / Accepted: 4 August 2025 / Published: 5 August 2025
(This article belongs to the Special Issue Innovative Solutions for Producing High-Quality Silage)
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Versions Notes

Abstract

The purpose of this study was to investigate the effect of different additives on the microbial composition, fermentation quality, and bacterial community structure of big-bale Leymus chinensis silage. An experiment was set up with four treatment groups: a control (C) group, Lacticaseibacillus rhamnosus (L) group, molasses (M) group, and L. rhamnosus + molasses (LM) group, with three replications per group, and L. chinensis silages were fermented for 20 and 40 days. The lactic acid, acetic acid, 1,2-propanediol, and propionic acid contents increased, and pH, butyric acid, 1-propanol, and ethanol contents decreased in the L, M, and LM groups compared to the C group. In the LM group, the number of lactic acid bacteria was the highest, while the pH was the lowest. Enterobacter and Paucibacter were the main dominant genera in the C group. The addition of L. rhamnosus and molasses increased the relative abundance of Lactobacillus, Weissella, and Enterococcus. Lactobacillus abundance correlated positively (p < 0.01) with Lactococcus, Enterococcus, and Weissella and correlated negatively with Enterobacter and Paucibacter. Conversely, Enterobacter and Paucibacter showed a strong positive correlation (p < 0.01, R = 0.55) during fermentation. Lactobacillus, Enterococcus, and Weissella were positively associated (p < 0.01) with acetic and lactic acid levels, while Enterobacter abundance was correlated positively (p < 0.05, R = 0.43) with 1,2-propanediol content. In summary, the addition of both L. rhamnosus and molasses improved the fermentation quality and bacterial community structure of big-bale L. chinensis silage. In addition to inhibiting harmful microorganisms, this combination improved the fermentation products of big-bale L. chinensis silage through microbial regulation.

1. Introduction

Forage is crucial for the sustainable development of the livestock industry. Leymus chinensis belongs to the perennial grass family and is one of the key forage species for the development of the forage industry in China [1]. It is widely distributed in grassland in Inner Mongolia due to its tolerance to complex environments (e.g., extremely cold regions at −47.5 °C and arid regions with soil humidity below 6%) [2,3,4]. L. chinensis is an important forage resource for extending the supply period of green forage because of its abundant young stems and leaves [5]. The use of big-bale silage in low-temperature regions has been highly favored in recent years. Compared with the traditional method of making hay, big-bale silage can effectively reduce forage loss and preserve the nutritional value of fresh material [6]. Therefore, using fresh L. chinensis as raw material in big-bale silage can not only improve the utilization rate of L. chinensis but also increase the content of nutrients for ruminants.
It is difficult to make L. chinensis silage because L. chinensis has a lower water-soluble carbohydrate content and lactic acid bacteria (LAB) count compared with other grasses [7]. Molasses, a nutritional additive, serves as a fermentation substrate for silage. Its addition accelerates the fermentation process and enhances the fermentation quality, the neutral detergent fiber, and acid detergent fiber digestibility of oat, king grass, soybean, and alfalfa silage [8,9,10]. The fermentation quality of silage is closely related to the strains of LAB it contains. L. rhamnosus is a homofermentative LAB with strong acid resistance. In an anaerobic environment, they can convert water-soluble carbohydrates in silage into organic acids, which reduce the pH value, inhibit spoilage microorganisms, and preserve nutrients [11]. We isolated several LAB strains with good fermentation products from oat, Caragana korshinskii, alfalfa, and Leymus chinensis silage. Wu et al. [12] found that the addition of L. rhamnosus and Lentilactobacillus buchneri to Caragana korshinskii silage not only improved the fermentation quality but also inhibited aerobic spoilage. According to size and shape, eighty-five LAB strains were isolated from big-bale L. chinensis silage stored for 180 days. L. rhamnosus is not only resistant to low temperatures but also has high acid resistance and an antibacterial effect on Pichia anomala and Aspergilus flavus, which cause silage spoilage. Nevertheless, information is lacking regarding the characteristics of L. rhamnosus isolated from big-bale L. chinensis silage and its effects on the fermentation products and bacterial community structure of big-bale L. chinensis silage. Hence, we identified and characterized an L. rhamnosus strain from big-bale L. chinensis silage using 16S ribosomal RNA gene sequencing and phenotyping. It is essential to study the alterations in fermentation quality and bacterial community structure during fermentation when L. rhamnosus is added to big-bale L. chinensis silage.
Even though some investigations have been carried out on fermentation products, bacterial communities, and neutral detergent fiber and acid detergent fiber digestibility in laboratory-scale L. chinensis silage [13,14], only a small number of studies have assessed the fermentation quality and bacterial community structure in big-bale L. chinensis silage during fermentation. Therefore, we examined the effects of L. rhamnosus (L), molasses (M), and L. rhamnosus + molasses (LM) on the microbial composition, bacterial community structure, and fermentation products of big-bale L. chinensis silage during fermentation using high-performance liquid chromatography integrated with high-throughput sequencing technology.

2. Materials and Methods

2.1. Experiment Material and Ensiling Preparation

On 21 August 2024, we collected L. chinensis in the early flowering stage from the experimental field of the Inner Mongolia Dairy Sheep Breeding Technology Company (120°14′–121°39′ E, 43°12′–44°37′ N) in Huatugula Town, Inner Mongolia. Using a cutting machine, the freshly harvested L. chinensis was cut into sizes of 15 to 25 mm to obtain a dry matter level of about 328 g/kg. The experiment consisted of a control (C) group, molasses (M) group, L. rhamnosus (L) group, and L. rhamnosus + molasses (LM) group. In the L group, L. rhamnosus cultures were prepared through 96 h of anaerobic incubation in MRS broth at 30 °C and diluted using sterile physiological saline. Thereafter, L. rhamnosus was added at a concentration of 1 × 106 CFU/g. In the M group, molasses was added at a concentration of 20 g/kg. In the LM group, L. rhamnosus (1 × 106 CFU/g) and molasses (20 g/kg) were added together. In the C group, an equal volume of purified water was added. After fully mixing the L. rhamnosus and molasses with the fresh L. chinensis, the samples were tightly wrapped in eight layers of plastic film to ensure the anaerobic fermentation of the big bales (40 cm diameter × 40 cm length), each of which weighed approximately 200 kg. These bales were stored in three layers outdoors at 23 °C in sunny weather, with each treatment replicated three times. The fermentation of L. chinensis silage was examined at 20 and 40 days.

2.2. Examination of Fermentation Characteristics, Microbial Populations, and Chemical Composition Profiles

In total, 25 g of the silage samples was blended with 225 mL of purified water for 1 min. The resulting homogenate was then filtered through a 0.22-μm membrane, yielding water extracts. These extracts served to measure the concentrations of butyric, lactic, acetic, and propionic acids, as well as the pH value. A glass electrode pH meter (PHS-320; Boqu Ltd., Shanghai, China) was used to measure the extracts’ pH values. The levels of ammonia nitrogen (NH3-N) were determined using the phenol–hypochlorite method [15]. Meanwhile, ion-exclusion polymeric HPLC with an RI detector was employed to assess the fermentation qualities [16].
On a clean bench, a series of dilutions was carried out to quantify various microorganisms. Mold and yeast were enumerated on potato dextrose agar (C010287, Haibo Ltd., Qingdao, China) at pH 3.4; enterobacteria were enumerated using violet-red bile agar (HBBD0114, Haibo Ltd., Qingdao, China); and lactic acid bacteria were enumerated on de Man, Rogosa, and Sharpe agar (HB0384; Jiya Ltd., Shanghai, China). Colony counts were determined by assessing viable microorganisms in the samples.
The dry matter (DM) content of fresh material and Leymus chinensis silage was determined by drying samples at 65 °C for 48 h, then pulverizing them through a 1 mm sieve with a Wiley mill (SM300; Chile Ltd., Shanghai, China). Water-soluble carbohydrates (WSCs), crude protein (CP), detergent fiber (ADF), and neutral detergent fiber (NDF) were analyzed according to the standard procedures outlined by Wu et al. [16] and Van Soest et al. [17].

2.3. Bacterial Sequencing Analysis

In total, 40 g of fresh material and L. chinensis silage was added to 160 mL of sterile phosphate-buffered saline at pH 7.4, then oscillated at 125 rpm for three hours using an electronic oscillator. Subsequently, the mixture was filtered through two-layer gauze, and the filtrate was centrifuged at 4 °C and 12,000× g for 15 min. The supernatants were discarded, and the pellets were stored on dry ice. Biomarker Technologies (Beijing, China) performed DNA extraction, metagenomic sequencing, and PCR amplification. Data processing and Illumina MiSeq sequencing were also conducted. UPARSE version 7.1 clustered operational taxonomic units (OTUs) at a 97% similarity threshold [18]. Chimeric sequences were removed, and the Ribosomal Database Project classifier (version 2.2) was used to classify and analyze feature OTU sequences (at a 0.7 confidence level) against the 16S rRNA databases (Silva v138) [19]. Finally, the raw sequence data were submitted to the NCBI Sequence Read Archive (accession number: PRJNA1283056).

2.4. Statistical Analyses

The effects of storage time and addition on the microbial composition, bacterial community structure, and fermentation products of fermented L. chinensis were analyzed using the John’s Macintosh Project (JMP) software (version 15.2.0; SAS Institute, Tokyo, Japan). Results were estimated via two- and one-way analyses of variance (ANOVA) to assess the effects of storage time and additive, respectively. Tukey’s test was used for multiple comparisons, with statistical significance defined as p < 0.05. Additionally, a free online biomarker analysis platform (https://www.biocloud.net/ (accessed on 16 December 2015)) was utilized to evaluate microbial diversity, analyze correlations between bacterial communities and fermentation products, and explore bacterial co-occurrence networks.

3. Results and Discussion

3.1. Chemical and Microbial Composition of Fresh Leymus Chinensis

As shown in Table 1, the DM content of the fresh L. chinensis was 328 g/kg, which was lower than the DM content of 436 g/kg reported by Wu et al. [20]. The CP content was observed to be 92.21 g/kg DM, which is lower than that reported by Liu et al. [21]. To ensure that good-quality silage was obtained, the content of WSC could not be less than 60 g/kg DM. The WSC content of the fresh L. chinensis used in this study was 42.98 g/kg DM. Without additives to assist in fermentation, it may be difficult to achieve the desired preservation results. The number of LAB attached to the surface of fresh material (FM) was one of the indicators used to assess whether exogenous LAB were added or not. In this study, the LAB count was 3.62 log cfu/g FM, and the enterobacteria and yeast counts were 5.62 log cfu/g FM and 5.34 log cfu/g FM, respectively. We found that the LAB count in the fresh L. chinensis was less than 5.0 log cfu/g FM, and the enterobacteria and yeast counts were higher than the LAB count. This suggests that the addition of L. rhamnosus and molasses to big-bale L. chinensis silage may be beneficial for faster and better silage fermentation.

3.2. Fermentation Characteristics and Microbial Counts of Big-Bale Leymus Chinensis Silage Treated with/Without Lacticaseibacillus Rhamnosus and Molasses

As shown in Table 2, after 20 days of fermentation, a higher pH and lower total organic acid content resulted in poor silage quality in the C group. The acetic acid and lactic acid contents were 5.67 and 10.78 g/kg of DM in the C group, respectively. The LAB count, lactic acid, acetic acid, and propionic acid contents gradually increased, and the butyric acid content decreased in the M, L, and LM groups compared to the C group. The C group had 1,2-propanediol and 1-propanol contents of 0.67 and 3.14 g/kg DM, respectively. It has been reported that some strains of heterofermentative LAB can convert lactic acid to 1,2-propanediol and acetic acid during short-term storage [22]. 1-propanol and butyric acid often exist in small amounts in silage [23]. The addition of molasses provided sufficient WSCs for the growth of heterofermentative LAB in the silage [9]. Hence, the acetic acid content was higher in the M and LM groups than in the C and L groups. After 40 days of fermentation, the LM group had a pH < 3.9 and higher lactic, acetic, and propionic acid contents than the C, L, and M groups. This indicated that the combined addition of L. rhamnosus and molasses to big-bale L. chinensis silage synergistically produces more beneficial organic acids, thus further improving the fermentation quality of big-bale L. chinensis silage. After 20 and 40 days of fermentation, the ethanol content and the enterobacteria and yeast counts in the L, M, and LM groups decreased compared to those in the C group, highlighting the positive effects of L. rhamnosus and molasses on inhibiting ethanol fermentation and the growth of harmful microorganisms. Lower enterobacteria counts and yeast counts were observed in the LM group, further confirming that the combined addition of L. rhamnosus and molasses was more effective in improving the fermentation quality of big-bale L. chinensis silage. The interaction effect of these two factors has a significant impact on the microbial count and fermentation parameters. This result indicates that a deeper understanding of these interactions could help optimize practical applications, determining the optimal storage period for specific additive treatments to maximize fermentation quality.

3.3. Bacterial Diversity and Bacterial Community Structure of Big-Bale LEYMUS Chinensis Silage Treated with/Without Lacticaseibacillus Rhamnosus and Molasses

A Venn plot was utilized to visually present the quantity of common and unique OTUs (operational taxonomic units) across different treatment groups (Figure 1A). All groups had 64 common OTUs and 42,782 exclusive OTUs. The distribution of these exclusive OTUs was as follows: fresh material, 4991; 20-day fermentation, 17,130; and 40-day fermentation, 20,661.
Figure 1B shows the distribution and differences in bacterial communities between the different sample groups in big-bale L. chinensis silage treated with L. rhamnosus and molasses. There is a clear separation of bacterial communities between the fresh samples and the big-bale L. chinensis silage samples. In our previous research, similar results were also obtained when molasses and L. rhamnosus were added to big-bale alfalfa silage [24]. In addition, compared with the M40 group, the bacterial community structures of the LM40 and the L40 groups had a high degree of similarity. This may be because both groups were inoculated with L. rhamnosus. These results indicate that the inoculation of L. rhamnosus can significantly change the bacterial community structure of big-bale L. chinensis silage.
The most prominent genera of the bacterial community before and after the fermentation of L. chinensis are shown in Figure 2A. Enterobacter and Paucibacter were detected as the dominant genera before the fermentation of L. chinensis. Their relative abundance decreased significantly during the ensiling process, especially in silage with the addition of L. rhamnosus and molasses. Lactobacillus, Enterococcus, Enterobacter, and Weissella were highly abundant in the L, M, and LM groups. After 20 and 40 days of fermentation, poor fermentation quality was observed in the control group, which could be related to Enterobacter and Paucibacter. Enterobacter are facultative anaerobes that compete with LAB for fermentation substrates during the fermentation of L. chinensis (Figure 2C). The action mechanism of Paucibacter is similar to that of Enterobacter, and both can cause the deterioration and nutrient loss of silage [25]. In addition, we found that Enterobacter growth was inhibited in highly acidic environments, which is consistent with the findings of Bach et al. [26].
Lactobacillus is one of the beneficial bacteria in silage (Figure 2B). In the present study, Lactobacillus (29.9%) and Lactococcus (8.4%) peaked in the LM group after 20 days of fermentation. Lactobacillus was also the most abundant genus at 22.7% and 20.5% in the M and L groups. The increase in the abundance of Lactobacillus and Lactococcus significantly increased the lactic acid level, which was consistent with the lower pH and higher lactic acid levels noted in previous results [24]. Xu et al. [27] determined that the prolonged fermentation of silage causes bacteria to lack available fermentation substrates, ultimately leading to a decrease in the number of lactic acid bacteria. Therefore, in the L. chinensis silage, Lactobacillus showed higher levels at 20 days of fermentation compared to 40 days of fermentation. Moreover, the abundance of Lactobacillus was higher than that of Enterobacter and Paucibacter in all additive groups compared to the C group. This may be due to the ability of bacteriocins produced by Lactobacillus to reduce the number of undesirable microorganisms [28]. Fermentation for 20 and 40 days resulted in a higher abundance of Weissella and Enterococcus in the M and LM groups. Weissella is a heterofermentative bacterium that can adapt to acidic environments with a pH < 3.9 and is closely related to silage quality [29]. During silage fermentation, Weissella converts soluble carbohydrates into large amounts of lactic acid and acetic acid. After 20 days of fermentation, the relative abundance of Weissella in the L and LM groups was lower than in the M group. This may be because the addition of molasses increased the amount of available fermentation substrates, enhancing the competitiveness of Weissella in the microbial community. This also explains why the acetic acid content was higher in the M group.
Enterobacter and Paucibacter were the dominant bacteria in the C group. After 20 days of fermentation, the L, M, and LM groups exhibited lower Enterobacter abundance compared to the C group. The abundance of Paucibacter accounted for 8.1% of the total bacterial abundance in the C group. Paucibacter abundance in the L and M groups was 2.1% and 2.3%, while its abundance in the molasses and L. rhamnosus combined group was 1.6%. This indicates that the combined addition of molasses and L. rhamnosus inhibits Paucibacter more effectively than the addition of molasses or L. rhamnosus alone. In conclusion, compared with the C group, the M, L, and LM groups had a high abundance of Lactobacillus and a low abundance of Enterobacter and Paucibacter, which improved the fermentation products of big-bale L. chinensis silage.

3.4. Co-Occurrence Networks of Bacterial Community of Big-Bale Leymus Chinensis Silage with/Without Lacticaseibacillus Rhamnosus and Molasses

The co-occurrence network of a bacterial community is shown in Figure 3. In silage, the co-occurrence network is one of the most important characteristics of its bacterial ecosystem [30]. After 20 and 40 days of fermentation, harmful microorganisms (such as Enterobacter and Paucibacter) in the C group likely decreased the fermentation quality. The abundance of Lactobacillus correlated positively (p < 0.01) with Lactococcus (R = 0.90), Enterococcus (R = 0.88), and Weissella (R = 0.73), while it negatively correlated (p < 0.01) with Enterobacter (R = −0.66) and Paucibacter (R = −0.49). Conversely, Enterobacter and Paucibacter showed a positive correlation during fermentation (p < 0.01; R = 0.55). Overall, the co-occurrence network shows LAB in synergistic or competitive relationships with important microorganisms in big-bale L. chinensis silage.

3.5. Correlation Analysis Between the Bacterial Community and pH and Fermentation Products

The interaction between microorganisms and their metabolites is key to the successful fermentation of L. chinensis. Pahlow et al. [31] reported that homofermentative LAB (e.g., Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactococcus lactis) produce organic acids, mainly lactic acid, by fermenting sugar. Wu et al. [20] discovered that heterofermentative LAB (e.g., Lactobacillus buchneri and Weissella cibaria) converted WSC into acetic acid, 1,2-propanediol, and lactic acid during silage fermentation. Enterobacter species can ferment sugars to ethanol, 2,3-butanediol, lactic acid, and acetic acid during ensiling [32]. Figure 4 illustrates Spearman’s correlations between major bacterial genera and pH, lactic acid, 1-propanol, butyric acid, 1,2-propanediol, acetic acid, propionic acid, and ethanol after 20 and 40 days of fermentation. Lactobacillus, Enterococcus, and Weissella are correlated positively (p < 0.01) with acetic and lactic acid concentrations and negatively correlated (p < 0.05) with pH, butyric acid, and 1,2-propanediol levels. By contrast, Bacteroides and Pediococcus showed negative correlations (p < 0.05) with acetic and lactic acid concentrations but positive correlations (p < 0.05) with pH and 1,2-propanediol. Additionally, Paucibacter positively correlated (p < 0.05) with pH and 1,2-propanediol. Notably, Enterobacter abundance was positively associated (p < 0.05; R = 0.43) with the 1,2-propanediol content. These results indicate complex relationships between fermentation products and bacterial communities in big-bale L. chinensis silage. In the future, we will need to conduct further mechanistic studies on these results to verify potential causal relationships.

4. Conclusions

L. rhamnosus and molasses additives enhance the quality of big-bale L. chinensis silage, reduce dry matter loss, increase the relative abundance of Lactobacillus, Weissella, and Enterococcus, and decrease the relative abundance of Enterobacter and Paucibacter. The incorporation of L. rhamnosus and molasses, either individually or in combination, represents a highly effective strategy for the production of big-bale L. chinensis silage. These additions resulted in a substantial increase in organic acid accumulation while effectively inhibiting the proliferation of enterobacteria and yeast. In summary, through high-performance liquid chromatography integrated with high-throughput sequencing technology, this study shows that adding L. rhamnosus and molasses improved the fermentation quality and bacterial community structure of big-bale L. chinensis silage, providing a theoretical basis for big-bale L. chinensis silage improvement. Moreover, these research results can also be promoted and applied in other regions.

Author Contributions

Conceptualization, B.W. and X.C.; methodology, M.F., Y.B., T.W., K.L., S.W. and F.G.; formal analysis, Y.B., H.M., H.W., X.C. and Y.S.; investigation, T.W., M.F., Y.B., H.M., H.W. and Y.S.; data curation, K.L., S.W., F.G., H.M., H.W. and Y.S.; writing—original draft preparation, B.W.; writing—review and editing, H.M., H.W. and Y.S.; visualization, S.W., M.F., Y.B., H.M., H.W. and Y.S.; supervision, F.G., T.W., K.L., H.M., H.W. and Y.S.; project administration, X.C., H.M., H.W. and Y.S.; funding acquisition, B.W., H.W., H.M. and Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Tongliao Science and Technology Planning Project [grant number TL2023YF028]; the Natural Science Foundation of Inner Mongolian [grant number 2025MS03011, 2024MS03002, 2024QN0372]; the Innovation and Entrepreneurship Program for Returned Overseas Scholars in Inner Mongolia [grant number 2024LXCX005, 2024LXCX002]; the National Natural Science Foundation of China [grant number 32260848]; the Basic Scientific Research Business Expenses of Directly Affiliated Universities in Inner Mongolia Autonomous Region [grant number GXKY25Z042, GXKY25Z043]; and the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region [grant number NJYT22053].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 01888 g001
Figure 1. (A) Venn diagram and (B) spatial distribution. Principal component analysis illustrated the operational taxonomic units (OTUs) of big-bale Leymus chinensis before and after fermentation. FM represents fresh material; C, L, M, and LM denote the control group, Lacticaseibacillus rhamnosus group, molasses group, and Lacticaseibacillus rhamnosus + molasses group, respectively. The numbers after C, L, M, and LM denote fermentation times.
Figure 1. (A) Venn diagram and (B) spatial distribution. Principal component analysis illustrated the operational taxonomic units (OTUs) of big-bale Leymus chinensis before and after fermentation. FM represents fresh material; C, L, M, and LM denote the control group, Lacticaseibacillus rhamnosus group, molasses group, and Lacticaseibacillus rhamnosus + molasses group, respectively. The numbers after C, L, M, and LM denote fermentation times.
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Figure 2. Bacterial community at the genus level (A) and error bar plot (B,C) of big-bale Leymus chinensis before and after fermentation. FM represents fresh material; C, L, M, and LM denote the control group, Lacticaseibacillus rhamnosus group, molasses group, and Lacticaseibacillus rhamnosus + molasses group, respectively. The numbers after C, L, M, and LM denote fermentation times.
Figure 2. Bacterial community at the genus level (A) and error bar plot (B,C) of big-bale Leymus chinensis before and after fermentation. FM represents fresh material; C, L, M, and LM denote the control group, Lacticaseibacillus rhamnosus group, molasses group, and Lacticaseibacillus rhamnosus + molasses group, respectively. The numbers after C, L, M, and LM denote fermentation times.
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Figure 3. In the correction networks of the bacterial community at the genus level, circles represent microorganism genera, with their size indicating abundance. Lines depict correlations between genera: thickness signifies the strength of these correlations, while red and green colors denote positive and negative correlations, respectively.
Figure 3. In the correction networks of the bacterial community at the genus level, circles represent microorganism genera, with their size indicating abundance. Lines depict correlations between genera: thickness signifies the strength of these correlations, while red and green colors denote positive and negative correlations, respectively.
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Figure 4. Correlation analysis between the bacterial community and pH and fermentation products. LA, lactic acid; BA, butyric acid; AA, acetic acid; PA, propanoic acid; E, ethanol; P, 1,2-propanediol; and B, 1-propanol. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; and ***, p < 0.001.
Figure 4. Correlation analysis between the bacterial community and pH and fermentation products. LA, lactic acid; BA, butyric acid; AA, acetic acid; PA, propanoic acid; E, ethanol; P, 1,2-propanediol; and B, 1-propanol. *, 0.01 < p < 0.05; **, 0.001 < p < 0.01; and ***, p < 0.001.
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Table 1. Chemical and microbial compositions of pre-ensiling Leymus chinensis.
Table 1. Chemical and microbial compositions of pre-ensiling Leymus chinensis.
Leymus chinensis
Dry matter (g/kg)328 ± 5.32
pH5.68 ± 0.36
Crude protein (g/kg DM)92.21 ± 1.72
Neutral detergent fiber (g/kg DM)365.83 ± 21.53
Acid detergent fiber (g/kg DM)203.86 ± 8.52
Water-soluble carbohydrate (g/kg DM)42.98 ± 1.71
Lactic acid bacteria (log cfu/g)3.62 ± 0.73
Yeast (log cfu/g)5.34 ± 0.45
Enterobacteria (log cfu/g)5.62 ± 0.57
Data are the mean of duplicate analyses.
Table 2. Fermentation characteristics and microbial counts of big-bale Leymus chinensis silage treated with/without Lacticaseibacillus rhamnosus and molasses.
Table 2. Fermentation characteristics and microbial counts of big-bale Leymus chinensis silage treated with/without Lacticaseibacillus rhamnosus and molasses.
20 Days 40 Days 2-Way ANOVA
CLMLMSECLMLMSEZTZ × T
Dry matter (g/kg)379.27383.69379.25382.492.42380.29382.75386.68382.781.65NSNSNS
pH5.53A4.03B3.81C3.79C0.124.58a3.97b3.79c3.73c0.17******
Lactic acid (g/kg DM)10.78C14.72B16.67A16.97A0.7414.63c18.72b20.57a21.78a1.03******
Acetic acid (g/kg DM)5.67C7.23B9.48A8.36B0.426.31c8.36b7.83b9.38a0.48******
Propionic acid (g/kg DM)3.75C4.76B4.59B5.37A0.364.27c5.24b5.38b6.32a0.57******
Butyric acid (g/kg DM)2.43A0.85B0.78B0.46C0.282.12a0.82b0.88b0.93b0.38****NS
1,2-Propanediol (g/kg DM)0.67C0.84B1.35A1.31A0.160.84c1.23b1.31b1.82a0.43******
1-Propanol (g/kg DM)3.14A2.26B1.83C1.45C0.333.28a2.26b1.62c1.34c0.37******
Ethanol (g/kg DM)2.43A1.86B1.14C1.23C0.431.82a0.74b0.82b0.95b0.41******
Lactic acid bacteria (log cfu/g)6.16C7.21B7.82A7.87A0.356.32b7.44a7.76a7.82a0.28**NS**
Enterobacteria (log cfu/g)6.37A5.58B4.74C4.83C0.315.29a4.55b4.04c3.83c0.43******
Yeast (log cfu/g)6.24A5.82B5.17C5.03C0.245.73a5.24b4.64c4.16d0.32******
Values are means from triplicate silage. For the same preservation period, values with different lowercase (a–d) or uppercase (A–C) letters are significantly different (p < 0.05). Z, T, and Z × T denote the effects of inoculations, preservation periods, and their interaction, respectively. Significance levels are indicated as ** (p < 0.01) and NS (p ≥ 0.05). C, L, M, and LM denote the control group, Lacticaseibacillus rhamnosus group, molasses group, and Lacticaseibacillus rhamnosus + molasses group, respectively. The numbers after C, L, M, and LM denote fermentation times.
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Wu, B.; Cao, X.; Fu, M.; Bao, Y.; Wu, T.; Liu, K.; Wen, S.; Gao, F.; Wang, H.; Mei, H.; et al. Exploring the Fermentation Profile, Bacterial Community, and Co-Occurrence Network of Big-Bale Leymus chinensis Silage Treated with/Without Lacticaseibacillus rhamnosus and Molasses. Agronomy 2025, 15, 1888. https://doi.org/10.3390/agronomy15081888

AMA Style

Wu B, Cao X, Fu M, Bao Y, Wu T, Liu K, Wen S, Gao F, Wang H, Mei H, et al. Exploring the Fermentation Profile, Bacterial Community, and Co-Occurrence Network of Big-Bale Leymus chinensis Silage Treated with/Without Lacticaseibacillus rhamnosus and Molasses. Agronomy. 2025; 15(8):1888. https://doi.org/10.3390/agronomy15081888

Chicago/Turabian Style

Wu, Baiyila, Xue Cao, Mingshan Fu, Yuxin Bao, Tiemei Wu, Kai Liu, Shubo Wen, Fenglin Gao, Haifeng Wang, Hua Mei, and et al. 2025. "Exploring the Fermentation Profile, Bacterial Community, and Co-Occurrence Network of Big-Bale Leymus chinensis Silage Treated with/Without Lacticaseibacillus rhamnosus and Molasses" Agronomy 15, no. 8: 1888. https://doi.org/10.3390/agronomy15081888

APA Style

Wu, B., Cao, X., Fu, M., Bao, Y., Wu, T., Liu, K., Wen, S., Gao, F., Wang, H., Mei, H., & Song, Y. (2025). Exploring the Fermentation Profile, Bacterial Community, and Co-Occurrence Network of Big-Bale Leymus chinensis Silage Treated with/Without Lacticaseibacillus rhamnosus and Molasses. Agronomy, 15(8), 1888. https://doi.org/10.3390/agronomy15081888

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