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Effects of different wet distillers’ grains ratios on fermentation quality, nitrogen fractions and bacterial communities of total mixed ration silage

BMC Microbiology volume 25, Article number: 31 (2025) Cite this article

Abstract

Objective

Wet distiller’s grains (WDG) are rich in crude protein, yet challenging to preserve. Nevertheless, incorporating WDG into total mixed ration (TMR) silage holds promise for enhancing fermentation quality. This study investigated the effects of varying WDG proportions on nitrogen composition, fermentation quality, and microorganisms in TMR silage.

Methods

Three TMR formulations were prepared: (1) 0% WDG (T0), (2) 15% WDG (T15), and (3) 30% WDG (T30) were ensiled for 7, 15, 30 and 60 days.

Results

After 7 days of ensiling, butyric acid was detected in T0 and T15 groups, while T30 exhibited significantly lower levels (p < 0.05). Both T15 and T30 treatments led to improved V-scores of TMR silage. Non-protein nitrogen (NPN) production was slower in T30, with significant increases observed in NPN levels for T0 and T15 after 30 days (p < 0.05). However, the abundance of Clostridium was extremely low in the present study. Protein degradation and and butyric acid production may be attributed to Weissella.

Conclusion

The fermentation quality of TMR silage is always decreasing during storage, so its storage time should be minimized. Incorporating 30% WDG reduced abundance of Weissella, resulting in less protein degradation and better fermentation quality in TMR silage.

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Introduction

Wet distillers’ grains (WDG) are the primary by-products of liquor production, have an annual output of up to 20 million tons in China. In addition to containing protein, starch, and dietary fiber, they are also rich in bioactive substances such as polyphenols and peptides, which can serve as valuable livestock feed [1]. However, their high acidity and moisture pose challenges for long-term preservation [2], potentially leading to environmental pollution [3]. Total mixed ration (TMR) silage, utilizing anaerobic fermentation, offers a solution for complete feed formulation and storage, ensuring formation accuracy and enhancing storage stability [4]. WDG in ruminant diets enhance feed efficiency and nutrient utilization, promoting overall animal health and productivity [5]. Furthermore, WDG can inhibit harmful microbes responsible for protein degradation, thereby improving the overall nutritional value of the feed [6].

Processing high-moisture poor-palatability agricultural by-products into TMR silage not only diversifies feed sources but also reduces environmental impact, with potential for commercial distribution [7]. In general, the optimum moisture content for TMR fermentation is 35-60% [8]. Since WDG has a high moisture content, it can provide additional moisture to low-moisture feedstocks such as hay. However, the alcoholic distillation process consumes most of the carbohydrates from the grain, so WDG is not a good source of fermentation substrate. Using WDG to produce TMR silage can effectively regulate moisture levels and provide additional fermentation substrates through other raw materials.

Although microorganisms play a crucial role in fermentation, some undesirable bacteria such as clostridia can reduce feed quality by producing butyric acid and degrading proteins [9, 10]. Nutrient loss during the ensiling process are significant concerns. Protein degradation in TMR silage may lead to the conversion of protein to non-protein nitrogen (NPN), which ruminants cannot fully utilize, resulting in protein loss [11]. While prior studies have employed WDG to produce high-quality TMR silage [12, 13], the effects of WDG components on protein quality and microbial communities remain unexplored. Our previous research demonstrated that increasing alfalfa silage content in TMR promotes acidic environment formation, enhancing fermentation quality [14]. Given WDG’s substantial lactic acid content, similar effects may occur. This study hypothesizes that WDG addition may expedite acidic environment formation, thereby reducing protein degradation in TMR silage. Therefore, compared with other studies focusing on fermentation quality, this study focused on the nitrogen composition and protease activity of TMR silage. The microbial community was also determined to further explore the mechanism of protein degradation affected by different WDG levels.

It is concluded that the optimal proportion of wet distillers’ grains can enhance the utilization and value of agricultural by-products while ensuring fundamental nutritional requirements, thereby offering a valuable reference for optimizing feeding strategies in small-scale farms.

Materials and methods

Preparation of TMR silage

The corn silage was obtained from the Guizhou University Guanling Experimental Station, and WDG was obtained from the Moutai Distillery in Renhuai City, Guizhou Province. The main raw material used to make liquor is sorghum. The chemical composition and fermentation quality of corn silage and WDG are detailed in Table 1.

Table 1 Chemical composition and fermentation quality of corn silage and wet distillers’ grains
Full size table

The mixed concentrate, comprising mainly corn meal, soybean meal, and rapeseed meal, was acquired from the Guanling Experimental Station. The ingredient composition and chemical composition of the TMR are provided in Table 2. WDG, based on dry matter (DM), constituted 0% (T0), 15% (T15), and 30% (T30) of the TMR, respectively. The components of TMR need to be well mixed according to the proportions in Table 2. Approximately 500 g of fresh samples were placed into specialized silage bags at the size of 35 cm × 45 cm and vacuum sealed. A total of 75 bags (3 WDG ratios × 5 storage periods × 5 replicates) were sampled after 7, 15, 30, and 60 days of fermentation for further analysis. The 75 bags were randomly selected according to the standard of Robinson et al. [15]. Specifically, the three WDG ratios were randomly distributed in equal proportions among 75 bags of other ingredients prepared in advance. The 75 bags of samples were stored individually, and 5 samples of each ratio were selected and opened randomly at different storage period.

Table 2 Ingredient and chemical composition of the total mixed ration
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Fermentation characteristics analysis

To analyze fermentation characteristics, a subsample of 20 g of silage was measured and placed in a 300 mL conical flask. Subsequently, 180 mL of distilled water was added, and the samples were refrigerated at 4 ℃ for 24 h. Then, the extracts were filtered through 4 layers of cheesecloth and Whatman filter paper (Xinhua Co. Ltd., Hangzhou, China). After filtration, the filtrate was collected for multiple analyses, including pH, ammonia-nitrogen (NH3-N) content, organic acids concentrations, and ethanol level. pH was measured using a PHS-3 C acidimeter (Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China). NH3-N content was determined using the sodium hypochlorite and phenol method, and the specific value was obtained using colorimetric method [16]. Organic acids, such as lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA), were analyzed according to the method developed by Zhao et al. [17]. The sample was passed through a 0.2 μm filter membrane and determined by high-performance liquid chromatography (HPLC, Thermo Fisher Scientific, Inc., Waltham, USA; column: Shodex RS Pak KC-811, Showa Denko KK, Japan; detector: diode array detector, Thermo Fisher Scientific, Inc., Waltham, USA; eluent: 1.0 mL min-1, 3 mM HClO4; column temperature: 55 °C; detection wavelength: 210 nm). Ethanol analysis was conducted following the method used by Tao et al. [18]. The V-score was calculated based on the method proposed by Chen et al. [19]. In essence, the NH3-N /total nitrogen (TN) ratio, along with the contents of AA, PA, and BA, were utilized as rating indicators. Specific scoring formulas were applied to each index due to their varying contents. The scores for all indexes were then aggregated to derive a total score out of 100, which categorized silage quality as good (above 80), fairly good (60–80), or poor (below 60).

Chemical composition analysis

A portion of approximately 200 g of TMR silage was subjected to oven-drying at 65 ℃ for 48 h to determine its DM content. The dried samples were then passed through a 1-mm sieve (FW100, Tester Instruments Co., Ltd., Tianjin, China) for subsequent chemical composition analysis. Neutral detergent fiber (aNDF) and acid detergent fiber (ADF) were assessed following the method outlined by Van Soest et al. [20]. For aNDF determination, sodium sulfite and α-amylase were utilized, with both aNDF and ADF contents reported inclusive of residual ash. Water soluble carbohydrates (WSC) were quantified using the anthrone sulfate colorimetric method as described by Weatherburn et al. [21]. The TN was analyzed via the Kjeldahl procedure [22], and the crude protein (CP) content was derived by multiplying TN by a factor of 6.25.

Analysis of nitrogen fraction and proteinase activity

The NPN content was assessed following the procedure outlined by Licitra et al. [23]. For the determination of amino acid nitrogen (AA-N), the method was described by Broderick et al. [24]. Peptide nitrogen (Peptide-N) was calculated according to the formula: Peptide-N (%TN) = NPN (%TN) - AA-N (%TN) - NH3-N (%TN) [25].

A subsample weighing 10 g of silage was combined with 50 mL of pre-chilled 0.1 M sodium phosphate buffer (pH 6.0, containing 5 mM sodium thiosulfate). Following homogenization, the resulting mixture was filtered through four layers of gauze and then centrifuged at 8000 × g, 4 ℃ for 10 min. The supernatant obtained served as the crude enzyme extract and was stored at -80 ℃ until further analysis of aminopeptidase, carboxypeptidase, and acidic proteinase activities [26]. The enzyme activities were determined using enzyme-linked immunosorbent assay kits (ADANTI Biotechnology Co., Ltd., Wuhan, China).

Analysis of microbial communities

Following the methodology outlined by Xie et al. [7]. DNA was extracted from the silage samples, and the total DNA of microorganisms was accurately quantified. The V3-V4 region of the bacterial 16 S rDNA gene sequence was amplified via PCR using universal primers: 341 F (5’-CCCTACACGACGCTCTTCCGATCTG-3’) and 805R (5’-GACTGGAGTTCCGGCACCCGAGAATTCCA-3’). PCR amplification conditions were set as follows: initial denaturation at 94 °C for 3 min, followed by denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, repeated for 26 cycles, with a final extension at 72 °C for 10 min. The PCR products were verified using 2% agarose gel electrophoresis and subsequently submitted to Novogene (Novogene Bioinformatics Technology Co., Ltd. Beijing, China) for Illumina MiSeq high-throughput sequencing.

Alpha diversity and beta diversity were computed by standardizing to the same random sequences. Alpha diversity metrics, such as Shannon, Simpson, Chao1, and observed OTUs, were employed to assess the complexity of species diversity with samples. Beta diversity was evaluated through principal coordinate analysis (PCA) using UniFrac metrics, and statistical comparisons among groups were performed using ANOSIM. Both alpha diversity and beta diversity analyses were conducted using QIIME2 scripts. Spearman’s correlation matrices were computed following the approach described by Qiu et al. [27]. Sequence data have been deposited in NCBI’s Sequence Read Archive under Bio-Project accession number PRJNA 1,097,618.

Statistical analysis

Initial organization and visualization of the basic data were carried out using Excel 2010. Statistical analysis of experimental data was conducted using SPSS 26.0 software, which facilitated one-way or two-way analysis of variance. Duncan’s multiple range test was employed to discern statistically significant differences among the means. Throughout all analyses, statistical significance was defined as p < 0.05.

Results

Fermentation quality of TMR silage

Table 3 illustrates the shifts in fermentation characteristics throughout the ensiling process of TMR silages. The interaction between WDG ratios and ensiling period significantly affected (p < 0.001) the pH; lactic acid, acetic acid, propionic acid, butyric acid NH3-N and ethanol levels. As fermentation progressed, pH levels significantly decreased (p < 0.05), while lactic acid and NH3-N exhibited significant increases (p < 0.05). The addition of WDG resulted in higher levels of propionic acid and lower levels of butyric acid in the T30 group than in the T0 group (p < 0.05). The NH3-N content increased significantly on both 7th day and 15th day (p < 0.05). Ethanol levels in the T30 group increased at a faster rate than the other two groups. However, the ethanol content in all TMR silage was low and did not exceed 5 g kg− 1 (DM). The V-score in Fig. 1 is obtained by combining a variety of indicators from Table 3, so it provides a more comprehensive picture of the fermentation quality of silage. According to Fig. 1, the V-scores of T15 and T30 groups surpassed those of the T0 group. Even after 60 days of fermentation, the V-score of T15 and T30 groups remained high (≥ 60).

Table 3 Changes in fermentation characteristics throughout the ensiling process of total mixed ration silages
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Fig. 1
figure 1

The variation in V-score during the ensiling of total mixed ration (TMR) silage. V-score is used to indicate silage quality based on volatile fatty acid and ammonia-nitrogen contents. T0, T15, and T30 represent TMR with 0%, 15%, and 30% wet distillers’ grains (dry matter), respectively. Bars indicate the standard errors of the means. Significant differences between means within the same treatment (a, b, c, d) or within the same storage period (A, B, C) are denoted by different superscripts (p < 0.05)

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Chemical composition of TMR silage

Table 4 presents the nutrient composition of TMR silages across various storage durations. The interaction between WDG ratios and ensiling days significantly influenced (p < 0.001) the levels of DM, WSC, and ADF. Notably, the WSC content of T0 and T15 groups experienced a significant decline during the initial fermentation stage (p < 0.001), although the trend was not observed in the T30 group.

Table 4 Nutrient composition of total mixed ration silages across varying storage durations
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Nitrogen fraction and proteinase activity

The dynamics of protease activity and nitrogen fraction contents throughout the ensiling process are shown in Fig. 2. The rate of carboxypeptidase inactivation in T30 group decelerated after 7 days of fermentation. In the T30 group, carboxypeptidase retained an activity level of 69.19 U g− 1 DM on the 30th day of silage. Aminopeptidase activity exhibited an initial decrease followed by an increase with extended silage time. After 7 days of ensiling, the NPN content in the T30 group was notably lower than that in the other two groups. Throughout the ensiling period, NPN and AA-N contents in all treatment groups gradually rose and then stabilized. Although there was a non-significant decrease from 0 to 7 days, subsequent increases were observed from 15 to 45 days (p < 0.05). By the 45 days of ensiling, peptide nitrogen content in the T30 group was significantly lower than in the other groups (p < 0.05).

Fig. 2
figure 2

The dynamics of proteinase activity (a) and the levels of nitrogen fractions (b) during the ensiling of total mixed ration (TMR) silage. Error bars represent the standard errors of the means. DM, dry matter; TN, total nitrogen; NPN, non-protein nitrogen; Peptide-N, peptide nitrogen; AA-N, amino acid nitrogen; NH3-N, ammonia-nitrogen. T0, TMR with 0% wet distillers’ grains (DM); T15, TMR with 15% wet distillers’ grains (DM); T30, TMR with 30% wet distillers’ grains (DM)

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Impact on bacterial diversity during fermentation

Figure 3 illustrates changes in overall bacterial relative abundance and important bacterial genera in the TMR silage. Initially, the predominant bacteria in WDG raw material were Proteobacteria (82.65%) and Firmicutes (7.60%), with smaller amounts of Actinobacteria (8.08%) and Cyanobacteria (0.49%). After 7 days of fermentation, the bacterial micro-ecological community shifted primarily to Firmicutes, with minor presence of Proteobacteria, Cyanobacteria, and Bacteroidetes. This trend persisted at 30 and 60 days, with Firmicutes (79.80%) prevailing as the primary phylum. At the genus level, bacterial structures in all treatment groups were similar during fermentation, with Lactobacillus being predominant. The abundance of Lactobacillus fluctuated over storage duration, and Weissella abundance in the T15 and T30 groups was significantly lower than that in the T0 group (p < 0.05).

Fig. 3
figure 3

Variations in the overall bacterial relative abundance (a) and significant bacterial genera (b) in total mixed ration (TMR) silage. Significant differences between means within the same treatment (a, b, c) or within the same storage period (A, B) are indicated by different superscripts (p < 0.05). Error bars represent the standard error of the means. T0, TMR with 0% wet distillers’ grains (dry matter, DM); T15, TMR with 15% wet distillers’ grains (DM); T30, TMR with 30% wet distillers’ grains (DM)

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The alpha diversity of bacterial communities during ensiling is displayed in Fig. 4a. All samples exhibited Good’s coverage exceeding 99.9%, indicating comprehensive coverage of bacterial communities. Prior to ensiling, the T30 group showed a higher Shannon index (p < 0.05). On days 7 and 15, the T30 group had a lower Chao1 index (p < 0.05). The addition of WDG significantly increased the Shannon index (p < 0.05). Figure 4b presents the results of PCA during ensiling. Microbial composition before fermentation was relatively similar. Microbial community structures at T15 and T30 appeared similar during ensiling.

Fig. 4
figure 4

Alpha diversity indices (a) and PCA plots (b) of total mixed ration silage throughout ensiling. Error bars represent the standard errors of the means. T0, 0% wet distillers’ grains; T15, 15% wet distillers’ grains; T30, 30% wet distillers’ grains

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The heatmap of Spearman correlation analysis of bacterial community composition with fermentation parameters and nitrogen fractions is depicted in Fig. 5. The abundance of Weissella showed a significant positive correlation with NPN and butyric acid contents at several time points during the overall ensiling process. While the abundance Lactobacillus showed the opposite trend.

Fig. 5
figure 5

Heatmap depicting Spearman correlation analysis between bacterial community composition, fermentation parameters, and nitrogen fractions. NPN, non-protein nitrogen; Peptide-N, peptide nitrogen; AA-N, amino acid nitrogen; NH3-N, ammonia nitrogen. *p < 0.05; **p < 0.01

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Discussion

The pH serves as a crucial indicator of silage feed quality, with desirable silage typically possessing a pH below 4.0 [28]. In this study, despite initial pH variations among the treatment groups, all reached around 4.2 after 60 days of ensiling. The pH of forage silage feed is influenced by various factors, including forage types and their chemical composition, particularly the content of soluble sugars [18]. The brewing process utilizes non-structural carbohydrates fully, resulting in low levels of soluble sugars available for fermentation.

In this study, the utilization of WDG replaced traditional concentrated feed components like cornmeal, bran, soybean meal, and rapeseed meal, resulting in decreased substrate levels available for lactic acid bacteria utilization in the T15 and T30 groups prior to fermentation. Nonetheless, the high lactic acid content in WDG could potentially create a more acidic environment for TMR silage directly. Thus, the T30 group had a lower pH on day 0. Similarly, acetic acid and propionic acid in the groups with WDG were significantly higher than in the T0 group at several time points, which may be due to the presence of some acetic acid and propionic acid in the WDG itself on the one hand, and to microbial activity on the other. The acetic acid is derived pre-dominantly from the action of heterofermentatvie lactic acid bacteria, propionibacteria and enterobacteria on sugars, and the propionic acid production originates partly from propionibacteria activity [29]. However, the abundance of associated bacteria in this study was not substantially affected by WDG, so we hypothesized that the former was the main reason for the differences in volatile fatty acids contents among the groups. Yuan et al. [25] suggested lactic acid production in silage with WDG addition leads to rapid acidification, inhibiting undesirable aerobic microorganism growth in early stages and reducing butyric acid generation. Alternatively, residual ethanol in WDG might exhibit antibacterial effects on aerobic microorganisms. However, the ethanol content detected in our study was relatively low, indicating that the observed difference in butyric acid might not be attributable to ethanol. It is noteworthy that ethanol growth was faster in the T30 group in this study. This may be due to the fact that the microorganisms remaining in the WDG are more adept at utilizing carbohydrates to produce ethanol. However, the community abundance of fungi such as yeast was not measured in this study, so this conjecture needs to be re-examined in the future. The NH3-N content in this study did not show a good pattern among the three treatments, which may be due to the variation of protease levels in each storage period. The V-score serves as an indicator that excludes lactic acid influence while integrating volatile fatty acids content and protein degradation, offering a comprehensive evaluation of silage quality [30]. In our investigation, introducing WDG in the later stage of storage elevated the V-score of TMR silage. By the 15th day of storage, TMR silage lacking WDG addition couldn’t attain a V-score of 60 due to increased butyric acid production. Conversely, incorporating 30% WDG delayed butyric acid production, thereby enhancing the fermentation quality of short-term stored TMR silage.

The WSC content in WDG is relatively low due to consumption during the distilling process. Corn meal, soybean meal and rapeseed meal replaced by WDG in groups T0 and T15 contained more WSC could be used as a substrate for acid production during fermentation. Therefore, the WSC content in group T30 did not show a decreasing trend over time as in the other two groups. Similarly, TMR silages with various formulations had different rates of nutrient availability during storage, which may be the main reason for the different rates of DM change in different groups. Since ADF is a difficult part to utilize in silage, its content is generally more stable during ensiling [31]. Changes in ADF content may be due to variations in DM content, and the rate of change in DM contents are different. Therefore, the underlying reason for the significant interaction of the WDG ratios and ensiling days on WSC, DM and ADF contents in the present study could be the different rates of microbial conversion of nutrients in different formulations.

In our study, the incorporation of WDG led to a reduction in NPN formation in TMR silage. However, nitrogen component analysis indicated that WDG primarily decreased the formation of amino acid nitrogen and peptide nitrogen. Peptides and free amino acids can either be synthesized into microbial protein by rumen microorganisms and utilized by animals or continue to be deaminated to form NH3-N [32]. Excessive nitrogen is excreted as urea in urine under conditions of energy deficiency or when the degradation rate of peptides surpasses the synthesis rate. Compared to the formation of NH3-N in TMR silage, the likelihood of amino acid nitrogen and peptide nitrogen participating in the rumen-urea cycle is lower, suggesting reduced protein waste [33].

Fermentation has the capacity to modify both the community structure and abundance within silage. Firmicutes emerged as the predominant phylum across the different treatment groups. During the initial stage of silage fermentation, Firmicutes and Proteobacteria were the dominant microorganisms. These bacteria possess the capability to stimulate the production of lignocellulose-degrading enzymes in other microorganisms, thereby providing additional energy sources for microbial activities [34]. Prior to fermentation, predominant genera in TMR silage included Lactobacillus, Weissella, Ralstonia, Devosia, and other genera. Throughout the fermentation process, dominance shifted to Lactobacillus and Weissella, of which the former had the highest abundance. Weissella, an obligate heterofermentative bacteria, is recognized as an initial colonizer in the fermentation process, capable of metabolizing sugars into lactic and acetic acids. As pH decreases, Weissella growth tends to diminish [35,36,37]. However, in our study, Weissella abundance slightly increased on the 60th day of fermentation, possibly due to pH not reaching sufficiently low levels. Furthermore, Weissella (e.g. Weissella cibaria, Weissella hellenica, and Weissella kandleri) carrying a higher number of carbohydrate- active enzyme genes for degrading arabinoxylan and cellulose [38]. As fermentable sugars in silage decrease, Weissella may have a stronger competitive advantage because they are able to utilize more complex carbon sources. This could also be a possible reason for the increased abundance of Weissella in the later stages of TMR silage. The addition of WDG also had an effect on the abundance of the predominant bacteria, and PCA showed that the two groups with WDG added were more similar in their bacterial community structure. Overall WDG addition decreased the abundance of Weissella and increased the abundance of Lactobacillus in the later stages of fermentation. This may be related to the strong acidity brought by WDG at the beginning of fermentation, as Lactobacillus is more adapted to an acidic environment than Weissella [35, 36].

Typically, protein degradation and butyric acid generation in silage are attributed to clostridia [39]. However, in our study, Clostridium abundance was found to be below 1.00% in all treatment groups during ensiling. Li [40] found a significant positive correlation between Sphingobacterium and NPN formation in alfalfa silage. However, Sphingobacterium also had a low abundance in this study and did not change significantly with treatment, so it may not be a major cause of protein degradation either. Correlation analyses showed positive correlations between Weissella abundance and NPN/butyric acid levels at multiple ensiling periods. Cai et al. found that silage inoculated with Weissella had significantly higher NH3-N and butyric acid levels, demonstrating that Weissella can not improve silage quality and may result in fermentation losses [41]. Furthermore, Weissella also has protein hydrolyzing activity [42]. Since Weissella was the most abundant bacteria in this study except Lactobacillus, it may be the main causative factor for the increased butyric acid and NPN levels in the T0 group.

During ensiling, protein degradation primarily undergoes two processes: plant proteases predominantly hydrolyze true proteins, while microorganisms primarily deaminate free amino acids [43]. These processes reflect the relative activity of protein hydrolysis and deamination Yuan et al. [25]. In our study, the assessment of protease activity in TMR silage revealed that carboxypeptidase and acidic protease activities exhibited a relatively slow decline during ensiling. Subsequently, carboxypeptidase and acidic protease activities gradually decreased after 15 days of ensiling and remained relatively low thereafter. This finding aligns with previous research by Guo et al. [26]. In the T0 group of our study, acidic protease exhibited higher activity in the early stages of anaerobic fermentation, whereas aminopeptidase showed higher activity in the later stages. The addition of WDG did not cause a significant decrease in protease activity, so it is difficult to explain the inhibition of NPN production in the T15 and T30 groups from the perspective of protease activity. However, from the microbial point of view, the addition of WDG decreased the abundance of Weissella in the TMR silage, which in turn reduced the production of NPNs such as AA-N and Peptide-N, and decreased the level of butyric acid.

Considered in conjunction with the V-score, TMR silage is always accompanied by fermentation losses during storage and fermentation quality decreases over time, so the storage time of TMR silage should be minimized. The addition of 30% WDG to TMR silage both reduces the conversion of protein to NPN and improves fermentation quality. At the same time, WDG is much cheaper than the other concentrates it replaces, so the T30 treatment also makes good economic sense.

Conclusion

The fermentation quality of TMR silage is always decreasing during storage in this study, so its storage time should be minimized. Incorporating 30% wet distillers’ grains (WDG) into TMR silage has demonstrated several beneficial effects. It promoted an acidic environment, which effectively inhibited butyric acid production and consequently enhanced the fermentation quality of TMR silage. Furthermore, the inclusion of WDG also suppressed protein degradation. The beneficial effects of WDG addition may stem from its ability to reduce the abundance of Weissella.

Data availability

Sequence data that support the findings of this study have been deposited in the NCBI’s Sequence Read Archive under Bio-Project accession number PRJNA 1097618.

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Funding

This research was funded by the Guizhou University Doctoral Fund [grant numbers (2022) 31 and (2019) 54], the GZMARS-Forage Industry Technology System of Guizhou Province (GZMCCYJSTX-01), and the National Key R&D Program of China (2022YFD1300900).

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Authors and Affiliations

  1. Collage of Animal Science, Guizhou University, Guiyang, Guizhou, China

    Ermei Du, Ning Mao, Shihao Liu, Hanyu Zhang, Meiling Fan, Hong Sun, Yulong Zheng, Qiming Cheng, Chunmei Wang, Ping Li & Yixiao Xie

  2. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang, Guizhou, China

    Ermei Du, Ning Mao, Shihao Liu, Hanyu Zhang, Meiling Fan, Hong Sun, Yulong Zheng, Qiming Cheng, Chunmei Wang, Ping Li & Yixiao Xie

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  1. Ermei Du
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Contributions

Yixiao Xie designed the study. Ermei Du drafted the manuscript. Yulong Zheng, Qiming Cheng, and Chunmei Wang supervised the study. Ping Li, and Hong Sun contributed valuable insights. Ning Mao, Shihao Liu, Meiling Fan and Hanyu Zhang contributed to data collection. All authors have reviewed and approved the final manuscript for publication.

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Correspondence to Yixiao Xie.

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Du, E., Mao, N., Liu, S. et al. Effects of different wet distillers’ grains ratios on fermentation quality, nitrogen fractions and bacterial communities of total mixed ration silage. BMC Microbiol 25, 31 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03750-w

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BMC Microbiology

ISSN: 1471-2180

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