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. 2025 Jan 23;25:99. doi: 10.1186/s12870-025-06063-2

Effects and function of citric acid on fermentation quality and microbial community in sugarcane tops silage with high and low water-soluble carbohydrate content

Qichao Gu 1,2,3, Jie Zhang 1,2,3, Bo Lin 1,2,3, Hao Ding 1,2,3, Qi Yan 1,2,3, Caixiang Wei 1,2,3, Yipei Yao 1,2,3, Ruizhanghui Wang 1,2,3, Caixia Zou 1,2,3,✉
PMCID: PMC11755813  PMID: 39849361

Abstract

Sugarcane tops silage (STS), as a source of roughage for ruminants, is rich in water-soluble carbohydrate (WSC) content, which significantly affects silage quality. Citric acid (CA) is a low-cost natural antimicrobial agent that can inhibit undesirable microbes and improve silage quality. The objectives of this study were to investigate the effects of CA on the chemical composition, fermentation quality, microbial communities, and metabolic pathways of STS with high and low WSC contents before or after aerobic exposure. Fresh sugarcane tops with low-WSC [143.05 g/kg dry matter (DM)] and high-WSC (249.99 g/kg DM) contents were treated with and without CA and then ensiled for 125 days, followed by aerobic exposure for 4, 8, and 16 days. The results showed that high-WSC STS had lower crude protein (CP) content and higher DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents, whether treated with CA or not. CA-inoculated silage exhibited decreased DM loss and enterobacteria (EB) counts compared to the control. High-WSC STS treated with CA had higher WSC content and lower yeast count than those without CA inoculation. During the 0–16 days of aerobic exposure, the propionic acid and butyric acid contents in CA-inoculated silage were almost unchanged and ranged from 0 to 1 g/kg DM. Meanwhile, the ethanol content was almost unchanged and ranged from 0 to 1 g/kg DM only in low-WSC STS, irrespective of CA addition. Before aerobic exposure, CA inoculation decreased the abundances of undesirable microbes (e.g., Clostridium_sensu_stricto_12 and Paecilomyces) and animal pathogens, while amino acid metabolism was lower in high-WSC STS regardless of CA treatment. After aerobic exposure, CA inoculation increased the abundance of bacteria with antibacterial effects, including Paenibacillus and Bacillus. Moreover, the metabolism of energy and nucleotides was lower in high-WSC STS treated with CA, and the animal pathogens was lower in low-WSC STS treated with CA. In conclusion, CA inoculation could be effective in decreasing nutrients loss, improving fermentation quality, inhibiting harmful microorganisms, and modulating the metabolic pathways of microorganisms in STS with high and low WSC contents prior to and after aerobic exposure.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06063-2.

Keywords: Citric acid, Water-soluble carbohydrate, Silage quality, Antimicrobial, Microbial communities, Function prediction

Introduction

Citric acid (CA), a natural organic compound, is considered the most economically feasible product for microbiological production [1]. CA can provide an additional fermentation substrate for lactic acid bacteria (LAB) during anaerobic fermentation, such as Lactobacillus [2–5]. The anaerobic metabolites of CA are D-lactic acid, acetic acid, and 2,3-butanediol, while the aerobic metabolite is succinic acid [6]. Many previous studies have confirmed that adding CA to silage can improve its fermentation quality through mechanisms such as inhibiting the growth of harmful bacteria (e.g., Enterobacter and Pseudomonas) and fungi (e.g., yeasts and molds) in silage both before and after aerobic exposure [2, 3, 5, 7], limiting proteolysis [7–9], decreasing dry matter losses [8], reducing acetic acid content [10–13], and increasing lactic acid content [7–16]. Previous research on forage silage found that CA addition increased the DM, crude protein (CP), and water-soluble carbohydrate (WSC) contents [10, 12, 14, 17, 18] and decreased the neutral detergent fiber (NDF) content [13, 15]. Moreover, CA can also inhibit amino acid metabolism through inhibiting the activities of related microbes (e.g., Enterobacter) [2]. Notably, Mahdizadeh et al. reported that CA could effectively remove Mycoplasma bovis, a pathogen that causes diseases such as pneumonia and mastitis in cattle [19].

Sugarcane tops are rich in WSC and other nutrients [20, 21], and have great potential for development into high-quality silage. The WSC content of raw materials is one of the key factors affecting silage quality, and the ideal range of WSC content for producing high-quality silage ranges from 60 to 80 g to/kg DM [22]. Furthermore, pastures (e.g., sugarcane) containing excess WSC will not only stimulate the growth of anaerobic yeasts during ensiling and will not be completely inhibited by low pH but also compete with LAB for the utilization of WSC, producing ethanol and other substances, reducing the recovery of DM [23]. In particular, after exposure to air, yeasts utilize a large amount of residual WSC in the silage, as well as lactic acid as a nutrient substrate, to grow rapidly, causing the temperature and pH to rise, and molds begin to reproduce in large quantities, which in turn leads to aerobic deterioration of silage [24]. However, the WSC content of fresh sugarcane tops exceeded the optimal WSC range for producing high-quality silage. Based on this, we preliminarily compared the effects of low and high-WSC contents (143.35 vs. 258.42 g/kg DM) of fresh sugarcane tops on silage quality and found that higher DM loss and butyric acid content were in STS with low-WSC content and high-WSC content before aerobic exposure, respectively. Meanwhile, in both WSC contents, STS had a large proliferation of undesirable microorganisms (e.g., Enterobacter and yeast), resulting in silage deterioration after exposure to air [25]. Recent studies have shown that exogenous additives (such as organic acids) can reduce nutrient loss and inhibit the growth of harmful microorganisms during the aerobic exposure stage to improve silage quality [26]. However, whether CA can improve the quality, inhibit the growth of harmful microorganisms, and regulate the bacterial metabolism of STS with varying WSC contents remains unknown. To address these questions, the objectives of the present study were to investigate the effects of CA on the fermentation quality, microbial community, and their functions during aerobic exposure of STS with high and low-WSC contents.

Materials and methods

Raw materials and ensiling

Sugarcane tops with two different levels of WSC contents were harvested from the forage breeding ground of Fusui Experimental Base of Guangxi University, among which the high-WSC sugarcane tops (Measured WSC content: 249.99 g/kg DM) produced from sugarcane variety GUC-34 (a high-sugar-accumulating variety) and the low-WSC sugarcane tops (Measured WSC content: 143.05 g/kg DM) produced from sugarcane variety 15-6049 (a low sugar-accumulating variety). The other chemical compositions of the two fresh sugarcane tops are listed in Table S1.

Fresh sugarcane tops were chopped to a length of approximately 2 cm with a forage cutter (model zfd5570, Zheng Feng Machinery Company, Jining, China). The treatments in this study were as follows: (1) low-WSC sugarcane tops with the addition of 10 g/kg fresh weight (FW) distilled water (LCK); (2) low-WSC sugarcane tops with the addition of 10 g/kg FW CA (LCA); (3) high-WSC sugarcane tops with the addition of 10 g/kg FW distilled water (HCK); and (4) high-WSC sugarcane tops with the addition of 10 g/kg FW CA (HCA). There were four replications per treatment. The CA was obtained from the Tianjin Beichen Fangzheng Reagent Factory (Tianjin, China). After mixing, the material of each treatment group was manually packed into a 2.5-L plastic bottle (15 × 22 cm) with a plastic cover, and the compaction density was over 600 kg/m3 FM. Then, the plastic bottles were stored at ambient temperature (10–35 °C) for 125 days. After ensiling, the samples were opened and collected for analysis of the aerobic stability, chemical composition, fermentation quality, microbial population, and microbial community.

Aerobic stability test

The method described by Nishino and Touno [27] was used to determine the aerobic stability of the STS. Briefly, a silage sample (150 g) from each treatment was weighed and placed into a 500-mL plastic bottle, which was sealed with a single layer of gauze to reduce cross-contamination of the silage and ensure air circulation. After 4, 8, and 16 days of aerobic exposure, silage samples (20 g) were collected for fermentation quality and microbial analyses.

Chemical composition, fermentation profile, and microbial population analysis

The DM content of each sample (200 g) was determined by air-oven drying at 65 °C for 72 h. Then, the samples were crushed through a 1-mm sieve and stored in zip-lock bags for subsequent chemical analysis. Total nitrogen (TN) was calculated using the Kjeldahl method, with CP = TN × 6.25 [28]. The ash content was measured via high-temperature furnace burning (550 °C for 5 h) in a muffle furnace [28]. The acid detergent fiber (ADF) and NDF contents were measured using an ANKOM 200I fiber analyzer (ANKOM Technology, Macedon, NY, USA) [29]. WSC content was determined using anthrone sulfuric acid colorimetry [30]. DM losses during ensiling were determined as the dry weight differences before and after 125 days of ensiling.

Each silage sample (20 g) was added to 180 mL of Ringer’s solution, mixed, and allowed to stand for 30 min at 4 °C, then filtered with double-layer gauze, and the filtrate was collected to determine the fermentation parameters and microbial counts. The pH of the filtrate was immediately measured using a pH meter (Mettler-Toledo Delta 320; Mettler-Toledo, Greifensee, Switzerland). The filtrate was centrifuged (12 000 ×g, 15 min, 4 °C) to assess the organic compounds (lactic acid, acetic acid, propionic acid, butyric acid, and ethanol) and ammonia nitrogen (AN). The lactic acid and AN contents were measured following the method described by Zhang et al. [20]. The volatile organic compound contents (acetic acid, propionic acid, butyric acid, and ethanol) were determined using a gas chromatograph (Agilent 7890 A, Agilent Technologies, CA, USA; sample size 1 µL and split ratio 10:1) equipped with a flame ionization detector and an HP-INNOwax column (19091 N-213, 30.0 m × 320 μm × 0.5 μm, Agilent Technologies, CA, USA), based on the methods described by Ren et al., with some modifications [30].

According to the methods described by Gu et al. [21], the filtrates were continuously diluted for microbial counts (from 10− 1 to 10− 6). LAB were grown on De Man, Rogosa, and Sharpe agar (MRS, Beijing Land Bridge Technology, Beijing, China) and incubated at 35 °C for 48 h in an anaerobic incubator. The yeasts and molds were counted on potato dextrose agar (Beijing Land Bridge Technology, Beijing, China) at 25 °C for 72 h. The number of enterobacteria (EB) was counted on violet red bile agar (Beijing Land Bridge Technology, Beijing, China) at 37 °C for 48 h.

DNA extraction

Microbial DNA from each silage sample (including silage samples after 0 and 16 d of aerobic exposure) was extracted according to the methods of Zhang et al. [20]. The integrity of the extracted DNA was checked using 1% agarose gel electrophoresis, and the DNA concentration and purity were determined with a NanoDrop 2000 ultraviolet–visible (UV-vis) spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Polymerase chain reaction (PCR) amplification and UV light gel documentation

The extracted microbial DNA was sent to Guangxi Pufei Information Technology Co. Ltd. (Guangxi, China) for 16 S RNA and ITS1 gene amplicon sequencing on a NovaSeq 6000 platform. Amplification sequencing of bacterial 16 S RNA was performed using primers 341 F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Amplification of fungal ITS1 was performed using primers ITS5-1737 F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R (5′-GCTGCGTTCTTCATCGATGC-3′). The PCR products were mixed in equimolar ratio and purified using the QIAquick Gel Extraction Kit (Qiagen, Dusseldorf, Germany). The amplified products were resolved by electrophoresis on 1% agarose gel and visualized under a UV gel documentation system (Thermo Scientific, Wilmington, DE, USA).

Analysis of microbial diversity

The alpha diversity of the bacterial and fungal communities was analyzed using the software platform QIME 1.7.0. Functional profiles of bacterial communities were predicted based on 16 S rRNA gene sequencing data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) [31]. Fungal ecological guilds were predicted based on ITS gene sequencing data using FUNGuild [32]. Linear discriminant analysis effect size (LEfSe) software (version 1.0) was employed to perform LEfSe analysis.

Statistical analysis

Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, Illinois, USA). One-way analysis of variance (ANOVA) was performed on the chemical composition, microbial population, and prediction of microbial function of the STS, and a general linear model (GLM) was used for two-way analysis of variance on WSC levels, additives, and their interaction. Tukey’s honest significant difference (HSD) test was used to identify significant differences between the two groups. Statistical significance was set at P ≤ 0.05. Graphing was conducted in RStudio software (4.0.3). Principal coordinate analysis (PCoA) was performed using the Vegan package. All other maps in the current study were drawn using the “ggpolt2” package.

Results

Chemical characteristics and microbial population of STS

The chemical composition and microbial population of the STS are shown in Table 1. Regardless of whether CA was added, the DM, NDF, and ADF contents of STS in the HCK and HCA groups were significantly higher than those in the LCK and LCA groups (P < 0.001), while the CP content of STS in the HCK and HCA groups was significantly lower than that in the LCK and LCA groups (P < 0.001). The WSC content of STS was significantly affected by the CA and WSC levels (P ≤ 0.05). At the same WSC level, the WSC content in the HCA group was significantly higher than that in the HCK group (P < 0.001). The DM loss of STS was also significantly affected by the CA and WSC levels (P ≤ 0.05). After the addition of CA, DM loss in the LCA and HCA groups was significantly lower than that in the LCK and HCK groups (P < 0.001). The ash content in STS was significantly affected by the WSC level (P = 0.001) and the interaction between the CA and WSC levels (P = 0.002). Moreover, the ash content in the LCK group was significantly higher than that in the LCA, HCK, and HCA groups (P ≤ 0.05). However, the CA or WSC levels had no effect on the CF content and LAB count of STS (P > 0.05). Notably, CA and WSC levels had a significant effect on yeast counts (P ≤ 0.05). Moreover, at the same WSC level, the yeast count in the HCA group was significantly lower than that in the HCK group (P = 0.001). In addition, EB were not detected in the LCA and HCA groups, irrespective of WSC levels. No mold was enumerated in any of the groups.

Table 1.

Chemical composition and microbial population of STS

Items Treatments P-value
LCK LCA HCK HCA SEM C A C ×A
Chemical composition, g/kg DM
DM 287.83b 291.65b 306.91a 303.36a 2.53 < 0.001 0.948 0.100
CP 67.59a 70.34a 46.73b 44.15b 3.63 < 0.001 0.961 0.134
CF 376.03 374.76 391.33 396.10 8.96 0.386 0.932 0.884
WSC 61.51bc 94.84ab 81.17b 116.55a 7.74 0.007 0.001 0.816
NDF 599.12b 629.48b 749.98a 760.09a 26.48 0.005 0.592 0.787
ADF 306.56b 313.63b 449.94a 439.94a 25.42 0.005 0.968 0.817
ADF 287.83b 291.65b 306.91a 303.36a 25.43 0.005 0.507 0.475
Ash 67.13a 64.50b 62.35c 64.12bc 0.66 0.001 0.246 0.002
DM loss 13.76a 12.68b 10.62c 9.14d 0.54 < 0.001 < 0.001 0.154
Microbial population, log10 cfu/g FM
LAB 5.12 4.94 4.25 5.23 0.16 0.172 0.082 0.028
Yeast 3.18b 3.14b 4.67a 3.71b 0.24 0.004 0.050 0.062
Mold ND ND ND ND - - - -
EB 0.66 ND 0.74 ND - - - -
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LCK, low-WSC sugarcane tops treated with no additive; LCA, low-WSC sugarcane tops treated with 10 g/kg FW citric acid; HCK, high-WSC sugarcane tops treated with no additive; HCA, high-WSC sugarcane tops treated with 10 g/kg FW citric acid. DM, dry matter; CP, crude protein; CF, crude fiber; WSC, water-soluble carbohydrate; NDF, neutral detergent fiber; ADF, acid detergent fiber; LAB, lactic acid bacteria; EB, enterobacteria. SEM, standard error of the mean. C, water-soluble carbohydrate levels; A, additive; C × A, the interaction between water-soluble carbohydrate contents and additive. ND, not detected. a–cMeans within a row without a common superscript letter differ significantly at P < 0.05

The fermentation parameters of STS prior to and after aerobic exposure

The pH and AN dynamics of STS during aerobic exposure are shown in Fig. 1A and B. The pH of low-WSC STS was consistently lower than that of high-WSC STS on days 0–8 of aerobic exposure, whether CA-treated or untreated (Fig. 1A). Unlike the pH of the LCK group, which remained stable throughout the aerobic exposure, that of LCA rapidly increased over 6.0 on days 8–16 of aerobic exposure. In contrast, the pH trend remained stable throughout the 16 days of aerobic exposure in the HCA group, while it rapidly increased over 6.0 on days 8–16 of aerobic exposure in the HCK group (Fig. 1A). However, a rapid increase to over 6.0 in the LCA group on the 8–16 days of aerobic exposure, while in the HCA group, it remained stable (around 4.2) throughout the 16 days of aerobic exposure (Fig. 1A). Additionally, the AN content of the LCA and HCA groups was lower than that of the LCK and HCK groups, while all the groups contained less than 0.4 g/kg DM during 16 days of aerobic exposure (Fig. 1B).

Fig. 1.

Fig. 1

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The dynamics of the fermentative characteristics of STS during aerobic exposure. pH (A) NH3-N (B) lactic acid (C) acetic acid (D) propionic acid (E) butyric acid (F) ethanol (G). LCK, LCA: low-WSC sugarcane tops treated with no additive and 10 g/kg FW citric acid; HCK, HCA: high-WSC sugarcane tops treated with no additive and with 10 g/kg FW citric acid

The volatile organic compound dynamics, including lactic acid (C), acetic acid (D), propionic acid (E), butyric acid (F), and ethanol (G), of STS during aerobic exposure are shown in Fig. 2. In both the LCA and HCA groups, lactic acid content decreased on days 8–16 of aerobic exposure (Fig. 1C), while the lactic acid content increased in the LCK group between days 8 and 16 after exposure. On days 4–8 of aerobic exposure, the acetic acid content decreased in all groups (Fig. 1D). Moreover, on days 4–8 of aerobic exposure, propionic acid content decreased in the LCK and HCK group on days 4–8 of aerobic exposure (Fig. 1E). However, propionic acid content in the LCA and HCA groups remained stable at 0 g/kg DM throughout the 16 d of aerobic exposure (Fig. 1E). Additionally, butyric acid levels significantly increased on days 4–8 of aerobic exposure in the HCK group and decreased on days 8–16 of aerobic exposure (Fig. 1F). However, butyric acid content in the LCK, LCA, and HCA groups remained stable at 0–1 g/kg DM during 16 days of aerobic exposure (Fig. 1F). Moreover, on days 0–8 of aerobic exposure, the ethanol content continued to decrease to a minimum only in the LCA group (Fig. 1G). However, ethanol content greatly increased in the LCK, HCK, and HCA groups on days 0–4 of aerobic exposure, and decreased to a minimum value of 0 g/kg DM on days 4–16 of aerobic exposure (Fig. 1G).

Fig. 2.

Fig. 2

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Venn analysis of operational taxonomic units (OTUs) for STS. Bacterial OTUs of STS before (A) and after aerobic exposure (B). Fungal OTUs of STS before (C) and after aerobic exposure (D). Arabic numerals indicate days of aerobic exposure. LCK, LCA: low-WSC sugarcane tops treated with no additive and 10 g/kg FW citric acid; HCK, HCA: high-WSC sugarcane tops treated with no additive and with 10 g/kg FW citric acid

Microbial diversity of STS prior to and after aerobic exposure

Venn analysis (Fig. 2) showed that there were 500 common bacterial OTUs among all the groups, while there were 453, 54, 80, and 221 bacterial OTUs in the LCK, HCK, HCA, and LCA groups, respectively, on day 0 of aerobic exposure. On day 16 of aerobic exposure, all groups contained 232 common bacterial OTUs, while 167, 112, 64, and 36 particular bacterial OTUs were present in the LCK, HCK, HCA, and LCA groups, respectively (Fig. 2A). For fungi, there were 123 common fungal OTUs among all the groups, and 225, 86, 33, and 59 particular fungal OTUs in the LCK, HCK, HCA, and LCA groups, respectively, on day 0 of aerobic exposure. On day 16 of aerobic exposure, all groups contained 109 common bacterial OTUs, while 69, 161, 26, and 18 particular bacterial OTUs were present in the LCK, HCK, HCA, and LCA groups, respectively (Fig. 2B).

According to the principal coordinate analysis (Fig. 3), the bacterial communities were disengaged from each other between different WSC levels and treatments, except for the treatment group where CA was added after 16 days of aerobic exposure (Fig. 3A). Similarly, the fungal communities were disengaged from each other between the different WSC levels and treatments, whether before or after aerobic exposure (Fig. 3B).

Fig. 3.

Fig. 3

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Principal coordinate analysis of the bacterial (A) and fungal community (B) in STS. Phylum (C) and genus (D) levels of bacterial communities in STS. Phylum (E) and genus (F) levels of fungal communities in STS. Arabic numerals indicate days of aerobic exposure. LCK, LCA: low-WSC sugarcane tops treated with no additive and 10 g/kg FW citric acid; HCK, HCA: high-WSC sugarcane tops treated with no additive and with 10 g/kg FW citric acid

Microbial communities of STS prior to and after aerobic exposure

The bacterial community dynamics of STS at the phylum and genus levels are shown in Fig. 3C and D, respectively. At the phylum level, Firmicutes and Proteobacteria were the predominant phyla in all groups before aerobic exposure, and these two phyla were also predominant in the LCK, HCK, and LCA groups on day 16 of aerobic exposure (Fig. 3C). However, in the HCA group, Firmicutes and Actinobacteria were the two dominant phyla on day 16 of aerobic exposure (Fig. 3C). At the genus level, Lactobacillus in the LCK, LCA, and HCA groups was the predominant genus, while Pantoea was the top first genus in the HCK group before aerobic exposure (Fig. 3D). Notably, Clostridium_sensu_stricto_12 was also dominant in the LCK and HCK groups before aerobic exposure (Fig. 3D). Moreover, on day 16 of aerobic exposure, Bacillus was the first dominant genus in the LCK, LCA, and HCA groups, whereas in the HCK group, the first dominant genera were still Pantoea. Furthermore, Lactobacillus levels were higher in the LCA and HCA groups before aerobic exposure (Fig. 3D). Clostridium_sensu_stricto_12 decreased in the LCA and HCA groups, both before and after 16 days of aerobic exposure (Fig. 3D). In contrast, the relative abundance of Bacillus increased in the LCA and HCA groups after 16 d of aerobic exposure (Fig. 3D).

The fungal community dynamics of STS at the phylum and genus levels are shown in Fig. 3E and F, respectively. At the phylum level, Ascomycota was the most dominant phylum in the LCK group, whereas in the LCA, HCK, and HCA groups, the most dominant phyla were Zygomycota before aerobic exposure (Fig. 3E). In contrast, on day 16 of aerobic exposure, Ascomycota was the most dominant phylum in the LCK, LCA, and HCA groups, whereas Zygomycota was the most dominant phylum in the HCK group (Fig. 3E). At the genus level, Meyerozyma was the first dominant genus in all groups, and Paecilomyces was the most dominant genus, only found in the LCK and HCK groups before aerobic exposure (Fig. 3F). Moreover, the relative abundances of Meyerozyma and Paecilomyces decreased in the LCA and HCA groups compared to those in the LCK and HCK groups before aerobic exposure (Fig. 3F). Meanwhile, the relative abundance of Saccharomyces increased in the LCA and HCA groups compared to that in the LCK and HCK groups before aerobic exposure, and it was also increased in HCA compared to that in the HCK group after 16 days of aerobic exposure. Moreover, the relative abundance of Pichia decreased in the LCA and HCA groups compared to that in the LCK and HCK groups before aerobic exposure, and it continued to decrease in HCA compared to that in the HCK group after 16 days of aerobic exposure. Furthermore, on day 16 of aerobic exposure, Paecilomyces was the most dominant genus found in LCK and LCA treatments, while it was changed in high-WSC STS, among which Monascus was the most dominant genus in the HCK group, and Wickerhamomyces was the most dominant genus in the HCA group (Fig. 3F).

Microbial biomarkers of STS prior to and after aerobic exposure

Differences in bacterial communities among the groups were determined using LEfSe analysis (Fig. 4A and B). Before aerobic exposure, Clostridium_sensu_stricto_12 was higher in the LCK group, Bradyrhizobium was higher in the LCA group, Pantoea and Enterobacter were higher in the HCK group, and Lactobacillus was higher in the HCA group (Fig. 4A); On day 16 of aerobic exposure, Bradyrhizobium and Pseudomonas were higher in the LCK group, Paenibacillus was higher in the LCA group, Pantoea, Enterobacter, Clostridium_sensu_stricto_12, and Sporolactobacillus were higher in the HCK group, and Bacillus was higher in the HCA group (Fig. 4B).

Fig. 4.

Fig. 4

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Identification of the communities that have significant differences among the groups. Difference between bacterial communities on STS before (A) and after aerobic exposure (B). Difference in the fungal community of STS before (C) and after aerobic exposure (D). Arabic numerals indicate days of aerobic exposure. LCK, LCA: low-WSC sugarcane tops treated with no additive and 10 g/kg FW citric acid; HCK, HCA: high-WSC sugarcane tops treated with no additive and with 10 g/kg FW citric acid

The differences in fungal communities among the groups were also studied using LEfSe analysis (Fig. 4C and D). Before aerobic exposure, Bacteroidia was higher in the LCK group, Wickerhamomyces and Monascus were higher in the LCA group, Paecilomyces and Aspergillus were higher in the HCK group, and Saccharomyces and Mucor were higher in the HCA group (Fig. 4C); On day 16 of aerobic exposure, Defluviitaleaceae_UCG_011 was higher in the LCK group, Paecilomyces was higher in the LCA group, and Monascus, Meyerozyma, Candida, Pichia, and Blakeslea were higher in the HCK group, and Wickerhamomyces and Saccharomyces were higher in the HCA group (Fig. 4D).

Function prediction of microbial communities in STS prior to and after aerobic exposure

The potential functions of bacterial communities in STS with WSC levels and CA before and after aerobic exposure were predicted using PICRUSt2 software, and the top five functions, including amino acid metabolism, carbohydrate metabolism, energy metabolism, nucleotide metabolism, and metabolism of cofactors and vitamins, were selected for statistical analysis (Fig. 5A and B). The relative abundance of amino acid metabolism significantly decreased in the HCK and HCA groups before aerobic exposure (P < 0.05). The relative abundance of amino acid metabolism was significantly lower in the HCK group after 16 d of aerobic exposure (P < 0.05). Whether before or after 16 days of aerobic exposure, the relative abundance of energy and nucleotide metabolism in the HCK group was significantly higher than that in the HCA group, and the relative abundance of carbohydrate metabolism and the metabolism of cofactors and vitamins were significantly lower in the LCK group (P < 0.05).

Fig. 5.

Fig. 5

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The predicted function of microbial communities (on the second level). Bacterial community phenotypes of STS before (A) and after aerobic exposure (B). Fungal community functional guild of STS before (C) and after aerobic exposure (D). Arabic numerals indicate days of aerobic exposure. LCK, LCA: low-WSC sugarcane tops treated with no additive and 10 g/kg FW citric acid; HCK, HCA: high-WSC sugarcane tops treated with no additive and with 10 g/kg FW citric acid. a–c indicate significant differences between groups at P < 0.05

The following top five functional abundances of fungal communities of STS with WSC levels and CA before and after aerobic exposure were also selected for statistical analysis: animal pathogens, plant pathogens, Undefined Saprotroph, Animal Pathogen-Undefined Saprotroph, and Animal Pathogen-Endophyte-Lichen Parasite-Plant Pathogen-Soil Saprotroph-Wood Saprotroph (Fig. 5C and D). The relative abundance of animal and plant pathogens significantly decreased in the LCA and HCA groups before aerobic exposure (P < 0.05). Furthermore, the relative abundance of animal pathogens was significantly higher in the HCA group (P < 0.05), and the relative abundance of plant pathogens significantly decreased in the LCA and HCA groups after 16 days of aerobic exposure (P < 0.05).

Discussion

Fermentation quality of STS

The chemical composition and microbial population of STS are shown in Table 1. In the present study, the HCK and HCA treatments had lower CP contents. There may still be a large number of acid-tolerant proteolytic bacteria that can utilize the abundant sugar substrates remaining in the silage for growth and reproduction [33]. Similar results were reported in sorghum silage and grass-clover silage with high WSC contents [34, 35]. In the present study, the DM content was higher in the HCK and HCA treatments. Similar results were also found in sorghum and millet silage with high WSC contents [34]. This is mainly due to the higher DM content in the high-WSC sugarcane tops before ensiling (Table S1). The HCK and HCA treatments also yielded higher NDF and ADF contents in the current study. This was mainly because the easily consumed nutrients in silage, such as WSC, promoted the increase of structural carbohydrates such as NDF and ADF in silage after being decomposed and utilized in large quantities. Amer et al. also reported that compared to regular-WSC sorghum silage, high-WSC sorghum silage contained higher NDF and ADF contents, but the difference was not significant [34]. In the present study, higher WSC content was only observed in the HCA treatment. Similar findings were obtained in alfalfa silage treated with 12 g/kg fresh weight (FW) CA and 12–60 g/kg FW CA residue [7, 9]. This may be due to the higher WSC content of the fresh high-WSC sugarcane tops or the antimicrobial effect of CA, which can reduce certain microorganisms that utilize sugar substrates [4]. In this study, the ash content was higher in the LCA treatment compared to the HCK and HCA treatments. Previous research on low-WSC sorghum and millet silage obtained comparable results [34]. This may be related to the lower organic matter content (higher ash content) in the fresh low-WSC sugarcane tops (Table S1). Additionally, in this study, yeast counts were lower in the HCA group. Lower yeast counts were also observed in mulberry leaf silage treated with 20 g/kg FW CA on days 3, 7, and 14 and paper mulberry silage treated with 2 g/kg, 5 g/kg, and 8 g/kg FW CA [18]. However, the results were inconsistent with those of previous studies [8, 9], which reported that the yeast increased in all CA-treated alfalfa silage compared to untreated silage. This may be because (1) high-WSC sugarcane tops can provide more available sugar substrates to promote lactic acid fermentation and produce more lactic acid to inhibit yeast reproduction [33], and (2) CA directly inhibits the growth of yeast [7]. Similar to our study, lower EB counts were observed in alfalfa silage treated with 12 g/kg FW CA residue [11]. This was attributed to the antibacterial effect of CA against EB [36]. No mold was enumerated in any of the STS. This is consistent with the study of Zhang et al. [20], who also reported that mold was not detected in natural STS. This is related to silage pH; when the pH is < 5, fungal growth is inhibited. Moreover, in this study, the DM loss in CA-treated STS was significantly lower. It is possible that the antibacterial CA or the high lactic acid content produced in silage inhibited the reproduction of these harmful microorganisms [7, 33, 36]. These results show that adding CA to low-WSC or high-WSC STS can reduce nutrient loss and counts of harmful microorganisms.

Moreover, in this study, the pH of LCA treatment was more likely to exceed 6.0 in the late stage of aerobic exposure. Similar results were also found in alfalfa silage with CA on days 4–6 of aerobic exposure and in Napier grass silage with CA on days 0–6 of aerobic exposure [10–13]. The increase in silage pH may be due to the lack of sugar substrates in the silage as the aerobic exposure time increases, and some microorganisms capable of aerobically degrading lactic acid (e.g., lactate-assimilating yeasts) degrade lactic acid in large quantities [33, 37]. However, in the present study, the pH of the LCK treatment remained stable (around 4.0) until day 16 after exposure. Similarly, Xia et al. also reported that the pH remained 3.9–4.2 in whole-plant corn silage with low WSC content on days 0–9 of aerobic exposure [38]. It is possible that in STS, low WSC content is suitable for the survival of some acid-producing bacteria (e.g., Lactobacillus and Acetobacter) under aerobic conditions [20, 39], thereby effectively maintaining the silage pH. Furthermore, in the current study, the pH of the HCA treatment remained stable (approximately 4.2) throughout the 16 d of aerobic exposure. Similar results were observed in alfalfa silage with 36 and 60 g/kg FM CA residue on days 4–6 of aerobic exposure and alfalfa silage with 12 g/kg FM CA residue on days 0–2 of aerobic exposure [10, 11]. A possible reason is that CA and the large amount of residual carbohydrates in high-WSC STS can serve as substrates for LAB, and their metabolites are all organic acids (e.g., lactic acid) regardless of their exposure to air [6, 33]. In contrast, the pH of the HCK treatment was increased at the late stage of aerobic exposure in the present study. Comparable results were found in sugarcane silage with high WSC content after 8 days of aerobic exposure [40]. The increased silage pH may be attributed to the fact that more residual carbohydrates are present in the high-WSC STS, which can provide substrates for yeast under prolonged aerobic exposure [41]. Additionally, in the present study, the AN content of CA-treated STS was always lower during aerobic exposure. Similar results were found in alfalfa silage with 12 g/kg FM CA residue on days 0–2 of aerobic exposure [10]. This indicated that CA effectively plays a role in inhibited protein hydrolase activity [2, 7–9]. While, in our study, the AN contents were no more than 0.4 g/kg DM in all STS during 16 days of aerobic exposure. This is mainly related to sugarcane tops, which are low-protein forages.

The lactic acid content of CA-treated STS decreased during the late stage of aerobic exposure. Similarly, Tao et al. also reported that lactic acid content decreased in alfalfa silages with CA residue on days 2–8 of aerobic exposure [10]. This may be because, with prolonged aerobic exposure time, the residual carbohydrates for lactic acid production decreased in the STS or some aerobic yeasts (e.g., lactate-assimilating yeasts) proliferated in the forage silage with CA [9–11, 37]. However, the lactic acid content of the LCK treatment increased at the late stage of aerobic exposure. This may have occurred because, although the STS contained low WSC content, Lactobacillus, which can utilize residual sugar to produce lactic acid, remained the most abundant bacterial genus on days 6–14 of aerobic exposure [21]. Moreover, in this study, the acetic acid content in all STS decreased to a minimum value during middle and late aerobic exposure. These results are in agreement with those of Tao et al. [10–13], who reported that the acetic acid content also decreased in alfalfa silage with or without CA residue on days 4–6 of aerobic exposure and in Napier grass silage with CA residue on days 0–4 of aerobic exposure. Acetic acid in silage can inhibit the growth and reproduction of mold and other spoilage microorganisms, thus improving the aerobic stability of silage [42]. In contrast, in this study, propionic acid content in CA-treated STS was always 0 g/kg DM throughout the 16 days of aerobic exposure. Nishino et al. pointed out that propionic acid has a stronger antibacterial ability than acetic acid in silage [43]. Additionally, in present study, the butyric acid content in CA-treated STS was maintained at 0–1 g/kg DM during 16 days of aerobic exposure. This is primarily attributed to the presence of large amounts of lactic acid in the silage or the addition of CA, both of which have strong inhibitory effects on Clostridium multiplication [2, 44]. It is worth noting that butyric acid is closely related to the aerobic spoilage of silage, and large amounts of butyric acid can easily lead to the aerobic deterioration of silage. In the present study, the butyric acid content of the HCK treatment increased on day 8 but decreased between days 12–16. Sun et al. also reported that butyric acid in Leymus chinensis silage (fresh materials cut to a length of 4–5 cm) with molasses increased on days 1–3 of aerobic exposure and decreased on days 3–7 of aerobic exposure [45]. The sudden increase in butyric acid after aerobic exposure may have occurred because Clostridium utilizes the large amount of sugar substrates remaining in silage to proliferate, and its metabolites are mainly butyrate under aerobic conditions [33]. The butyric acid may subsequently decrease because as the aerobic exposure time of the silage is prolonged, bacteria that can secrete antibiotics (e.g., Bacillus) proliferate rapidly with sufficient sugar substrate [21], and some species of these bacteria can inhibit the proliferation of Clostridium. Additionally, in the present study, the ethanol content decreased to a minimum only in LCA treatments in the early and middle stages of aerobic exposure. This may be because CA supplementation inhibits the growth of alcohol-producing fungi, such as yeasts and molds [46]. Therefore, further microbiological analyses of STS before and after aerobic exposure are required.

Bacterial communities of STS

The number of common bacterial OTUs and specific bacterial OTUs—except for the HCK treatment—decreased after aerobic exposure. This is because of the inhibition of the growth of anaerobic bacteria in silage under aerobic conditions. The specific bacterial OTUs only increased in HCK treatment under aerobic conditions, which may be related to the increased sugar substrates remaining in the silage, which in turn provides a breeding ground for aerobic harmful bacteria. It is possible that CA has antibacterial and bactericidal functions [47], and the specific bacterial OTUs were lower in CA-treated STS, whether before or after aerobic exposure. Similar results were also found in CA-treated king grass silage [3, 5]. In contrast, possibly due to the difference in silage raw materials, the specific bacterial OTUs increased in CA-treated cassava foliage silage [48]. The principal component analysis demonstrated that the bacterial community could be impacted by WSC content and CA in silage before aerobic exposure.

Firmicutes and Proteobacteria were the predominant phyla in all groups before aerobic exposure, and these two phyla were also predominant in STS (except the HCA treatment) after 16 d of aerobic exposure. Gavande et al. point out that the facultative anaerobic Proteobacteria, and aerobic and anaerobic Firmicutes were mainly associated with the degradation of cellulose [49]. However, Firmicutes and Actinobacteria were the dominant phyla only in the HCA treatment on day 16 of aerobic exposure. It has been demonstrated that aerobic cellulose-degrading Actinobacteria can secrete soluble cellulases and hemicellulases to degrade cellulose [50]. This could be because under aerobic conditions, high-WSC contents STS can provide more residual substrates for the aerobic Actinobacteria to utilize.

Before aerobic exposure, Lactobacillus was the most dominant genus in the CA-treated STS, and LCK treatment, whereas Pantoea was the most dominant genus in the HCA treatment. Lactobacillus usually dominates silage and is an important microorganism that promotes lactic acid metabolism [51]. Pantoea, whose role in silage is still unclear, can compete with LAB for organic substrates to produce butyrate, leading to the deterioration of silage quality [52]. Ogunade et al. suggested that Pantoea reduces protein loss in silage [53]. Moreover, Clostridium_sensu_stricto_12 was also dominant in the LCK and HCA treatments before aerobic exposure. Clostridium_sensu_stricto_12 is considered an undesirable microorganism that can cause protein degradation, DM loss, and butyric acid production in silage [54]. Moreover, after aerobic exposure, Bacillus was the first dominant genus in CA-treated STS and the LCK treatment, while the first dominant genera were Pantoea in the HCK treatment. Lara et al. found that some Bacillus species can extend silage storage time by producing bacteriocins under aerobic conditions [55]. Furthermore, Lactobacillus was increased in CA-treated STS compared to untreated STS before aerobic exposure. Similar results were obtained for mulberry leaf silage [2], king grass silage [3], Amomum villosum silage [4], and mixed silage containing king grass and rice straw [5] treated with CA. This may be because CA can serve as an energy source for LAB to promote their growth or it can inhibit the bacterial flora that competes with LAB [56, 57]. Considering the high concentrations of butyrate and NH3-N in the HCK treatment compared to CA-treated STS prior to and after aerobic exposure, we speculate that Pantoea is an undesirable bacterium that promotes the aerobic deterioration of the HCK treatment. Importantly, due to the antibacterial effect of CA, the relative abundance of Clostridium_sensu_stricto_12 decreased in the CA-treated STS both before and after aerobic exposure. Similarly, the relative abundance of Clostridium also decreases in fermented seed-used pumpkin leaves [58]. For the same reason, the relative abundance of Pantoea decreased in the HCA treatment compared to that in the HCK treatment, both prior to and after aerobic exposure. Additionally, the relative abundance of Bacillus was increased in the CA-treated STS after aerobic exposure. This is possibly due to the fact that at oxygen transfer conditions, some species of Bacillus can be the use of CA as the carbon source to produce serine alkaline proteases [59]. This indicated that high- or low- WSC content of STS treated with CA could inhibit the reproduction of harmful bacteria and improve aerobic stability.

To further evaluate the differences in bacteria among the groups, we used LEfSe analysis. In the present study, whether in the LCK or HCK treatments, undesirable bacteria such as Clostridium_sensu_stricto_12 and Enterobacter were more likely to proliferate before aerobic exposure. This may explain the higher nutrient loss in the control silage group. Moreover, Bradyrhizobium, which has a strong nitrogen-fixing ability, was the most differentially abundant bacterium in LCK and LCA treatments prior to and after aerobic exposure, respectively. Hence, this silage obtains more CP, as reported in our study. Furthermore, Lactobacillus was the most differentially abundant bacteria in HCA treatment. This may be because, under a sufficient carbon source substrate, CA can promote the proliferation of LAB [60]. Notably, after aerobic exposure, whether in the LCK or HCK treatment, it was easier for some bacteria with antibacterial effects to accumulate. Among these, Paenibacillus and Bacillus were more abundant in the LCA and HCA treatments, respectively.

Amino acids, carbohydrates, energy, nucleotides, cofactors, and vitamin metabolism are the main metabolic pathways that cause changes in fermentation and chemical characteristics in silage [61]. Amino acids metabolism abundance was notably lower in the HCK and HCA treatments before aerobic exposure. This finding was consistent with the lower AN content in high-WSC STS (Fig. 1B), possibly because the lower pH (Fig. 1B) inhibited undesirable microorganisms such as Clostridium_sensu_stricto_12 and Enterobacter. After aerobic exposure, the amino acid metabolism abundance was also the lowest in the HCK treatment. Considering the change in the AN content during aerobic exposure (Fig. 1B), the reason for this result may be that the undesirable aerobic bacteria almost consumed the remaining available nitrogen in the silage in the late stages of aerobic exposure. Therefore, carbohydrate metabolism is mainly gluconeogenesis and glycolysis, which are related to LAB abundance and usable carbohydrates in silage [61–63]. Therefore, in this study, carbohydrate metabolism abundance was lowest in the LCK treatment, whether prior to or after aerobic exposure. Moreover, energy and nucleotide metabolism abundance was higher in the HCK treatment than in the HCA treatment, both prior to and after aerobic exposure. Energy metabolism mainly involves bacteria metabolizing WSC into organic acids, among which high-quality silage can be obtained if LAB metabolizes WSC to produce lactic acid [64]. Kilstrup et al. pointed out that most metabolic reactions are related to the utilization of nucleotides [65] or the regulation of metabolites by bacteria and that nucleotide metabolism is also related to LAB [61, 66]. These results may be due to the antibacterial effect of CA, which limits the metabolic effects of certain bacteria in silage before aerobic exposure, whereas the massive reproduction of aerobic bacteria (for example, LAB) utilizes CA and weakens its effects, turning the overall metabolism into one dominated by the metabolism of LAB after aerobic exposure. Our study also confirmed that after aerobic exposure, Lactobacillus abundance was higher in the HCA treatment. The metabolism of cofactors and vitamin abundance were notably lowest in the LCK treatment, whether before or after aerobic exposure. Studies have shown that LAB can promote the production of vitamins during silage fermentation [61, 67]. However, Clostridium_sensu_stricto_12 and Bacillus were the dominant bacteria in the LCK treatment.

Fungal communities of STS

The number of common fungal OTUs and specific fungal OTUs (except for the HCK treatment) decreased after aerobic exposure. This is possibly due to the inhibition of the growth of anaerobic fungi in the silage under aerobic conditions. The increase in specific fungal OTUs of the HCK treatment might be related to the more sugar substrates remaining in the silage during aerobic exposure, which in turn provides a breeding ground for aerobic fungi, such as molds and yeasts. Therefore, these specific fungal OTUs may be the main cause of silage spoilage in high-sugar raw materials. Moreover, the specific fungal OTUs were lower in CA-treated STS, whether before or after aerobic exposure. This was related to the antifungal properties of CA [46]. Principal component analysis demonstrated that the fungal community could be affected by the WSC content and CA in silage prior to and after aerobic exposure.

Ascomycota and Zygomycota were the most dominant phyla in STS. Similar results were also reported by Gu et al. [21]. Ascomycota is usually the main group involved in cellulose decomposition in silage and is also the most diverse and species-rich phylum in the fungal kingdom. Zygomycota is also a member of the fiber-degrading group and is considered a true fungus that contains chitin in its cell wall [68]. These results may be attributed to the availability of substrate in the silage before and after aerobic exposure and the antifungal effect of CA.

Meyerozyma was the dominant genus in all groups prior to aerobic exposure. Meyerozyma is the dominant genus found in corn stalks and sugarcane tops after anaerobic fermentation [20, 69, 70]. Meyerozyma spp. are yeasts found in natural ecosystems that have strong antifungal activity that inhibits mold growth and/or mycotoxin production [71, 72]. However, possibly given the antibacterial effect of CA, the relative abundance of Meyerozyma was lower in CA-treated STS. In this study, Paecilomyces was the most dominant genus found only in the LCK and HCK treatments before aerobic exposure. Anderson et al. have reported that some species of Paecilomyces in silage were shown to be capable of producing patulin [73], while the accumulation of patulin may lead to poor silage quality. Saccharomyces is tolerant of CA, and it can use CA, as well as sugars, as an energy source for growth under both aerobic and anaerobic conditions [74]. This explains why the relative abundance of Saccharomyces is higher in CA-treated STS before aerobic exposure. In contrast, the relative abundance of Pichia is higher in CA-treated STS before aerobic exposure. This may also be related to the antibacterial effect of CA. Moreover, after aerobic exposure, Paecilomyces was the most dominant genus in low-WSC STS, and Monascus was the most dominant genus in the HCK treatment. The genera of molds, including Monascus and Paecilomyces, have been found in aerobically spoiled silages [75]. Monascus can compete with LAB to utilize carbohydrates (e.g., glucose) and organic acids (e.g., Lactic acid) as carbon sources and produce a variety of enzymes that degrade organic matter in silage [76]. In contrast, in the HCA treatment, Wickerhamomyces was the most dominant genus after aerobic exposure. Wickerhamomyces, the dominant fungus in silage after aerobic exposure, was also reported in king grass silage [77], and some species of Wickerhamomyces are known to secrete mycotoxins with a fungicidal effect [78]. This may be due to the interaction between the additive and differences in raw material sugar content, leading to the lactic acid content in silage. Possibly due to the availability of more substrates (residual sugar and CA), the relative abundance of Saccharomyces was higher in the HCA treatment compared to the HCK treatment after aerobic exposure. Meanwhile, the relative abundance of Pichia was lower in in the HCA treatment compared to the HCK treatment after aerobic exposure. This was mainly due to the lower silage pH (4.36 vs. 6.22). Saccharomyces and Pichia are often the main causes of the aerobic deterioration of silage [33].

We also used LEfSe analysis to evaluate differences in fungi among the groups. Bacteroidia and Defluviitaleaceae_UCG_011 are opportunistic pathogens [79, 80]. However, prior to or after aerobic exposure, pathogens were the most differentially abundant fungi in the LCK treatment. In contrast, in high-WSC sugarcane tops, untreated silage with CA, mycotoxin-producing fungi (e.g., Paecilomyces and Aspergillus) were the most differentially abundant fungi before aerobic exposure. However, probably because of the presence of abundant sugar substrates after aerobic exposure, many yeasts (e.g., Meyerozyma, Candida, and Pichia) were the most differentially abundant fungi in the HCK treatment. In contrast, Wickerhamomyces, as an antibacterial fungus, was the most differentially abundant fungus in the LCA treatment, but after aerobic exposure, the mycotoxin-producing fungi Paecilomyces were the most differentially abundant fungi. It is worth noting that Saccharomyces, as a potential probiotic, was the most differentially abundant fungus in the HCA treatment, both before and after aerobic exposure. Saccharomyces is tolerant to CA and can use CA as well as sugars as an energy source for growth under aerobic or anaerobic conditions [74]. Meanwhile, after aerobic exposure, Wickerhamomyces, a fungal pathogen [81], was also the most differentially abundant fungus in the HCA treatment.

In general, saprotrophs are not conducive to silage fermentation, causing spoilage and reducing the nutritional quality of the silage [82]. In this study, animal pathogen and plant pathogen abundance decreased in CA-treated STS before aerobic exposure, and after aerobic exposure, plant pathogens also decreased. This may be because CA inhibits the growth of the saprophytic fungi. The animal pathogen was highest in the HCA treatment after aerobic exposure. This may be related to the massive proliferation of the aerobic fungal pathogen Wickerhamomyces after long-term aerobic exposure. Notably, the presence of saprophytic fungi can lead to the accumulation of mycotoxins [83], which threaten the health of animals.

Conclusion

This study analyzed the effects of CA on the fermentation quality, microbial community, and microbial functions during the aerobic exposure of STS with high and low-WSC contents. The results showed that CA inoculation decreased DM loss in STS and inhibited the production of butyric and propionic acids throughout the 16 days of aerobic exposure. Moreover, CA inoculation decreased the abundances of undesirable bacteria and fungi before aerobic exposure, while after aerobic exposure CA inoculation increased the abundance of bacteria with potential antimicrobial effects. CA inoculation improved the metabolic pathways of bacteria to obtain more nutrients and reduced animal pathogens in STS to prevent animal diseases before aerobic exposure. Furthermore, compared to low-WSC STS, high-WSC STS contained more DM, NDF, and ADF and less CP, whether treated with CA or not. Notably, the HCA treatment exhibited more WSC, lower yeast counts, and no EB compared with the HCK treatment. Additionally, in the LCA treatment alone, ethanol content decreased in the early and middle stages of aerobic exposure, and animal pathogens decreased after 16 days of aerobic exposure. Our results provided an important basis for regulating the silage quality of forages rich in WSC using CA.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (13.2KB, docx)

Acknowledgements

The authors are grateful to Dr. Chengwu Zou of the College of Agriculture, Guangxi University, for providing the experimental materials.

Abbreviations

CA

Citric acid

DM

Dry matter

CP

Crude protein

WSC

Water-soluble carbohydrates

CF

Crude fiber

NDF

Neutral detergent fiber

ADF

Acid detergent fiber

LAB

Lactic acid bacteria

EB

Enterobacteria

AN

Ammonia nitrogen

TN

Total nitrogen

DNA

Deoxyribonucleic acid

RNA

Ribonucleic acid

OTU

Operational taxonomic units

PcoA

Principal coordinate analysis

LEfSe

Linear discriminant analysis effect size

Author contributions

QG conducted the study and prepared the first manuscript draft. JZ, BL, HD, and QY revised the manuscript draft. CW, YY, and RW performed data collection and statistical analysis. CZ contributed to securing financial support and designing the study. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Key Research and Development Program of China [grant number 2022YFD1300602], the National Natural Science Foundation of China [grant numbers 31860661 and 312360851], and the Innovation Project of Guangxi Graduate Education [grant number YCBZ2023050].

Data availability

The datasets generated or analysed during the current study are available from the corresponding author upon reasonable request (zou.cx@gxu.edu.cn). Bacterial and fungal raw sequence data were deposited in the sequence read archive at NCBI (https://www.ncbi.nlm.nih.gov/) under the accession numbers PRJNA968153 and PRJNA968154, respectively.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (13.2KB, docx)

Data Availability Statement

The datasets generated or analysed during the current study are available from the corresponding author upon reasonable request (zou.cx@gxu.edu.cn). Bacterial and fungal raw sequence data were deposited in the sequence read archive at NCBI (https://www.ncbi.nlm.nih.gov/) under the accession numbers PRJNA968153 and PRJNA968154, respectively.

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