Skip to main content

Fermentation quality and bacterial community of delayed filling stylo silage in response to inoculating lactic acid bacteria strains and inoculating time



Filling silos generally takes much time in practice, which may negatively affect silage fermentation and feed intake. To clarify the effects of inoculating time, lactic acid bacteria (LAB) strains and filling time on the silage fermentation and microbes of stylo (Stylosanthes guianensis) and its silage, ensiling was quickly performed (quickly filling, QF) with or without LAB (Lactobacillus plantarum SXC48, Lb. plantarum CCZZ1 and Enterococcus faecalis XC124), and was performed after stylo inoculated with or without LAB was placed for 1 day (delayed filling, DF1) and 2 days (DF2).


Delayed filling significantly increased pH, buffering capacity, microbial counts and lactic acid, acetic acid and NH3–N contents of stylo prior to ensiling. Inoculating Lb. plantarum SXC48 and CCZZ1 improved the fermentation quality of QF silage, indicated by more lactic acid, as well as lower pH and butyric acid content. Inoculating time significantly affected the pH and lactic acid content of silages. For the DF2 silages, inoculating SXC48 at filling was better than at chopping, while inoculating CCZZ1 had good fermentation quality, regardless of inoculating time. The results of 16S rRNA sequencing indicated that delayed filling enhanced the bacterial diversity of materials and silage, and inoculating significantly changed the composition of silage microbes. Kosakonia, Pseudomonas and Pantoea jointly dominated the fresh material, while Pantoea and Lelliottia jointly dominated the DF2 material. For the QF silages, inoculating SXC48 and CCZZ1 increased the relative abundance of Lactobacillus from 16.4% in the control silage to 76.5% and 82.0%, respectively. Pantoea and Lelliottia jointly dominated the DF silages. Inoculating SXC48 and CCZZ1 also increased the relative abundance of Lactobacillus in the DF stylo silages.


Under the present research conditions, delayed filling increased the lactic acid content and reduced the acetic acid, propionic acid and NH3–N contents of stylo silage, however, increased the bacterial diversity and relative abundance of undesirable bacteria, such as Pantoea and Lelliottia. The inoculating effectiveness varied with the LAB strains and inoculating time. Inoculating strian SXC48 at filling was better than at chopping, while inoculating strian CCZZ1 at both chopping and filling obtained the similar benefit.

Graphical Abstract


Stylo (Stylosanthes guianensis) is a very important tropical legume and feed source for local livestock because of its high crude protein content [1]. Feeding cattle with stylo-contained diet significantly improved the dry matter (DM) and N intake [2], and feeding pigs with stylo replacing soybean increased the weights of lung, large intestine and stomach [3]. Wilting is difficulty in the tropical and subtropical regions due to wet and rainy; therefore, ensiling may be a practical way to preserve forage [4]. Ensiling is an approach for long-term preservation of forage crops under the anaerobic conditions. Adding stylo silage to the diet could enhance goats production [5], and benefit the total tract apparent digestibility, N retention and energy digestibility in pigs [6]. In general, the silage well-preserved has lower pH and higher lactic acid content [4]. However, Liu et al. [7] and Pitiwittayakul et al. [8] reported that stylo silage had poor fermentation quality with high pH and NH3–N content. The factors limiting the fermentation of legume were relatively low concentration of water-soluble carbohydrates (WSC), high buffering capacity and less epiphytic LAB, compared to grasses [9]. Therefore, additives have been widely used to enhance the preservation of stylo silage. Nevertheless, chemical additives have some shortage, such as high cost and poor safety [10]. LAB have been used to promote rapid and efficient fermentation during ensiling through producing more lactic acid [11].

Shortening the initial aerobic phase in silage making is necessary [12]. Several researches [4, 13, 14] reported that the chopped materials exposed to the air for several days might delay the onset of fermentation, encouraging growth of undesirable microbes, such as yeasts. Then, the built-up yeasts partly remained latent after sealing until the silo was opened for feed-out [15,16,17], which might lead to aerobic deterioration. However, for large-scale silage production, ensiling is difficult to be completed in a short time, at least several days even 1 week [18]. In addition, poor management, such as leaving chopped materials in wagons or piles, or rainfall at harvesting, will prolong the filling process and increase exposure period. The fermentation quality and nutrition losses of silage are easily influenced by delayed filling [13, 14, 18]. Although some losses are unavoidable, good management practices can reduce them, such as using additives [19]. Arbabi et al. [20] found that adding buffered propionic acid-based additive in whole-plant corn exposed to air for 1 d and 2 d before ensiling prevented a decrease in DM digestibility of silage. Mills and Kung [13] also reported that adding buffered propionic acid-based additive affected the chemical compositions and yeast counts of whole-plant barley exposed to air for 1 d before ensiling, and prevented the reduction in in vitro digestion of silage, irrespective of adding either before or after exposure to air. Besides, the result of Cai et al. [16] showed that inoculating Lactobacillus plantarum did not improve the fermentation quality of silage sealed with 1 d delay. However, little is known about the inoculating time on the silage fermentation and microbial community of silage, and how bacterial community on forage crops change during aerobic exposure before ensiling.

Therefore, the objectives of this study were to investigate the effects of inoculating time, LAB strains and filling time on (1) the characteristics and microbial population of stylo before filling, and (2) the fermentation quality and bacterial diversity of stylo silage.

Materials and methods

Materials and silage preparation

Stylo was obtained from an experimental field at South China Agricultural University (23°260 N, 113°150 E, Guangzhou, China) in July 2021. The stylo harvested at the flowering stage was chopped into approximately 1 to 2 cm lengths using a mechanical chopper (9Z-0.4, Shentong Heavy Industry Co., Ltd., Zhengzhou, China). The chopped materials were thoroughly mixed and treated according to the experimental design. The experiment was designed as a 3 × 4 × 2 factorial study in a completely randomized design with three replicates per treatment. Ensiling time included quick filling (QF) within 6 h of harvesting, delayed filling after placed for 1 day (DF1) and 2 days (DF2). To test the differences in the fermentation quality among different strains, inoculating LAB strains (isolated by our laboratory) included Lb. plantarum SXC48 (SXC48), Lb. plantarum CCZZ1 (CCZZ1) and Enterococcus faecalis XC124 (XC124) at 105 cfu/g FM, and the equal amount of sterile water was added without LAB strain as the control (CK). Inoculating time was at chopping and at filling, the treated materials were packed into plastic film bags (30 cm × 20 cm, Mingkang Packing Co. Ltd, Zhongshan, China), then degassed and sealed using a vacuum sealer (Mainfold Vacuum Sealer DZ-280/2SD, Yijian Packaging Machinery Co. Ltd, Dongguan, China). The bags were kept at an ambient temperature of 28–35 °C and were opened after 60 d of ensiling to analyze the fermentation quality and microbial community composition.

Chemical composition analyses

Pre-ensiling stylo samples were dried in a forced air oven at 70 °C for 48 h to determine dry matter (DM) content, and ground to pass a 1.0 mm mesh screen for chemical analyses. The crude ash and crude protein were analyzed by the methods 942.05 and 984.13 of AOAC [21], respectively. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were performed following the procedure of van Soest [22] with an ANKOM A200i fiber analyzer (ANKOM Technology, Macedon, NY, USA) and were expressed exclusive of residual ash. The WSC content was measured using anthrone colorimetry [23]. The buffering capacity was determined by the method of McDonald et al. [4].

Fermentation quality analyses

Twenty grams of samples were sampled and homogenized with 80 mL deionized water, and kept in a refrigerator at 5 °C overnight as described by Zhang et al. [24]. Then, the material was filtered, and the filtrate was used to measure the fermentation products of silage, including pH, ammonia nitrogen (NH3–N) and organic acids. The pH value was measured using a glass electrode pH meter (FiveEasy Plus, Mettler Toledo Co., Ltd, Shanghai, China). The NH3–N content was analyzed using a Kjeldahl apparatus [25], and the concentrations of organic acids including lactic acid, acetic acid, propionic acid and butyric acid were measured by high-performance liquid chromatography (HPLC) method as described by Zhang et al. [24]. using Eleven Organic Acids on Transgenomic COREGel 87H3 column (Shodex, Japan), RID-10A detector (210 nm, SPD-20A, Shimadzu Research Laboratory Co., Ltd, Kyoto, Japan), eluent (0.1 mmol/L HP3O4, 1.0 mL/min), temperature (40 °C). The fermentation quality of silage was evaluated by the V-score evaluation system [26]. The V-score was calculated based upon the contents of NH3–N, acetic, propionic and butyric acid in silage using the formula in Table 1.

Table 1 Calculation of V-score

Microbial counts and bacterial diversity analyses

Ten grams of the sample was shaken well for 30 min with 90 mL of sterilized water, and serial dilutions (10−1–10−5) were made in sterile water. The LAB number was measured on de Man Rogosa Sharpe (MRS) agar incubated at 37 °C for 1–2 d under anaerobic conditions (Anaerobic Pack Rectangular Jar, 2.5 L, Mitsubishi Gas Chemical Company Inc., Tokyo, Japan). Aerobic bacteria, yeasts and molds were counted on nutrient agar and Rose Bengal agar incubated for 2–3 d at 30 °C under aerobic conditions, respectively. These media were obtained from Guangdong Huankai Microbial Sci. and Tech. CO. Ltd. (Guangzhou, China). Yeasts were distinguished from molds by observation of colony appearance. Colonies were counted as viable numbers of microorganisms in log10 cfu/g of FM.

Microbial DNA extraction from grass and silage samples was extracted with the TGuide S96 Bacteria DNA isolation kit (DP812, Tiangen, Beijing, China) according to manufacturer instructions. The 27F: AGRGTTTGATYNTGGCTCAG and 1492R: TASGGHTACCTTGTTASGACTT universal primer set was used to amplify the full-length 16S rRNA genes from the genomic DNA extracted from each sample by single molecule real-time (SMRT) sequencing technology. Both the forward and reverse 16S primers were tailed with sample-specific PacBio barcode sequences to allow for multiplexed sequencing. The polymerase chain reaction (PCR) program and procedures were performed as described by Mu et al. [27]. After purification and quantification, amplicons were sequenced using PacBio Sequel (Pacific Biosciences, Menlo Park, CA, USA). The raw reads generated from sequencing were filtered and demultiplexed using the SMRT Link software (version 8.0) to obtain the circular consensus sequencing (CCS) reads. The quality was filtered using the Cutadapt quality control process (version 2.7) through the recognition of forward and reverse primers. The UCHIME algorithm (v8.1) was used in detecting and removing chimera sequences to obtain the clean reads. Sequences with similarity ≥ 97% were clustered into the same operational taxonomic unit (OTU) by USEARCH (v10.0). Taxonomy annotation of the OTUs was performed based on the Naive Bayes classifier in QIIME2 using the SILVA database with a confidence threshold of 70%. Alpha diversity was calculated based on Shannon–Wiener, Simpson's diversity, Chao1 and rarefaction estimators and displayed by R software. Data were analyzed using the free online BMK Cloud Platform (www.

Statistical analyses

The effects of inoculating time, LAB strains and filling time on the chemical characteristics and microbes of stylo prior to ensiling, and on the fermentation parameters of stylo silage were analyzed with IBM SPSS 20.0 for Windows. The results were evaluated using analysis of variance (ANOVA). The means were compared for significance by Duncan’s multiple range method. Statistical significance was considered at the P < 0.05 level. An online platform ( was used to analyze the sequencing data of the bacterial community.


Characteristics and microbial population of stylo prior to ensiling

The contents of DM, crude protein and crude ash, pH and buffering capacity were significantly influenced by filling time, whereas the NDF, ADF and WSC contents were not. The DM, crude protein and crude ash contents, pH and buffering capacity of the DF1 and DF2 materials tended to increase, compared to the QF material. The buffering capacity of DF2 material was over 940 mEq/kg DM, which was significantly higher (P < 0.05) than that of the DF1 and QF materials. In addition, lactic acid and acetic acid were detected in the QF material, and they were increased by delayed filling. The NH3–N was not detected in the QF material, but was 46.5–55.8 g/kg TN in DF1 material and 70.4–88.5 g/kg TN in DF2 material, respectively. Inoculating significantly affected pH, buffering capacity, and the contents of crude protein, acetic acid and NH3–N. The buffering capacity, acetic acid and NH3–N contents of the DF1 and DF2 materials inoculated with SXC48 were higher than those of materials uninoculated and inoculated with CCZZ1 and XC124 (Table 2).

Table 2 Chemical compositions of stylo before ensilinga

Delayed filling significantly increased the amounts of microbes in pre-ensiling materials, while inoculating did not affect the amounts of aerobic bacteria, yeasts and molds. Inoculating significantly increased (P < 0.05) the LAB number of the DF1 and DF2 materials, compared to the uninoculated one (Table 3).

Table 3 Microbial counts of stylo before ensiling (lg cfu/g FM)a

Ensiling characteristics and microbial population of stylo silages

The fermentation quality of silage ensiled for 60 d is shown in Table 4. Inoculating Lb. plantarum SXC48 and CCZZ1 significantly decreased pH and NH3–N content, increased the lactic acid content of QF silage. Uninoculated DF2 silage had more lactic acid (30.89 g/kg DM), lower pH (4.73) and less NH3–N (134 g/kg TN) than the uninoculated DF1 and QF silages. Inoculating strains (except SXC48 before placing) decreased pH and increased lactic acid content of delayed filling silages compared to uninoculated one, and reduced the contents of propionic acid and butyric acid, especially inoculating at filling. However, the time of inoculating CCZZ1 had no significant effect on the fermentation quality of silage. The DF2 silage inoculated with SXC48 at filling had the lowest pH and the highest lactic acid content (Table 4).

Table 4 Fermentation quality and microbial composition of stylo silagesa

For QF silages, the V-scores of silages inoculated with SXC48 and CCZZ1 were higher (P < 0.05) than those inoculated with XC124 and uninoculated. For DF1 silages, inoculating strains (except SXC48 at chopping) significantly increased the V-scores of silage compared to uninoculated silages. The DF2 silages inoculated with SXC48 at filling, XC124 at chopping and CCZZ1 at either time had higher V-score than uninoculated silage (Fig. 1).

Fig. 1
figure 1

V-scores of stylo silages. (Different letters indicate significant difference among the treatments of same material)

Bacterial diversity of stylo and its silages

The alpha-diversity of the bacterial community in stylo before ensiling and its silages is summarized in Table 5. The coverage values of all samples were above 0.99. The bacterial community of DF2 materials before ensiling had higher (P < 0.01) Simpson and Shannon as well as lower (P > 0.05) OTUs and chao 1 than the QF materials, regardless of inoculating. For DF2 silages, Simpson and Shannon had an increasing trend compared to the QF silages.

Table 5 General information of sequence and bacterial diversity of stylo and its silagea

PCA 1, PCA 2 and PCA 3 were 38.8%, 20.8% and 31.7% of the total variance in this study, respectively. PCA illustrated that the bacterial community of DF2 materials before ensiling or silages differentiated apparently from that of corresponding QF materials. Inoculating also led to the clear separation of bacterial community of the QF silages, while had no significant effect on the bacterial community of DF2 silages. Moreover, the bacterial community of QF silages inoculated with SXC48 and CCZZ1 was separated from those of silages inoculated with XC124 and uninoculated (Fig. 2).

Fig. 2
figure 2

Cluster analysis of bacterial communities in stylo before ensiling and its silage as assessed by a Principal Coordinate Analysis

As shown in Fig. 3A, Proteobacteria was the dominant phylum (74.1–80.3%) in the bacterial community of QF and DF2 materials, followed by Bacteroidetes (15.4–25.0%). However, Proteobacteria (67.1–85.3%) and Firmicutes (13.8–31.8%) were the top two phyla in all silages except the QF silages inoculated with SXC48 and CCZZ1 that had remarkably higher relative abundance of Firmicutes (79.5% and 84.2%). The bacterial community was altered by delayed filling and ensiling. In Fig. 3B, the genera with the relative abundance of above 1% in the materials before ensiling were Kosakonia (21.7%, mainly Ko. cowanii), Pseudomonas (13.3%), Pantoea (12.3%), Chryseobacterium (3.7%), Lelliottia (2.0%), Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium (1.8%), Sphingobacterium (1.5%) and Paenibacillus (1.1%). The relative abundance of Kosakonia was greatly decreased to 2.6–3.7% after the materials were placed for 2 d. The DF2 materials were jointly dominated by Pantoea and Lelliottia, mainly Pa. ananatis and Le. jeotgali, respectively (Fig. 4). The relative abundance of Kosakonia decreased from 21.7% to 0.8–4.7% in QF silages and 2.5–4.5% in DF2 silages. Lelliottia and Lactobacillus were the top two genera in QF silages. Differently, Lelliottia was the dominant genus in the QF silages uninoculated and inoculated with XC124 (59.5% and 61.4%), while Lactobacillus was the dominant genus in the QF silages inoculated with SXC48 and CCZZ1 (76.5% and 82.0%), and Lb. plantarum was main species. The undesirable bacteria Lelliottia (27.9–40.2%) and Pantoea (20.5–35.4%) were the top two genera in the DF2 silages, while the relative abundance of Lactobacillus was only 7.2–10.0% (Figs. 3B and 4).

Fig. 3
figure 3

Bacterial community at the phylum (A) and genus (B) level of stylo before ensiling and its silages

Fig. 4
figure 4

Species-level microbes analyses of stylo before ensiling and its silages (FS, fresh stylo; CK, without inoculation; SXC48 and CCZZ1, inoculating Lb. plantarum SXC48 and CCZZ1; XC124, inoculating En. faecalis XC124)


Characteristics of stylo prior to ensiling

The contents of DM and WSC, and buffering capacity of forage crops prior to ensiling play key roles in good silage fermentation [4]. The DM, crude protein and WSC contents of raw stylo in this study were lower than the values reported by Rufino et al. [28], but were comparable with those reported by Wu et al. [29]. In this study, delayed filling significantly increased the DM content and buffering capacity, decreased the WSC content of materials before ensiling. This might be because the amount of materials piled was less, and the moisture greatly lost during placing. The microorganisms in the materials after placed significantly increased and produced more organic acids, resulting in an increase in buffering capacity and a decrease in WSC content. Mills and Kung [13] also reported that the WSC content of barley decreased by more than 50% as a result of exposure to air for 1 d.

Epiphytic LAB on the forage crops are also essential for its silage fermentation [30]. The LAB population (4.0 log cfu/g) in the present study was comparable to that reported by Wu et al. [29] (4.20 log cfu/g), but lower than 5.0 log cfu/g FM considered as adequate for the good fermentation of silage [31]. The LAB numbers of DF1 and DF2 materials increased to 5.0 log cfu/g, while the numbers of undesirable microorganisms such as aerobic bacteria, yeasts and molds all increased and were relatively high. This is consistent with the results reported by Cai et al. [16] and Pahlow et al. [15]. Moreover, aerobic bacteria dominated the microbial populations of delayed filling materials in this study, and reached 8–10 log cfu/g FM. Thus, lactic acid, acetic acid and NH3–N were detected in delayed filling materials. Such results also occurred in the pre-ensiled soybean curd residue placed for 2 days [17]. NH3–N indicated protein degradation by undesirable microorganisms, such as Enterobacter [32]. Inoculating SXC48 increased the NH3–N content of delayed filling materials, which might be that inoculating strain SXC48 did not inhibit the growth of harmful bacteria during aerobic exposure, resulting in more protein hydrolysis. Delayed filling would have a negative impact on the fermentation quality.

Fermentation quality and microbial population of stylo silage

Ensiling inhibits the activities of undesirable microorganisms mainly through producing lactic acid to reduce pH, so as to preserve nutrients [4]. In this study, relatively high pH (4.98) of the uninoculated QF silage indicated the low fermentation quality. Inoculating LAB promoted lactic acid production and reduced the NH3–N content of QF silage, thus accelerating pH decline, even though their final pH were still higher than the ideal pH below 4.20 for high-quality silage [4]. This is consistent with the results of Wu et al. [29] and Liu et al. [7], who reported that stylo silage had the high pH of 4.84–5.39, and inoculating Pediococcus pentosaceus and Lb. paraplantarum significantly improved its fermentation quality. Inoculating and delayed filling reduced the pH value and increased the lactic acid content to different degree of stylo silage, which was different from the findings that delayed sealing resulted in poor silage fermentation [33, 34] or inoculating was not effective for delayed sealing silage [16]. The reason might be that the materials piled were not thick, and delayed filling might play a similar role to wilting, promoting lactic acid fermentation of silage [35].

Butyric acid is unfavorable to the silage quality because of the nutritional losses during butyric acid fermentation by clostridial activity [33]. All uninoculated silages had more butyric acid than the inoculated silages except the inoculation of SXC48 at chopping, which were probably related to the promotion of lactic acid fermentation and inhibition of butyric acid fermentation by LAB strains [4].

Bacterial community of stylo and its silage

Analyzing bacterial community would contribute to reveal the changes of bacteria in the materials during aerobic exposure and further understand the silage fermentation. The coverage for all samples in the present study were over 0.99, indicating that sequencing abundance was large enough to reflect the profile of the bacterial community. The alpha-diversity indices revealed stylo silages had lower bacterial community richness and higher diversity relative to fresh materials. Moreover, the bacterial community diversity (Shannon and Simpson) in stylo and its silages were altered by delayed filling and inoculating in this study. The significant increase in bacterial diversity of delayed filling materials might be attributed to the remarkable growth of harmful microorganisms, since their growth and survival generally require moderate pH level and aerobic condition. This result was consistent with the report by Du et al. [36]. As microbes grow and compete during aerobic exposure or ensiling, inoculating LAB strains would shape the microbial community more desirable, resulting in the fermentation quality improvement [37]. Consistently, PCA analysis showed that the bacterial community of delayed filling materials and silages differentiated apparently from that of fresh material, and inoculating remarkably altered the bacterial community of silage.

In this study, Proteobacteria and Bacteroidetes were dominant in all DF2 materials before ensiling (> 1% abundance), while Proteobacteria and Firmicutes were the main phyla in all the silages. Quickly ensiling and inoculating SXC48 and CCZZ1 changed the dominant phylum from Proteobacteria to Firmicutes. The result of Zi et al. [9] also suggested that Firmicutes and Proteobacteria were dominant in all stylo silages, while the abundance of Proteobacteria was higher than that of Firmicutes in sucrose-treated silage. The presence of more Proteobacteria in silage indicates high pH value of silage as Proteobacteria prefer the neutral environment [38]. Moreover, gram-negative pathogenic Proteobacteria include a wide variety of pathogenic genera, such as Escherichia, Salmonella, Vibrio, and many others. The dominant phylum in the QF silages inoculated with strain SXC48 and CCZZ1, being the homofermentative LAB, was Firmicutes, which was generally considered desirable during ensiling, since they can inhibit the growth of Clostridia and decrease the content of NH3–N. Thus, inoculating strain SXC48 and CCZZ1 decreased the NH3–N content and improved the fermentation quality of QF silages.

The fresh stylo was jointly dominated by several genera, such as Kosakonia, Pseudomonas and Pantoea, while Pantoea and Lelliottia were main genera in the delayed filling materials. Bacteria such as Kosakonia and Pseudomonas in the DF1 and DF2 materials reduced, resulting in that the bacterial diversity changed during placing and then affected the silage fermentation. Kosakonia was also the main genera in stylo silage reported by He et al. [39] and Wang et al. [40]. Kosakonia is recently classified from the genus Enterobacter [41]. In this study, Kosakonia mainly included Ko. cowanii, which was rarely found in silage. Kumar et al. [42] reported Ko. cowanii promoted the plant growth. Lelliottia (mainly Le. jeotgali) dominated the QF silages uninoculated and inoculated with XC124, whereas Lactobacillus (mainly Lb. plantarum) was the overwhelming genus in the QF silages inoculated with SXC48 and CCZZ1. Lelliottia was also detected in stylo silage reported by Wu et al. [29], and it is separated from the genus Enterobacter and reclassified subsequently as a novel genus [43]. Thus, Lelliottia might play the similar function to Enterobacter in silage. The relatively higher pH and lower lactic acid content of the QF silages inoculated with XC124 and uninoculated might be attributed to more Lelliottia. Inoculating SXC48 and CCZZ1 made Lb. plantarum predominant to facilitate the fermentation of QF silages. Lelliottia and Pantoea jointly dominated the DF2 silages, and Pantoea was mainly Pa. ananatis. Pantoea is commonly found in stylo silage [9, 17, 44], and is undesirable microbes, because they compete the fermentation substrate with LAB [45]. However, some studies have claimed that Pantoea is beneficial to silage fermentation, owing to reduce the NH3–N content and pH of silage [42]. Pa. ananatis, a gram-negative bacterium, provides various beneficial characteristics, such as the growth promotion of their host plants and increased crop yield [46]. Tao et al. [47] reported Pa. ananatis had the ability to degrade the lignin. It is seldom reported in silage and the role of Pa. ananatis in silage fermentation needs to be further researched.


Under the present research conditions, delayed filling increased the pH, buffering capacity, DM content, microbes number, lactic acid content, acetic acid content and NH3–N content of stylo before ensiling. Delayed filling and inoculating LAB changed the bacterial community of stylo and its silages. Inoculating SXC48 and CCZZ1 largely increased the relative abundance of beneficial bacteria such as Lactobacillus and greatly reduced that of undesirable bacteria such as Kosakonia, Pantoea and Lelliottia in the QF silages. Delayed filling increased the lactic acid content and decreased NH3–N content of silage, but increased the bacterial diversity and relative abundance of Pantoea and Lelliottia. The inoculating effectiveness of delayed filling silage varied with the inoculating time and LAB strains, inoculating strian SXC48 at filling was better than at chopping, while inoculating strian CCZZ1 at both chopping and filling obtained the similar benefit.

Availability of data and materials

The data sets used or analyzed during the current study are available from the corresponding author on reasonable request.



Lactic acid bacteria

Lb. plantarum :

Lactobacillus plantarum

En. faecalis :

Enterococcus faecalis


Quickly filling


Delayed filling for 1 d


Delayed filling for 2 d


Dry matter


Fresh matter


Neutral detergent fiber


Acid detergent fiber


Water-soluble carbohydrates


Ammoniacal nitrogen


  1. Phaikaew C, Hare MD. Stylo adoption in Thailand: three decades of progress. Trop Grassl. 2005;39:216.

    Google Scholar 

  2. Pen M, Savage DB, Nolan JV, Seng M. Effect of Stylosanthes guianensis supplementation on intake and nitrogen metabolism of Bos indicus cattle offered a basal diet of mixed rice straw and tropical grass. Anim Prod Sci. 2013;53:8858–66.

    Article  Google Scholar 

  3. Kaensombath L, Neil M, Lindberg JE. Effect of replacing soybean protein with protein from ensiled stylo (Stylosanthes guianensis (Aubl.) Sw. var. guianensis) on growth performance, carcass traits and organ weights of exotic (Landrace x Yorkshire) and native (Moo Lath) Lao pigs. Trop Anim Health Prod. 2013;45:865–71.

    Article  PubMed  Google Scholar 

  4. McDonald P, Henderson AR, Heron SJE. The biochemistry of silage. 2nd ed. Marlow: Chalcombe Publications; 1991.

    Google Scholar 

  5. Bureenok S, Sisaath K, Yuangklang C, Vasupen K, Schonewille J. Ensiling characteristics of silages of Stylo legume (Stylosanthes guianensis), Guinea grass (Panicum maximum) and their mixture, treated with fermented juice of lactic bacteria, and feed intake and digestibility in goats of rations based on these silages. Small Rumin Res. 2016;134:84–9.

    Article  Google Scholar 

  6. Mutiaka BK, Boudry C, Picron P, Kiatoko H, Bindelle J. Feeding value of hays of tropical forage legumes in pigs: Vigna unguiculata, Psophocarpus scandens, Pueraria phaseoloides and Stylosanthes guianensis. Trop Anim Health Prod. 2014;46:1497–502.

    Article  Google Scholar 

  7. Liu Q, Chen M, Zhang J, Shi S, Cai Y. Characteristics of isolated lactic acid bacteria and their effectiveness to improve stylo (Stylosanthes guianensis Sw.) silage quality at various temperatures. Anim Sci J. 2012;83:128–35.

    Article  CAS  PubMed  Google Scholar 

  8. Pitiwittayakul N, Bureenok S, Schonewille JT. Selective thermotolerant lactic acid bacteria isolated from fermented juice of epiphytic lactic acid bacteria and their effects on fermentation quality of stylo silages. Front Microbiol. 2021;12: 673946.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Zi XJ, Liu Y, Chen T, Li M, Zhou HL, Tang J. Effects of sucrose, glucose and molasses on fermentation quality and bacterial community of stylo silage. Ferment Basel. 2022;8:191.

    Article  CAS  Google Scholar 

  10. Muck RE, Nadeau EMG, McAllister TA, Contreras-Govea FE, Santos MC, Kung L. Silage review: recent advances and future uses of silage additives. J Dairy Sci. 2018;101:3980–4000.

    Article  CAS  PubMed  Google Scholar 

  11. Bureenok S, Pitiwittayakul N, Yuangklang C, Vasupen K, Saenmahayak B, Kawamoto Y, et al. Evaluation of dried Mao Pomace (Antidesma Bunius Linn.) and lactic acid bacteria as additives to ensile stylo legume (Stylosanthes Guianensis CIAT184). J Anim Plant Sci. 2019;29:783–9.

    CAS  Google Scholar 

  12. Wilkinson JM, Davies DR. The aerobic stability of silage: Key findings and recent developments. Grass Forage Sci. 2013;68:1–19.

    Article  CAS  Google Scholar 

  13. Mills JA, Kung JRL. The effect of delayed ensiling and application of a propionic acid-based additive on the fermentation of barley silage. J Dairy Sci. 2002;85:1969–75.

    Article  CAS  PubMed  Google Scholar 

  14. Brüning D, Gerlach K, Weiss K, Südekum KH. Effect of compaction, delayed sealing and aerobic exposure on maize silage quality and on formation of volatile organic compounds. Grass Forage Sci. 2018;73:53–66.

    Article  Google Scholar 

  15. Pahlow G, Muck RE, Driehuis F, Oude Elferink SJWH, Spoelstra SF. Microbiology of ensiling. In: Buxton DR, Muck RE, Harrison JH, editors. Silage science and technology. Madison: American Society of Agronomy Inc, Crop Science Society of America Inc, Soil Science Society of America Inc; 2003. p. 31–93.

    Google Scholar 

  16. Cai Y, Du Z, Yamasaki S, Nguluve D, Tinga B, Macome F, et al. Influence of microbial additive on microbial populations, ensiling characteristics, and spoilage loss of delayed sealing silage of Napier grass. Asian-Aust J Anim Sci. 2020;7:1103–12.

    Article  Google Scholar 

  17. Wali A, Nishino N. Bacterial and fungal microbiota associated with the ensiling of wet soybean curd residue under prompt and delayed sealing conditions. Microorganisms. 2020;8:1334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kim SC, Adesogan AT. Influence of ensiling temperature, simulated rainfall, and delayed sealing on fermentation characteristics and aerobic stability of corn silage. J Dairy Sci. 2006;89:3122–32.

    Article  CAS  PubMed  Google Scholar 

  19. Borreani G, Tabacco E, Schmidt RJ, Holmes BJ, Muck RE. Silage review: Factors affecting dry matter and quality losses in silages. J Dairy Sci. 2018;101:3952–79.

    Article  CAS  PubMed  Google Scholar 

  20. Arbabi S, Ghoorchi T, Hasani S. The effect of delayed ensiling and application of an organic acid-based additives on the fermentation of corn silage. Asian J Anim Vet Adv. 2009;4:219–27.

    Article  CAS  Google Scholar 

  21. AOAC International. Official methods of analysis of AOAC international. 16th ed. Arlington: Association of Analytical Chemists; 2005.

    Google Scholar 

  22. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991;74:3583–97.

    Article  PubMed  Google Scholar 

  23. Murphy RP. A method for the extraction of plant samples and the determination of total soluble carbohydrates. J Sci Food Agric. 1958;9:714–7.

    Article  CAS  Google Scholar 

  24. Zhang JG, Kawamoto H, Cai YM. Relationships between the addition rates of cellulase or glucose and silage fermentation at different temperatures. Anim Sci J. 2010;81:325–30.

    Article  CAS  PubMed  Google Scholar 

  25. Broderick GA, Kang JH. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J Dairy Sci. 1980;63:64–75.

    Article  CAS  PubMed  Google Scholar 

  26. Society of utilization of self supplied feeds. In: The guidebook for quality evaluation of forage. 3th ed. Tokyo: Japan Grassland Agriculture and Forage Seed Association; 2009. p. 93–94

  27. Mu L, Wang QL, Cao X, Zhang ZF. Effects of fatty acid salts on fermentation characteristics, bacterial diversity and aerobic stability of mixed silage prepared with alfalfa, rice straw and wheat bran. J Sci Food Agric. 2021;102:1475–87.

    Article  PubMed  Google Scholar 

  28. Rufino LDD, Pereira OG, Agarussi MCN. Effects of lactic acid bacteria with bacteriocinogenic potential on the chemical composition and fermentation profile of forage peanut (Arachis pintoi) silage. Anim Feed Sci Technol. 2022;290: 115340.

    Article  CAS  Google Scholar 

  29. Wu S, Gao L, Chen D, Xue Y, Kholif AE, Zhou W, et al. Effects of forestry waste Neolamarckia cadamba leaf meal as an additive on fermentation quality, antioxidant activity, and bacterial community of high-moisture stylo silage. Front Environ Sci. 2022;10: 925400.

    Article  Google Scholar 

  30. Lin C, Bolsen KK, Brent BE, Hart RA, Dickerson JT. Epiphytic microflora on alfalfa and whole-plant corn. J Dairy Sci. 1992;75:2484–93.

    Article  CAS  PubMed  Google Scholar 

  31. Oliveira AS, Weinberg ZG, Ogunade IM, Cervantes AAP, Arriola KG, Jiang Y, et al. Meta-analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation, aerobic stability, and the performance of dairy cows. J Dairy Sci. 2017;100:4587–603.

    Article  CAS  PubMed  Google Scholar 

  32. Kung L, Shaver RD, Grant RJ, Schmidt RJ. Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. J Dairy Sci. 2018;101:4020–33.

    Article  CAS  PubMed  Google Scholar 

  33. Coblentz WK, Coffey KP, Chow EA. Storage characteristics, nutritive value, and fermentation characteristics of alfalfa packaged in large-round bales and wrapped in stretch film after extended time delays. J Dairy Sci. 2016;99:3497–511.

    Article  CAS  PubMed  Google Scholar 

  34. Serva L, Andrighetto I, Marchesini G, Contiero B, Grandis D, Magrina L. Prognostic capacity assessment of a multiparameter risk score for aerobic stability of maize silage undergoing heterofermentative inoculation (Lactobacillus buchneri) in variable ensiling conditions. Anim Feed Sci Technol. 2021;281: 115116.

    Article  CAS  Google Scholar 

  35. Araújo JAS, Almeida JCC, Reis RA, Carvalho CAB, Barbero RP. Harvest period and baking industry residue inclusion on production efficiency and chemical composition of tropical grass silage. J Clean Prod. 2020;266: 121953.

    Article  Google Scholar 

  36. Du Z, Sun L, Chen C, Lin J, Yang F, Cai Y. Exploring microbial community structure and metabolic gene clusters during silage fermentation of paper mulberry, a high-protein woody plant. Animal Feed Sci Technol. 2021;275: 114766.

    Article  CAS  Google Scholar 

  37. He L, Wang Y, Guo X, Chen X, Zhang Q. Evaluating the effectiveness of screened lactic acid bacteria in improving crop residues silage: fermentation parameter, nitrogen fraction, and bacterial community. Front Microbiol. 2022;13: 680988.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Brenner DJ, Krieg R, Staley JR. Bergey’s manual of systematic bacteriology. New York: Springer; 2005.

    Book  Google Scholar 

  39. He LW, Li S, Wang C, Chen XY, Zhang Q. Effects of vanillic acid on dynamic fermentation parameter, nitrogen distribution, bacterial community, and enzymatic hydrolysis of stylo silage. Front Microbiol. 2021;12: 690801.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Wang C, Pian RQ, Chen XY, Lv HJ, Zhou W, Zhang Q. Beneficial effects of tannic acid on the quality of bacterial communities present in high-moisture mulberry leaf and stylo silage. Front Microbiol. 2020;11: 586412.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Patz S, Becker Y, Richert-Poggeler KR, Berger B, Ruppel S, Huson DH, et al. Phage tail-like particles are versatile bacterial nanomachines–a mini-review. J Adv Res. 2019;19(Suppl 1):75–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kumar K, Verma A, Pal G, White JF, Verma SK. Seed endophytic bacteria of pearl millet (Pennisetum glaucum L.) promote seedling development and defend against a fungal phytopathogen. Front Microbiol. 2021;12:774293.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Yuk KJ, Kim YT, Huh CS, Lee JH. Lelliottia jeotgali sp. nov., isolated from a traditional Korean fermented clam. Int J Syst Evol Microbiol. 2018;68:1725–31.

    Article  CAS  PubMed  Google Scholar 

  44. Li M, Lv R, Zhang L, Zi X, Zhou H, Tang J. Melatonin is a promising silage additive: evidence from microbiota and metabolites. Front Microbiol. 2021;12: 670764.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Ávila CLS, Carvalho BF. Silage fermentation-updates focusing on the performance of microorganisms. J Appl Microbiol. 2020;128:966–84.

    Article  PubMed  Google Scholar 

  46. Usuda Y, Nishio Y, Nonaka G, Hara Y. Microbial production potential of Pantoea ananatis: from amino acids to secondary metabolites. Microorganisms. 2022;10:1133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tao J, Chen Q, Chen S, Lu P, Chen Y, Jin J, et al. Metagenomic insight into the microbial degradation of organic compounds in fermented plant leaves. Environ res. 2022;214: 113902.

    Article  CAS  PubMed  Google Scholar 

Download references


Not applicable.


This work was supported by the National Key R&D Program of China (Grant No. 2022YFE0111000) and the National Natural Science Foundation of China (Grant No. 31971764).

Author information

Authors and Affiliations



JT: methodology, visualization, data curation, and writing—original draft preparation; LH and RT: investigation, resources, and validation; JW and RT: software and formal analysis; JZ: conceptualization, reviewing and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Jianguo Zhang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors listed have read the complete manuscript and have approved submission of the paper.

Competing interests

No conflicts of interest declared by the authors.

Additional information

Publisher's Note

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

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tian, J., Huang, L., Tian, R. et al. Fermentation quality and bacterial community of delayed filling stylo silage in response to inoculating lactic acid bacteria strains and inoculating time. Chem. Biol. Technol. Agric. 10, 44 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: