Effects of Sucrose, Glucose and Molasses on Fermentation Quality and Bacterial Community of Stylo Silage

Abstract

To better understand the mechanism underlying water-soluble carbohydrates (WSC)-regulated silage fermentation, the bacterial community and fermentation quality of stylo (Stylosanthes guianensis) ensiled without (sterile distilled water) (CK) or with 10 g/kg sucrose (S), 10 g/kg glucose (G), and 10 g/kg molasses (M) were investigated. Each treatment was ensiled in three plastic bag silos for 30 days. The DM of stylo was 301.0 g/kg (fresh matter basis), while the contents of CP, WSC, NDF and ADF were 104.9, 12.3, 510.4 and 407.6 g/kg (dry matter basis), respectively. The bacterial community was characterized by using 16Sr DNA sequencing technology. The addition of WSC significantly altered both fermentation quality and the bacterial community of stylo silage. The additive treatment resulted in higher lactic acid (17.2 vs. 67.0 g/kgDM), lower pH (4.68 vs. 4.46), and lower acetic acid (37.0 vs. 28.9 g/kgDM) compared with the control. In addition, no propionic acid and butyric acid were detected in the additive treatment groups. These results indicated that WSC additives helped to produce greater quality stylo silage. Meanwhile, the Shannon index was higher, and the Simpson index was lower in the WSC additive-treated groups compared with the control, indicating that WSC improved microbial diversity. Furthermore, WSC treatments increased the abundance of acid-producing bacteria Megamonas and Bacteroides, decreased the abundance of Weissella, and inhibited the growth of the undesirable Enterobacter. Our results confirmed that sucrose, glucose and molasses have similar beneficial effects on both bacterial community and silage fermentation of stylo. Molasses was recommended to be used in stylo silage for economic benefit and resource utilization.

1. Introduction

Stylo (Stylosanthes guianensis) is an important legume forage and is widely distributed in tropical and subtropical regions. It has both a high biomass (15–22 t/ha per year, FM base) and abundant nutrients (15–16% crude protein, DM base), making it an ideal animal feed [1,2,3]. It has been reported that stylo supplemented in the rations of ruminants has positive effects on animal performance [4]. In addition, stylo is also used in pig feed and may improve the feed’s digestibility [5]. Normally, Stylo grows fast in the summer or rainy seasons, but its growth is significantly hindered in the winter, resulting in the lack of ruminants’ feed. High temperature and humidity in tropical regions of China limit the production of Stylo hay, thus blocking its intensive use in herbivorous livestock feeding. To overcome this obstacle, stylo is normally ensiled after harvest.

Ensiling is essential to ensure the supply of livestock feed throughout the year. However, stylo is a typical tropical forage with relatively low contents of water-soluble carbohydrates (WSC), which makes it hard to make high-quality silage [1]. Therefore, it is urgent to appropriately treat stylo for its ensiling and processing. Liu et al. (2012) isolated lactic acid bacteria (LAB) from stylo silage and found that these strains effectively improved fermentation quality [6]. Our previous study indicated that LAB treatment could improve the fermentation quality, chemical composition, and ruminal degradation of stylo silage [1]. Meanwhile, preparing stylo with other grass could improve silage quality and provide more balanced nutrition. Bureenok et al. (2016) reported that the quality of mixing silage of stylo with guinea was higher than that of the ensiled alone and that feeding the mixed silage improved the growth performance of goats [7]. In addition, Moringa oleifera (fresh leaf or hay) improved fermentation quality by altering the microbial community structure of stylo silage [8,9]. WSC is one of the major silage additives due to its promoting effect on the production of lactic acid by LAB, which as a result, drops the pH and then enhances the quality of silage.

WSC generally includes molasses, sucrose and glucose; molasses is a waste product of the sugar industry, glucose is a monosaccharide and sucrose is a disaccharide, and all have applications in silage modulation [10,11,12,13]. Sucrose can increase the abundance of Lactobacillus and Pseudomonas, and it also inhibits the growth of undesirable Enterobacter in mulberry leaf silage [12]. Meanwhile, Molasses enhances the amount of desirable Lactobacillus and inhibits the growth of undesirable microorganisms such as Clostridia and Enterobacter in soybean silage [13]. However, the effects of different WSC, which shape the silage microbial community and then improve fermentation quality, are varied, and stylo silage bacterial community structure is still unknown. We hypothesize that WSC in stylo could help to improve silage fermentation by shifting the silage microbial community during ensiling.

Therefore, in this study, the addition of sucrose, glucose and molasses was examined in stylo silage to explore their effects on bacterial community, fermentation, and then eventually, the silage quality was analyzed.

2. Materials and Methods

2.1. Preparation of Silage

Stylo was planted at our experimental base (109°58’ E, 19°52′ N, Chinese Academy of Tropical Agricultural Sciences, CATAS, Danzhou, China). The varieties were planted during the spring season (10 March 2019) at CATAS, and harvested on 10 July 2019 (vegetative stage); the first cut was at 30 cm (plant height was approximately 100 cm) from the ground surface. The harvested stylo was chopped into small pieces at about 2 cm. Four different treatments were conducted in our current study as follows: (1) no additive (CK), (2) 10 g/kg sucrose (S), (3) 10 g/kg glucose (G), and (4) 10 g/kg molasses (M), all on fresh matter basis. The added dosage was based on the result of some published studies [10,11,12,13]. Every treatment was carried out in triplicates. The application rate of sugar was on fresh matter basis. Briefly, 200 g of stylo was blended with additives before being placed and vacuumed in plastic bags (35 cm × 12 cm × 5 cm; Guozhong Packing Co., Ltd., Haikou, China). A total of 60 bags (four treatments × three replicates × 5 ensiling time points) were prepared and incubated at normal temperature (25 to 30 °C). The bags were opened for analyses of chemical composition and organic acid levels on days 1, 3, 7, 14 and 30. The composition of the bacterial community was analyzed on day 30.

2.2. Chemical Assay

Specimens were heated at 65 °C for 72 h, and dried materials were ground for chemical analysis. The contents of dry matter (DM), crude protein (CP), water-soluble carbohydrates (WSC), neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured as previously described [1]. The quality of silage fermentation was determined using distilled water extracts. Briefly, 50 g wet silage was blended with 200 mL distilled water, followed by incubation at 4 °C for 24 h and then filtration. Half of each extract sample was stored at −80 °C for analysis of the microbiota diversity. The pH was measured with a glass electrode pH meter. The organic acid was measured by high-performance liquid chromatography using a Sodex RS Pak KC-811 column (Showa Denko K.K., Kawasaki, Japan) and eloquent of 3 mole/L HClO₄ at a flow rate of 1.0 L/min and 40 °C and detected at 210 NM using DAD detector SPD-20A (Shimadzu Co., Ltd., Kyoto, Japan) as reported previously by Liu et al. [6].

2.3. Analysis of Microbial Community

The above-mentioned extracts were used for the molecular analysis of the microbiota. Microbial DNA was isolated from silage specimens with the E.Z.N.A.^® soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. The concentration and purity of extracted DNA were assessed by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, CA, USA), and DNA integrity was confirmed by electrophoresis on 1% agarose gel. Primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were adopted to amplify the V3-V4 hypervariable regions of the bacterial 16S rRNA gene using thermocycler PCR system (GeneAmp 9700, ABI, Foster City, CA, USA). After PCR products were purified and quantified, next-generation sequencing was carried out using Illumina MiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA), and paired-end reads of 250 bp were generated.

The assembly of tags was carried out using filtered reads according to the principles as follows: overlap between paired-end reads should be more than 10-bp overlap and less than 2% mismatch. The unique tags were obtained by removing redundant tags using mothur software (version 1.40.45) [14]. The abundance was then determined using the resultant unique tags. The high-quality reads were grouped into operational taxonomic units (OTUs) defined at a similarity of 97%. Diversity metrics were determined using the core-diversity plug-in within QIIME2 (https://docs.qiime2.org/2019.1/, accessed on 15 March 2020) [15]. The microbial diversity within an individual sample was assessed using the alpha diversity indices, including observed OTUs, Chao1 richness estimator, Shannon diversity index, and Simpson and ACE index [16]. Beta diversity was analyzed to assess the structural variation of microbiota across specimens, and then principal coordinates analysis (PCoA) and Unweighted Pair-group Method with Arithmetic Mean (UPGMA) were conducted [17]. Appropriate methods were employed to identify the bacterial strains with different abundances among samples and groups [18]. Unless specified above, parameters used in the analysis were set as default. The sequencing data were deposited in the Sequence Read Archive (SRA) under the accession number PRJNA637681.

2.4. Statistics

The effects of WSC additives, ensiling days and their interaction were evaluated by two-way analysis of variance using the general linear model procedure of SAS 9.3 software (SAS Institute Inc., Cary, NC, USA) according to the model for a 4 × 5 factorial treatment design. Differences were compared using Duncan’s multiple range test, and differences with p < 0.05 were considered statistically significant.

3. Results

3.1. Chemical Composition of Stylo

The DM of stylo was 301.0 g/kg (fresh matter basis), while the contents of CP, WSC, NDF and ADF were 104.9, 12.3, 510.4 and 407.6 g/kg (dry matter basis), respectively.

3.2. Soluble Sugar Improves the Fermentation Quality of Stylo Silage

The stylo silage’s fermentation characteristics during ensiling are listed in

Table 1. Fermentation quality of stylo silage.

Item	Treatment	Days of Ensiling					SEM	p-Value
		1	3	7	14	30		D	T	D*T
pH	CK	5.31 a	5.09 b	4.88 bA	4.74 bcA	4.68 cA	0.06	<0.01	<0.01	<0.01
	S	5.34 a	5.14 b	4.84 bcA	4.56 cB	4.48 cB
	G	5.21 a	5.01 b	4.73 cB	4.54 cdB	4.47 cB
	M	5.19 a	4.95 b	4.74 cB	4.55 dB	4.42 dB
Lactic acid	CK	8.8 dC	9.7 cdB	11.1 bcB	13.5 bB	17.2 aB	12.50	<0.01	<0.01	<0.01
	S	10.2 eB	19.0 dA	39.0 cA	53.8 bA	67.0 aA
(g/kg DM)	G	10.1 eB	20.7 dA	38.4 cA	54.4 bA	65.1 aA
	M	12.4 eA	24.7 dA	38.9 cA	56.9 bA	69.0 aA
Acetic acid	CK	8.7 e	11.3 d	16.3 cA	24.4 bA	37.0 aA	2.20	<0.01	<0.01	<0.01
	S	8.7 e	11.5 d	14.6 cAB	20.5 bB	27.8 aB
(g/kg DM)	G	8.9 e	12.9 d	16.9 cAB	21.1 bB	27.5 aB
	M	9.0 e	13.6 d	19.1 cAB	25.8 bAB	31.3 aAB
Propionic acid	CK	0.5 c	0.8 b	1.2 ab	1.3 ab	1.6 aA	0.39	<0.01	<0.01	<0.01
	S	ND	ND	ND	ND	0.0 B
(g/kg DM)	G	ND	ND	ND	ND	0.0 B
	M	ND	ND	ND	ND	0.0 B
Butyric acid	CK	0.2 c	0.4 b	0.6 ab	0.8 a	0.8 aA	0.21	–	<0.01	–
	S	ND	ND	ND	ND	0.0 B
(g/kg DM)	G	ND	ND	ND	ND	0.0 B
	M	ND	ND	ND	ND	0.0 B
Total acid	CK	18.2 eB	22.2 dC	29.2 cA	40.0 bB	56.6 aB	9.98	<0.01	<0.01	<0.01
	S	18.9 eB	30.5 dB	53.6 cA	74.3 bA	94.9 aA
(g/kg DM)	G	19 eB	33.6 dB	55.3 cA	75.5 bA	92.6 aA
	M	21.4 eA	38.3 dA	58.0 cA	82.7 bA	100.4 aA

CK, control; S, sucrose; G, glucose; M, molasses. DM, dry matter; ND, not detected; “–”, default. D, days of ensiling; T, treatment; D*T, interaction of ensiling time and treatment; SEM, standard error of means; Means with different letters in the same row (a–d) or column (A–D) differ (p < 0.05).

. The pH values in all silages in our study were significantly reduced (p < 0.05) after 7 days of ensiling, while no significant changes were observed in all silages on day 30 with the lowest pH. Meanwhile, the pH values of the G, S and M silages were significantly lower than that of the CK silage after ensiling (p < 0.05). The pH value of additive silages was close to 4.2, which ensured the good preservation of stylo silage. The level of lactic acid in additive-treated silages was largely increased during ensiling (p < 0.05), which was significantly higher than that of the CK group on day 30 (p < 0.05). No significant differences were observed among different additive-treated groups. The levels of acetic acid in all silages were significantly increased during ensiling (p < 0.05), and those of additives silages were lower than that of the CK group on day 30 (p < 0.05). In this study, the contents of propionic acid and butyric acid were slowly increasing during the ensiling process in the CK group but not detectable in additive-treated silages, indicating that the stylo was well-preserved after being treated with soluble sugar. The total acid content showed a similar trend as those of lactic acid and acetic acid, while additive-treated silage had higher total acid content than the CK group after ensiling. These results indicated that supplementation of soluble sugars promoted the fermentation quality and that different additive-treated silages in this study were similarly effective.

3.3. Effects of Soluble Sugars on the Microbial Community of Stylo Silages

On average, 75,611 clean tags and 62,803 effective tags were obtained in each silage sample from sequencing. Overall, 934,418 raw reads and 908,608 raw tags were generated. As shown in Figure 1, soluble sugar treatments affected the indices of OTUs, Ace, Chao 1, Shannon and Simpson of the microbial diversity and richness. 543 OTUs were observed in all stylo silages. Similar numbers of OUTs (540, 536, and 536) were observed in different soluble sugar treatment groups, but they were higher than that of the CK group (524) (Figure 1A). The treatment groups contained 514 common OTUs, while 2 specific OTUs were observed in the M group (Figure 1B). No significant differences were observed in the indices of richness (Chao 1 and ACE) between the CK group and the soluble sugar treatment groups (except ACE in CK group and G group) (Figure 1C,D). These indices indicated that soluble sugar slightly affected the richness of the stylo silage microbial community. In contrast to the richness indices, however, the Shannon index was higher, while the Simpson index was lower, in the additive-treated groups than those of the CK group, indicating that soluble sugar treatments led to higher diversity (Figure 1E,F). The higher OTUs number, Shannon index, together with the lower Simpson index suggested higher microbial abundance and higher diversity. These results suggested that soluble sugar treatment may raise microbial diversity. The principal coordinate analysis (PCoA) and cluster tree were employed to examine the correlations among the community structures of the silage microbial community. Clear separation and differences in bacterial community were found in all four groups (Figure 2A), while the CK group and the S group had closer genetic distance compared with the G and M groups (Figure 2B), indicating that the microbial composition was changed by soluble sugar treatments during the ensiling process. Overall, our results demonstrated that supplementing soluble sugar affects the microbial diversity and community structure of stylo silage.

Figure 3(A1,A2) describes the microbial community at the phylum level of all samples with each treatment. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria and Cyanobacteria were dominant in all treatments (>1% abundance). We also examined the bacterial structures of stylo silages at the genus level to further investigate the additives’ effects on the microbial community (Figure 3(B1,B2)). Enterobacter, Megamonas, Weissella, Pantoea, Bacteroides, Ruminococcus, Stenotrophomonas, Megasphaera, Faecalibacterium and Bifidobacterium were the top 10 dominant genus in all treatments. Furthermore, Weissella, Enterobacter, Pantoea and Stenotrophomonas were predominant in the CK group, representing 47.71%, 21.39%, 8.05% and 3.95% of the total sequences, respectively. While in the S group, Enterobacter, Megamonas, Weissella, Pantoea, Bacteroides, Ruminococcus, Stenotrophomonas, Megasphaera, Faecalibacterium and Bifidobacterium were the top 10 dominant genera, representing 14.04%, 10.55%, 9.68%, 7.73%, 4.19%, 3.84%, 2.72%, 2.46%, 2.34% and 2.20%, respectively. In the G group, Enterobacter, Megamonas, Weissella, Pantoea, Bacteroides, Ruminococcus, Megasphaera, Faecalibacterium and Bifidobacterium, Stenotrophomonas were the top 10 dominant genera, representing 16.96%, 14.71%, 9.77%, 8.03%, 5.46%, 5.06%, 3.41%, 3.28%, 2.92% and 1.91%, respectively. In the M group, Megamonas, Bacteroides, Enterobacter, Ruminococcus, Megasphaera, Weissella, Faecalibacterium, Bifidobacterium, Pantoea and Stenotrophomonas were the top 10 dominant genera, representing 20.10%, 7.91%, 7.47%, 7.40%, 4.75%, 4.53%, 4.44%, 4.15%, 3.39% and 1.13%, respectively. In addition, the number of unclassified species increased from 18.13% (CK) to 40.25% (S), 28.22% (G) and 34.75% (M).

Figure 4 shows the effects of sucrose, glucose and molasses on the relative abundance of bacteria in the most dominant phylum (A) and genus (B) in stylo silage. The community was shifted along with soluble sugar treatments: the abundances of Bacteroidetes and Actinobacteria were both increased (p < 0.05), while the abundance of Proteobacteria was decreased (p < 0.05) in additive-treated groups compared with the CK group. The abundances of Weissella, Enterobacter and Stenotrophomonas were decreased by additive treatments (except Pantoea in the G group) (p < 0.05), while the abundances of Megamonas, Bacteroides, Megasphaera, Faecalibacterium, Stenotrophomonas and Bifidobacterium were increased by the treatments (p < 0.05).

4. Discussion

Generally, the chemical composition of raw materials is an important factor to determine the silage fermentation quality, especially DM and WSC content. Guyader et al. [19] suggested that the DM content of well-preserved raw material of silage was 30–35%. Meanwhile, the WSC is another key factor that affects the quality of fermentation. In this study, the DM of stylo was consistent with the previous results, but the WSC content was obviously lower than the minimum requirement for well-preserved silage [1,3,8,9]. However, some previous studies have confirmed that stylo ensiled alone can not achieve high fermentation quality [1,3,8,9]. Therefore, it might be helpful to use additives, such as soluble sugars, to provide more substrates for lactic acid bacteria and promote lactic fermentation in order to obtain well-preserved stylo silage.

The silage pH is an important indicator of fermentation quality, and silage at pH 4.2 or lower is considered well-fermented [20]. The pH value of additive silages was close to 4.2, which ensured good preservation of stylo silage. A previous study has shown higher pH values at 5.0 or above in stylo silage, which used gallic acid additive and the chemical characteristics were different compared with this study [21]. The lactic acid in silage is the dominant product from fermentation, thus an important index to evaluate silage quality. The addition of soluble sugar may promote the reproduction and growth of LAB and lead to increased lactic acid content. This is consistent with previously reported results [10,11,12,13], demonstrating that sucrose, glucose and molasses can remarkably increase the content of lactic acid. As the main metabolite of acetobacter fermentation in silage, acetic acid is also a crucial index for the evaluation of silage quality. Higher acetic acid level leads to higher pH, which benefits the undesirable microorganism clostridium and then reduces the quality of fermentation [22]. In this study, the contents of propionic acid and butyric acid were not detectable in additive-treated silages, indicating that the stylo was well-preserved after being treated with soluble sugar. This is consistent with the results from mulberry leaf silage treated with sucrose [13]. These results indicated that supplementation of soluble sugars promoted the fermentation quality, and that different additive-treated silages in this study were similarly effective.

In the present study, Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were the dominant phylum in stylo silages. Xu et al. [23] and Dong et al. [24] have shown similar results in corn stover and red clover silage. Another study found that Firmicutes and Proteobacteria were the most abundant phyla in silages for hydrolysis and acidification, with more than 90% of increase after fermentation [9]. This might be caused by the low pH and the anaerobic condition of silage.

Furthermore, the soluble sugar changed the microbial composition of stylo silage at the genus level. The abundance of Megamonas was significantly increased and became dominant in the soluble sugar treatment groups. Megamonas is commonly found in the intestines of humans and animals [25,26,27]. Megamonas can ferment many types of carbohydrates and produce lactic acid, acetic acid and propionic acid [27]. In this study, the soluble sugar treatments resulted in higher levels of lactic acid and total acids but lower abundance of lactic acid bacteria, probably caused by the higher abundance of Megamonas. In addition, Bacteroides is an anaerobic bacterium, usually lives in the intestines and the respiratory tract of humans and animals and produces organic acids such as lactic acid, acetic acid and propionic acid [28,29]. Bacteroides, a bacterium rarely reported in silage, was found to be dominant in this study in the soluble sugar treatment groups with higher levels of both lactic acid and total acids, compared with the control group. Both Megamonas and Bacteroides are usually found in humans and animals but only rarely reported to be in silages. Therefore, it is not clear how their abundances were significantly increased in well-conserved stylo silages, and further study is required to explore the relevant mechanisms. Furthermore, Weissella’s abundance was decreased by soluble sugar treatments but remained as one of the predominant microbial species in stylo silage, similar to what was observed with the silage of forage soybean mixed with crop corn or sorghum [30]. Weissella is a lactic acid-producing bacterium and the dominant group in fresh forage or at the beginning of silage fermentation before being replaced by other lactic acid-producing microorganisms after ensiling [31,32,33]. This may be explained by the fact that Weissella can ferment fewer kinds of sugars compared with Megamonas, leading to weakened competitiveness to and being replaced by Megamonas. Enterobacter is one of the major undesirable microorganisms in silage [12,13,21]. Besides, Enterobacter is the main competitor of Megamonas for soluble sugar, thus its decline is beneficial to the lactic acid-producing microorganisms [34]. This may explain the relatively higher lactic acid levels in the soluble sugar-treated silages. Moreover, in this study, the abundances of Megasphaera, Faecalibacterium, Stenotrophomonas and Bifidobacterium were significantly enhanced in soluble sugar treatment groups with higher fermentation quality than those in the control group with lower fermentation quality. We speculated that the increases of these microbial species balanced the micro-ecosystem and improved the quality of silage fermentation.

It should be noted that, the full chemical compositions of silage were very important for the evaluation of fermentation quality. Unfortunately, in this study, the silage chemical compositions were not analyzed, and only discussed the role of WSC in stylo silage from the perspective of fermentation quality and microorganisms. Therefore, the implications of these results for silage production are limited. Besides, the 16Sr DNA sequencing technology was conducted for the microbial diversity study, and the result was only at the genus level, which is not accurate enough. Hence, in-depth investigations are needed to clearly understand their roles in silage fermentation, for instance, using single molecule, real-time (SMRT) sequencing or metagenomic sequencing.

5. Conclusions

The sucrose, glucose, and molasses treatment resulted in significantly increased lactic acid content, reduced pH, acetic acid, propionic acid and butyric acid content, then improved the quality of fermentation of stylo silage. Meanwhile, the WSC additives altered the stylo silage bacterial community, raising the abundance of acid-producing bacteria Megamonas, Bacteroides, Megasphaera, Faecalibacterium, Stenotrophomonas and Bifidobacterium, while decreasing the abundance of Weissella and inhibiting the growth of undesirable Enterobacter, which led to the improved fermentation of stylo silage. The results obtained from this study confirmed that sucrose, glucose, and molasses have similar beneficial effects on both the bacterial community and silage fermentation of stylo. Molasses was recommended to be used in stylo silage for economic benefits and resource utilization.