After mixing alfalfa with rice straw and wheat bran, the DM and WSC content was adjusted to 364 g/kg FW and 79.85 g/kg DW, which met the requirements for ideal DM (300-400 g/kg FW) and WSC (> 50 g/kg DM) content [15].
Effects of additives on fermentation and aerobic stability of alfalfa mixed silage
Enzyme-treated silages had higher pH and lower LA concentrations than the control during the initial 5 d of ensiling. Perhaps fibrolytic enzymes indirectly provided fermentable sugars, which were degraded from cell wall polysaccharides after a short lag [16]. In the previous study, applying cellulase in mixed silage of high-moisture amaranth and rice straw silage did not markedly accelerate LA fermentation during the initial 5 d of ensiling [1]. In mixed silage (corn and hulless-barley straw), LA accumulation was less efficient in cellulase- or xylanase-treated silages than in silages with added molasses [6]. However, after 15 d of ensiling, there was lower pH and higher LA concentrations in the E, X, and EX silages than C silage. Similarly, in a previous study, adding cellulase promoted production of LA, reducing pH in hybrid Pennisetum silage compared to the control after 60 d of ensiling [17]. A similar reduction in pH was obtained in xylanase-treated sugarcane silage after 60 d of ensiling [8]. Cellulase and xylanase hydrolyzed β- 1,4-glycosidic linkages in major plant polysaccharides, cellulose, and xylan, making WSC available for silage fermentation by LAB [18, 19]. In the present study, X silage had higher AA concentrations than C silages throughout ensiling. Furthermore, adding xylanase to sugarcane silage increased AA concentration as compared to the control [8]. We speculate that more xylose production is beneficial to heterofermentative LAB production. Both homo- and heterofermentative LAB can ferment various pentose sugars; the xylose is taken up by specific permeases and converted to D-xylose-5-phosphate which is then fermented to a mixture of LA and AA [20].
All 3 enzyme-treated silages had a significantly lower AN content than C silage on d 45 of ensiling, attributed to lower pH and higher AA concentration in enzyme-treated silages than in C silage. Furthermore, AN is usually an indicator of clostridia and enterobacteria, which are inhibited by low pH [21]. As AA reduces pH and inhibits growth of organisms that promote spoilage [8], it may have improved silage quality in this study. The EX silage had the lowest AN concentration after 3 d of ensiling. Similarly, fibrolytic enzymes in combination with cellulase and xylanase lowered AN concentration in corn silage compared to the control [6]. The superior effect of EX treatment in this study was attributed in part to synergistic effects of cellulase and xylanase on mixed silage. In this study, X and EX silages had lower PA concentrations than other treatments after 45 d of ensiling. Similarly, application of Pediococcus acidilactici and cellulases decreased PA in Caragana korshinskii Kom. silage after 60 d of ensiling. Generally, clostridial secondary fermentation can metabolize LA to propionic acid [22]. Reduced PA concentrations from application of cellulases may contribute to inhibited secondary fermentation, with better nutrient preservation [23], consistent with higher WSC and CP contents in X and EX silages after 45 d of ensiling.
In the study, greater WSC in EX silages than C silage was attributed to direct hydrolyzation of lignocellulose by cellulase and xylanase, releasing additional fermentable substrate. Cellulase accelerated LA fermentation and the decrease in pH, which were attributed to indirect supplementation of WSC by degradation of lignocellulose [24]. That C silage had the lowest DM content in this study may have been due to heterogeneous fermentation [25], consistent with Zhang et al. [26] who reported that adding cellulase markedly increased DM recovery in Caragana korshinskii Kom. silage. In this study, the CP content in C was lower than in X and EX silages, indicating more intensive proteolysis. Furthermore, fermenting with a fibrolytic enzyme in combination with cellulase and xylanase may inhibit protein degradation in whole-crop corn silage after 60 d of ensiling [27].
The lower NDF content in E, X, and EX treated silages than C silage during the initial 15 d of ensiling were attributed to: firstly, cellulase and xylanase effectively degrading cell wall carbohydrates in forage, thus reducing NDF and HC content in silage [28]; and secondly, the low pH promoting hydrolysis of the cell wall fraction, thereby reducing NDF and HC [29]. In that regard, cellulase lowered NDF and HC content as compared to control in hulless-barley straw mixed silage [30]. Xylan is the main component of hemicellulose, and the most important enzymes involved in hemicellulose degradation are xylanases and β-1-4 xylanases, which contributed to the lower HC contents in X silage than C silage.
Enzymes improved aerobic stability of silage. Perhaps the high AA concentration inhibited the growth of yeasts and molds to improve aerobic stability [27]. Similarly cellulase and xylanase improved aerobic stability in bermudagrass silage [31].
Effects of additives on microbial community dynamics during ensiling
The lower alpha diversity in enzyme-treated silages than C silage after 5, 15, and 45 d of ensiling was attributed to the dominance of LAB and lower pH in enzyme-treated groups. In that regard, there was low alpha diversity when the dominant LAB became relatively simple during ensiling [32]. The dominance of LAB accompanied by a decreased pH inhibited proliferation of undesirable microbes, reducing microbial diversity in alfalfa silage [33]. In the study, 45-d silages were separated from fresh, 5-, and 15-d silages, indicating clear differences in bacterial communities.
Before ensiling, Weissella, Acinetobacter, Pseudomonas, Stenotrophomonas, Sphingobacterium, and Chryseobacterium were the main epiphytic bacteria. All of those epiphytic bacteria were also detected (at the genus level) in fresh Italian ryegrass, corn stover, and paper mulberry [34-36]. In this study, the most dominant genus changed from Lactobacillus after 5 and 15 d of ensiling, and to Pseudomonas and Stenotrophomonas after 45 d of ensiling. It is well known that Lactobacillus is a rod-shaped LAB, with crucial roles in producing LA production and decreasing pH and it became the dominant bacteria in the natural fermentation of corn silage at the early stage of ensiling [37]. However, roles of Pseudomonas and Stenotrophomonas is silage are not well known.
Pseudomonas can inhibit pathogenic microorganisms during plant growth and continue to survive in an anaerobic environment [38, 39], whereas Stenotrophomonas maltophilia is a non-LAB related to lignocellulosic biomass degradation [40]. However, as Pseudomonas and Stenotrophomonas can degrade protein, they are considered undesirable bacteria in silage [41, 42]. In contrast, Ogunade et al. [43] and Ren et al. [44] reported negative correlations between AN concentration and relative abundance of Pseudomonas and Stenotrophomonas. Therefore, the roles of these 2 bacteria in silage need further study. Desirable bacteria Lactobacillus and Weissella were promoted soon after ensiling began, increasing LA production and lowering pH [45]. The RA of LA-producing bacteria (Lactobacillus and Weissella) was higher in enzyme-treated silages than C silage at d 5 and 15 of ensiling. The availability of WSCs provides ready fermentation substrates to enhance proliferation of Lactobacillus and Weissella [46]. In addition, molasses also enriched abundance of Lactobacillus and Weissella in soybean and amaranth silage, respectively [47, 48]. Cellulase addition expectedly increased the LAB counts as more WSC was available for microbial fermentation due to cell wall degradation [49]. Zhao et al. [2] reported that LAB was the dominant microorganism in mixed silage of soybean residue and corn stover with cellulase after 56 d of ensiling, and furthermore, that Lactobacillus dominated the bacterial community.
L. paralimentarius and L. parabrevis were higher in X and EX silages than in the other 2 treatments after 15 d of ensiling. L. paralimentarius are Gram-positive, catalase-negative, facultative heterofermenters, whereas L. paralimentarius was the main species observed in corn silage after 90 d of ensiling [32, 50]. L. parabrevis was also observed in whole-plant corn silage during 14 d of ensiling [51], and was positively related to AA concentration [52]. In the current study, the addition of xylanase increased AA concentration after 15 d of ensiling, and we speculate that L. paralimentarius and L. parabrevis used xylose to produce AA by heterofermentation. Weissella is obligative heterofermentative LAB that converts WSC into LA and AA during the early stage of ensiling [15]. In the current study, Weissella and W. cibaria had the highest abundance in E silage after 5 and 15 d of ensiling, which may have contributed to higher AA concentration in E silage than that in C silage after 15 d of ensiling. Bacilli, Bacillaceae, Bacillus, and Bacillus velezensis were higher in EX silage than in other 3 groups (based on LEfSe analysis) for 5-and 45-d silages. Bacillus improved fermentation quality and aerobic stability in alfalfa silage [53], can improve animal performance, and was defined as a fourth-generation silage inoculant [54]. Wang et al. [55] reported that adding pectinase increased the abundance of Bacillus in alfalfa silage. Bacillus velezensis had antagonistic properties towards toxigenic molds in silage conditions [56], indicating bioactive roles of EX in promoting growth of Bacillus velezensis to exert antifungal properties. Furthermore, C silage had high RA of spoilage-producing organisms (Acinetobacter sp, Erwinia sp, Lelliottia amnigena, and Sphingomonas sp). Lelliottia was reclassified as a novel genus from Enterobacter, an undesirable bacteria in silage [57]. Adding Moringa oleifera leaf to alfalfa silage could decrease the RA of Lelliottia [58]. Sphingomonas was considered to hydrolyze soluble protein in silage comprised of agricultural by-products [59].
Effects of additives on in vitro parameters
Cellulase and/or xylanase increased DM degradability, total VFA production, and gas production during in vitro fermentation of mixed silage. Cellulase reduced plant cell wall fractions and protein loss during ensiling, providing more digestible substrates for fermentation by rumen microbes and facilitating ruminal digestion [60]. According to Del Valle et al. [8], xylanase acts on the most digestible content of the NDF in sugarcane silage, increasing DM degradability. The in vitro DM degradability in cellulase-treated mixed silage of soybean residue and corn stover was significantly higher than control [2]. Volatile fatty acids produced by microbial fermentation in the rumen could be a main energy source for ruminants [61]. Furthermore, there was a strong positive relationship between in vitro DM degradability and total VFA production [62]. In this study, E, X, and EX-treated silage increased molar proportions of VFAs and resulted in high gas production, thereby decreasing pH, attributed to increased DM degradability. The lower value of ADF, the higher the digestibility of the feed and the higher the feeding value [63]. In the current study, EX silage had the highest DM degradability, total VFA production, and lowest pH, attributed to the lowest ADF content in EX silage after 45 d of ensiling. The type of fermented substrate, microbial population, and rumen environment influence the type of VFA produced in the rumen. Acetate, propionate and butyrate are key VFAs formed in the rumen, with small quantities of iso-butyrate, valerate, and iso-valerate [61]. In the study, the dominant VFA of ruminal fermentation was acetate, with no significant differences among treatments for concentrations of AA, PA, BA, iso-butyrate, valerate, or iso-valerate. However this contradicts a report of high concentrations of AA, PA and BA in agricultural waste-based complete feed silage given a cellulase enzyme treatment [64]. This response may be attributable to the consistency of the composition of the experimental diets [61]. The AN concentration in the study ranged from 24.06 to 25.85 mg/dL, within the target range (8.5~30 mg/dL) to maximize microbial protein synthesis [65]. Furthermore, enzyme treatments did not have any significant effect on AN concentration in the rumen. Similarly, there was no significant effect on rumen AN concentration in complete feed silage treated with cellulase [64].