Silage is a process in which saccharide is converted into organic acid-lactic acid by LAB anaerobic fermentation in a closed environment to reduce the pH and inhibit the growth and reproduction of other harmful microorganisms. This prevents the loss of nutritional value in feed. Good silage preservation requires a LAB count of > 105 CFU/g FM. In this study, the raw material had a low LAB content (< 5.00 log CFU/g FM), less than the number of beneficial microorganisms to support the success of the experiment. The data show that E. coli content was high, which indicates that alfalfa silage requires additives to ensure complete fermentation.
Adequate WSC is a key factor in forage silage, which provides sufficient nutrient substrate for LAB reproduction. When the WSC content reaches 60–80%, fermentation can be carried out normally . In this study, the WSC content of the alfalfa was 3.27% DM, which was lower than the WSC content of silage maize . It is possible that salt stress in soil inhibits the accumulation of WSC in fresh alfalfa . Therefore, the current WSC content is sufficient to ensure good preservation of the alfalfa with additives. This shows a relatively high protein content, which may be due to salt stress promoting protein accumulation in plants.
The duration of alfalfa ensiling has an important influence on silage fermentation quality and microorganisms . After 30 days of ensiling, CP and WSC decreased slightly, due to bacterial lactic acid fermentation. This is similar to the results of Maharlooei . It is known that cellulase has a degrading effect on macromolecular carbohydrates such as cellulose, hemicellulose, and lignin in the crude fiber of the stem, which degrade into small molecules of monosaccharides or polysaccharides, thereby rapidly enhancing the fermentation activity of the lactic acid bacteria. Cellulase has a significant effect on lactic acid content, pH, and NH3-N. As the ensiling duration increased, the microorganisms became increasingly active. Key nutrients were gradually being consumed, but the content of some, such as propionic acid and acetic acid, did not differ. It is also apparent that there is no spoilage in the silage process, and the rapid propagation of lactic acid bacteria, low pH, and the anaerobic environment inhibited the growth of mold. These findings suggest that salt-tolerant alfalfa has better nutritional quality after cellulase is added.
In this study, after 30 days of fermentation, the nutritional quality and fermentation quality of the silage did not change significantly. The change in quality on the surface is not too great. The main microorganisms in the silage of the salt-tolerant alfalfa are still the beneficial Firmicutes, but the main microorganisms had changed from Enterococcus at 30 days to Lactococcus at 60 days. After the addition of cellulase, the dominant genus shifted from Lactococcus at 30 days to Lactobacillus at 60 days . Jacxsens et al.  revealed that Pantoea agglomerans would be metabolized to produce acetic acid, propionic acid, and succinic acid. Enterobacteriaceae is also capable of producing sugar metabolism under anaerobic conditions. This is similar to the results of this study. The relative abundance of Enterobacteriaceae (especially Lactococcus and Pantoea) is higher in alfalfa silage under salt stress (Fig. 3), which may explain the increase in organic acids.
Lactic acid is the main factor leading to the pH drop in the silage environment, and pH is also an important indicator of whether anaerobic fermentation is complete. McDonald also showed that pH is an important indicator of the degree of fermentation and the quality of silage . In our experiment, the organic acid content at 60 d was significantly better than at 30 d, and the quality of alfalfa silage after salt stress treatment was better. However, after 30 and 60 days of fermentation, the cellulase-added silage had a higher WSC content, but its CP content was lower than in the CK. WSC is decomposed into lactic acid water by glycolysis (EMP) or the hexose phosphate (HMP) pathway. This may be because the addition of cellulase leads to a dramatic increase in the number of lactic acid bacteria and expands the effect of anaerobic fermentation . On the other hand, alfalfa has been identified as having high antibacterial activity , which may inhibit the growth of cellulase. In the current experiment, as ensiling duration was prolonged, the lactic acid content decreased and the pH value increased significantly. This may be because Lactobacilli can metabolize lactic acid in the absence of sugar . Therefore, the role of microbial bacteria in silage fermentation needs further study.
High-throughput sequencing can provide a wealth of data for exploring taxonomic classifications and activities of silage microbial bacteria . In this study, alpha diversity values indicate that the diversity of bacterial communities after silage is greater, which is consistent with the results of . St-Pierre found that Mencius, Bacteroides, Chloroflexi, and Proteobacteria are dominant phyla that play an important role in hydrolysis and acid production . Among these, Firmicutes is the main phylum in most grass silage . Proteobacteria are the most abundant bacteria in fresh alfalfa, and the content is above 90%. Bao also found that Proteobacteria is the main phylum of fresh alfalfa . In our study, the main genera after ensiling (e.g., Lactobacillus, Enterococcus, Lactococcus, and Weissella) were also significantly inhibited during the fermentation process. The growth of spoilage bacteria such as Proteobacteria is similar to the findings of Yanbing in experiments on corn and ryegrass .
LAB is an important member of the bacterial community and a key microorganism in silage, with major effects on silage quality. Lactobacillus, Lactococcus, and Enterococcus play key roles in the anaerobic fermentation of salt-tolerant alfalfa. Even Enterobacteriaceae and Pantoea play a role in it, and they also consume nutrients for their own growth and reproduction. Their relative abundance was lower than the LAB content, which also shows that the feed was well fermented. Under the influence of cellulase in silage, Lactobacillus rapidly multiplies and becomes another dominant bacterium in addition to Lactococcus and Enterococcus. Our research results on salinized silage microorganisms are different from those of previous studies. It has been reported that Lactococcus and Lactobacillus are the main genera of silage after fermentation . Therefore, the role of LAB silage fermentation requires further research to determine whether Lactobacillus is suitable for the growth and reproduction of alfalfa in saline soil. Among these genera, there may be LAB that is halophilic or salt-tolerant lactic acid bacteria.