Nutritional and Microbial Quantity of fresh Alfalfa
The nutritional composition of fresh alfalfa and the composition of its microbes are visible in Table 1. The nutrients of raw alfalfa directly affect the quality of later silage. And its nutrient indicators are calculated based on the dry matter of the raw materials. The dry matter (DM) content of the raw material is 29.9 g/kg, And the crude protein (CP) content of alfalfa is 21.9, Protein content is relatively good, which may also be due to salt stress promoting protein accumulation in plants. The number of Lactic acid bacteria (LAB) is higher than yesat. If the operation is normal, this quantity of LAB is enough to ferment with additives.
Nutritional and Fermentation Quality of alfalfa Silage
After 30 and 60 days of anaerobic fermentation, the nutritional quality and microbial quantity of alfalfa silage have changed significantly. After 30 days of fermentation, most of the indicators, CP and WSC, changed significantly, which reduced fermentation by 12.9% and 7.6% under cellulase (Table 2), This is due to your metabolic fermentation of lactic acid bacteria, respectively. From the nutritional indicators of 30 days and 60, most indicators have no significant difference, except for WSC. And regardless of CK and cellulase treatment, the WSC content at the same time has significant differences.
However, the changes in the treatment groups at the same silage time were consistent. From the nutritional point of view of saline-alkali mantle, its overall nutritional quality treatment is better. It indicates that the saline-alkali mantle can be used as a good feed to feed the animals after good silage fermentation.
In addition to understanding the nutritional quality of silage, the quality of its fermentation is also crucial. Through the fermentation indicators, we can understand what the secreted products of the main fermenting microorganisms are. We would see the fermentation quality of the saline-alkali silage in Table 3. Among them, butyric acid, which has not detected, is bad for livestock, which indicates that the silage has good palatability. Lactic acid is the main product of lactic acid bacteria in the process of fermentation and metabolism. Under the same cellulase treatment, the lactic acid content of the 30-day and 60-day silage was 2.66 and 3.48, respectively, an increase of 23.5%. This also explains the reason for the decline in WSC in Table 2. WSC is decomposed into lactic acid water by glycolysis (EMP) or hexose phosphate (HMP) pathway. And it is obvious that after the silage fermentation of the additive, the NH3-N and pH were significantly different from the control group (P < 0.01).
It can be distinguished from the silage fermentation index (Table 3). Cellulase has a significant effect on lactic acid content, pH and NH3-N. As the silage time prolongs, the life activities of its microorganisms become more and more active. The key nutrients are slowly being consumed, but some changes such as propionic acid and Acetic acid do 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 anaerobic environment inhibit the growth of mold. It also shows that the saline-alkali mantle will have better quality after being filled with additives.
Effect of Cellulase on Bacterial Community After 30 and 60 Days of Ensiling
In this study, high-throughput determinations of variable regions 3 and 4 of 16s rDNA were performed to calculate and evaluate bacterial diversity after salinization of saline-alkali mantle. As can be seen from Table 4, the coverage of all samples was greater than 99%. It indicates that the sequencing width is relatively comprehensive, and the microbial high-throughput data is sufficient to represent the characteristics of the bacterial microbial community. And, it is obvious that it is known. After a long period of fermentation, the number of OTUs is significantly reduced, which may also be that LAB has become the dominant dominant flora, inhibiting the growth and reproduction of other harmful microorganisms. According to the number of OTU and Chao index, the bacterial community abundance after using the additive and different silage time is different.
Principle component analysis (PCA) analyzes the similarity and difference of bacterial communities after alfalfa silage through different treated sample points. As shown in Figure 1, PCA1's contribution to the interpretation of total variance reached 67.73%, while PCA2 explained 13.53% of the total variance. Overall, there is a large difference between alfalfa raw materials and silage samples. And from the fermentation quality of the nutritional quality of Tables 2 and 3, there are differences in key indicators, such as WSC, CP and so on. The result is that they are most likely caused by these microbial flora.
After sequencing the DNA of the microbial community, we can know four phylums abundance contents have changed. It is also possible to understand the main role of the saline-alkali lining. The figure 2 shows the fresh alfalfa and silage microorganisms at the phylum level. The content of Firmicutes in the alfalfa raw material of saline-alkali is less than 0.1% of the total bacterial content. This is a very small amount. If no additives are added during anaerobic fermentation silage, the amount of silage beneficial bacteria is much smaller than that of the cellulase-treated treatment group.Before and after the silage, the microbes had similar species composition at the phylum level, and were composed of Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria. The only difference is the change in its content. This change in the species of different bacteria also represents the excitement of the microscopic world during the silage process.
Among them, the abundance of Proteobacteria was as high as 92.17%. However, after 30 days and 60 days of silage fermentation, the abundance of Proteobacteria decreased from 92.17% to 1.75-36.13%. Instead, the increase in firmicutes. It increased from 0.99% in raw material alfalfa to between 63.6% and 93.9%. And with the increase of silage time, whether it is the control group or the cellulase silage. The content of firmicutes has increased by 3%-13%. This also indicates that as the fermentation time is extended, the number of beneficial bacteria is also gradually increasing. And from the different experimental addition treatments, the firmicutes content of the silage added with cellulase is more rapid, so the 60-day firmicutes content is relatively low. This also indicates that the addition of cellulase can make silage fermentation more complete.
In pasture silage, some species are recognized as the main fermented lactic acid bacteria in the academic, such as Lactococcus, Enterococcus, and Lactobacteriaceae. As shown in Figure 3, only Pantoea's abundance is dominant in fresh alfalfa, and the advantage of LAB is that only insignificant Lactococcus and Enterococcus exist. It may also be that the lactic acid bacteria suitable for growth on the saline alfalfa are Lactococcus, Enterococcus. In the absence of additives, after 30 days and 60 days of silage, the dominant species became fermented feeds with Lactococcus and Enterococcus as the dominant lactic acid bacteria. And as time goes by, its dominant lactic acid bacteria does not change much. After adding the fermentation promoter cellulase, slightly different from the CK group, the dominant lactic acid bacteria increased Lactobacillus. It is undeniable that cellulase has a degrading effect, which degrades macromolecular carbohydrates such as cellulose, hemicellulose and lignin in the crude fiber of the stem into small molecules of monosaccharides or polysaccharides, thereby rapidly enhancing the fermentation activity of the lactic acid bacteria.
Through the inductive organization of silage sample microorganisms, 16S function prediction is carried out on the datas, through each OTU corresponding greengene id, from the eggNOG database to the description information of each COG, and its functional information, thereby obtaining functional abundance Spectrum (Figure 4). In the above, we know that alfalfa has undergone 30 days and 60 days of silage fermentation and its quality has changed. From 16S function prediction analysis, it is known that its microbial community mainly focuses on life activities between cells. It is the reproductive growth of these bacteria that leads to nutritional differences in the saline alfafa. The main difference is mainly in Energy production and conversion (C), Amino acid transport and metabolism (E), Carbohydrate transport and metabolism (G), Intracellular trafficking, secretion, and vesicular transport (U). These functions are related to microbial activity during silage. In particular, the differences in function G between these treatment groups, which is a clear distinction between bacteria and carbohydrates in plant nutrition. This also explains the difference between WSC in fresh alfalfa and post-silage WSC. LAB mainly ferments and metabolizes WSC, which will result in the production of carbohydrate transport and metabolism (G) and Amino acid transport and metabolism (E).