Chemical compositions and microbial populations in fresh alfalfa at initial and full flowering stages
It is well-known that the chemical compositions of forage are strongly influenced by the growth stage. This study described that the dry matter, water soluble carbohydrate, neutral detergent fiber, and acid detergent fiber contents increased as alfalfa matured. This may be because the ratio of leaf to stem declines with plant elongation and cell wall contents in stems increase with the maturity of alfalfa . In addition, the buffering capacity in alfalfa decreased with advancing maturity. McDonald and Henderson  reported that the buffering capacity of plants is mainly ascribed to the potassium, calcium and magnesium salts of organic acids. It is possible that the decline in buffering capacity may be linked with a reduction in the organic acid fraction with the maturity of alfalfa. Similarly, Greenhill  found that the least mature harvest of ryegrass, clover and Lucerne was more highly buffered than the advanced maturity, which could be due to the decrease in organic acids with maturity.
The growth stage of plant can obviously influence the microbial diversity and populations . In this study, the epiphytic lactic acid bacteria, yeast, Enterobacteriaceae and aerobic bacteria populations increased with the maturity of alfalfa. There are two possible explanations for this phenomenon. One is related to the accumulated water soluble carbohydrate contents as alfalfa matured. Lindow and Brandl  found that the availability of carbon-containing nutrients on leaves is critical in determining the epiphytic colonization, and bacterial community on forages was first limited by carbon availability and secondarily by nitrogen availability. The other reason is probably due to the increased matured tissues. Thompson et al.  reported that some nutrients released from matured tissues are important for microbial growth and microbial community diversity, which may be the reason for the differences in epiphytic microbiota at different growth stages.
Fermentation characteristics in alfalfa silages at initial and full flowering stages
The fermentation quality of silage is highly dependent on the growth stage of forage at harvesting . Present study exhibited that AL2 group had better fermentation quality than AL1 group with lower pH, NH3–N, ethanol and butyric acid concentrations, and higher lactic acid concentrations. This could be mainly attributed to the lower buffering capacity and moisture contents and higher water soluble carbohydrate contents in ALFM2 group compared with ALFM1 group. Guo et al.  concluded that suitable dry matter contents, sufficient water soluble carbohydrate contents and lactic acid bacteria populations, low buffering capacity and undesirable microbial populations are critical for successful ensiling.
Lactic acid is a metabolite produced by lactic acid bacteria during fermentation. The higher lactic acid contents in AL2 group than AL1 group were probably correlated with the higher water soluble carbohydrate contents and lactic acid bacteria populations in ALFM2 group, stimulating the lactic acid fermentation. Furthermore, the rate of pH decline is mainly determined by the organic acids produced during ensiling and buffering capacity of raw material. Hence, the lower pH in AL2 group than AL1 group during fermentation could be due to the rapid accumulation of lactic acid and lower buffering capacity in ALFM2 group, making it easier to change in pH.
In conserved silages, butyric acid and ethanol are both undesirable. Butyric acid was mainly produced by undesirable microorganisms which break down amino acid resulting in nutrient loss. The lower butyric acid contents in AL2 group may be correlated with the rapid drop of pH in AL2 group, inhibiting the propagation and metabolism of harmful microorganisms. Kung et al.  reported that the ethanol contents (> 30–40 g/kg DM) may be associated with the metabolism of yeast. Herein, the ethanol concentrations in AL1 and AL2 groups were both less than 25 g/kg DM, indicating the ethanol contents in alfalfa silages were mainly produced by other microbes, such as hetero-fermentative lactic acid bacteria.
The NH3–N content is a critical indicator for assessing the silage fermentation quality, reflecting the crude protein degradation through proteolysis by clostridium fermentation and plant protease activity . In the current study, the relatively higher NH3–N contents (> 140 g/kg TN) in AL1 and AL2 groups on day 60 could be ascribed to the insufficient acidic conditions at the initial fermentation stage. However, the AL2 group with an NH3–N content of 143 g/kg TN; less than 150 g/kg TN is reported by Mahanna and Chase  to be acceptable for well-preserved legume silage.
Bacterial community diversity, compositions and successions in fresh alfalfa and silages at initial and full flowering stages
Alpha diversity, including Shannon, Chao1, Ace, Sobs, Coverage and Simpson indices, describes the coverage, diversity, richness and evenness of entire bacterial community in samples. Herein, higher Shannon values and lower Simpson values in ALFM1 group than ALFM2 group indicated that higher bacterial diversity existed in ALFM1 group. In addition to the inherent changes in alfalfa with growth stage, Wang et al.  reported that the environmental factors, such as temperature, humidity and solar radiation, can also influence the epiphytic bacterial community of forage. After 60 days, higher Shannon values and lower Simpson values in AL1 group indirectly proved that AL2 group had better fermentation quality than AL1 group. Muraro et al.  concluded that a successful fermentation with a decline in diversity indexes, from raw material to final silage by establishing a parallel with changes occurring within a silo, where the complex bacterial community of fresh material is gradually replaced by lactic acid bacteria strains in silage when anaerobic condition is reached and pH decreases.
Noteworthy, the decreased abundance of Firmicutes and increased abundance of Proteobacteria with growth stage after 3 days seemed to imply that anaerobic condition was more difficult to realize in matured alfalfa silage. Conversely, lower pH and higher lactic acid concentrations were found in AL2 group compared to AL1 group on day 3. This is probably correlated with the species, interaction, activity and metabolic characteristics of epiphytic lactic acid bacteria strains in alfalfa at different growth stages. After 60 days, the higher abundances of Firmicutes and lower abundances of Proteobacteria in AL2 group than AL1 group may be because AL2 group had a more acidic environment than AL1 group, benefiting the growth and reproduction of Firmicutes and suppressing the multiplication of Proteobacteria .
Higher proportions of Weissella and Lactobacillus in AL2 group than AL1 group on day 3 could partly explain the result that AL2 group had lower pH and higher lactic acid concentrations after 3 days. Weissella are recognized as the initial colonizer microbes during fermentation and they could convert water soluble carbohydrates to lactic and acetic acids . Regarding Lactobacillus, they are often applied to occupy the predominant position at the beginning of fermentation, and guarantee good silage quality due to their capacity of producing lactic acid via consuming glucose .
After 60 days, higher abundances of Enterobacter and Enterobacteriaceae in AL1 group than AL2 group may result in higher NH3–N contents in AL1 group. Enterobacteriaceae and Enterobacter were both reported for their proteolytic activity, which can lead to inefficient lactic acid fermentation, higher NH3–N contents and pH [29, 30]. Herein, Enterobacter was the subdominant bacteria in AL1 group on day 60, likely responsible for the large amounts of NH3–N produced during fermentation . Santos et al.  described most Enterobacter frequently found in silage are nonpathogenic. Whereas, their development is unacceptable, because they are in competition with LAB for water soluble carbohydrates during fermentation. Silva et al.  also reported Enterobacter are detrimental to silage quality, because they can produce NH3–N and slow down the drop in pH during fermentation.
Correlation between fermentation products and bacterial community in alfalfa silages at initial and full flowering stages
The ALFM1 group was separated from ALFM2 group in PCoA plot, indicating that growth stage had great effects on the epiphytic bacterial community of forages. ANOSIM analysis presented greater discrepancies in bacterial communities between groups than within group. The positive correlation between Lactobacillus and lactic acid in RDA plot demonstrated that Lactobacillus play an important role in accumulating lactic acid and decreasing pH throughout the fermentation process .
In Spearman Correlation Heatmap plots, the positive correlation between the abundance of Weissella and acetic acid contents in AL1-3 group was in accordance with the fact that most Weissella species are obligate heterofermentative bacteria, generating acetic and lactic acids via converting WSC . The positive correlation between the abundance of Enterobacteriaceae and ethanol contents in AL1-60 group was consistent with the results of Sun et al. , who reported that Enterobacteriaceae could thrive in weak acidic and anaerobic conditions, fermenting water soluble carbohydrates and lactic acid to ethanol, succinic acids, acetic acid, or 2,3-butanediol.
Bacterial co-occurrence, co-occurrence network complexity, and stability in fresh alfalfa and silages at initial and full flowering stages
The separate bacterial network analysis was applied to assess the effects of growth stage and storage time on bacterial network complexity and stability of alfalfa silages at two growth stages. The results proved ensiling time possessed a more remarkable influence on bacterial community compositions than growth stage. Herein, longer storage time decreased the bacterial network complexity in alfalfa silages, indicated by simpler bacterial community structure in 60 day silages. This may be related to the acidic environment and inadequate fermentable substrates in 60 day silage, restricting the growth and reproduction of most microorganisms.
AL2 group had similar bacterial community stability (Degree, Degree Centrality, Closeness Centrality, Betweenness Centrality) with AL1 group on days 3 and 60. The higher bacterial community stability in AL1 group might be attributed to the inferior fermentation quality in AL1 group with lower amounts of lactic acid and higher pH, resulting in large amounts of undesirable bacteria. Regarding AL2 group, its higher bacterial community stability was probably due to the rapid drop of pH and quick production of lactic acid in AL2 group, resulting from the dominant role of beneficial lactic acid bacteria.
Predicted pathways of bacterial communities in fresh alfalfa and silages at initial and full flowering stages
Currently, an increasing number of highly accurate reference microbial genomes (> 50000) supply a tremendous resource of taxonomy-anchored functional information . Bioinformatic tools can use this resource to predict functional characteristics of microbial communities based on 16S rRNA gene profiles. Herein, PICRUSt2, which expands the capabilities of the original PICRUSt1 method to predict functional potential of a community based on marker gene sequencing profiles , was used to explore the effects of growth stage and storage time on functional profiles of bacterial communities in fresh alfalfa and silages at two growth stages.
On the Pathway level 1, higher proportions of environmental information processing, cellular processes, metabolism, organismal systems, and genetic information processing in AL1 group than AL2 group prior to ensiling might be linked with the higher diversity of epiphytic bacterial community in AL1 group. After ensiling, the higher proportions of metabolism and genetic information processing on day 3, and Cellular Processes on day 60 in AL1 group than AL2 group may reflect the flourishment of undesirable bacteria due to the less lactic acid production and higher pH environment.
On the Pathway level 2, higher proportions of amino acid and carbohydrate metabolism in silage samples confirmed the bacterial communities in silage possessed strong capacity to metabolize carbohydrates and amino acids during ensiling. The lower abundances of signal transduction and membrane transport in silages may be due to the production of organic acids and acidification during fermentation, inhibiting the signal transduction and membrane transport of bacterial community. The higher abundances of folding, sorting and degradation, translation, and replication and repair in AL2-60 group than other silage samples suggested that the beneficial LAB of bacterial community in AL2 still rapidly proliferated after 60 days, consistent with the higher lactic acid production in AL2 on day 60. Kilstrup et al.  showed nucleotides can be used by microbes to replicate and synthesize DNA and RNA, and supply energy for cellular process. The decreased abundances of cell motility, cellular community-prokaryotes, and environmental adaptation after ensiling were probably because the fermentation products (mainly organic acids) and acidic environment in silage were adverse to the bacterial cell motility, cellular community and their adaptive capacity.
On the Pathway level 3, the up-regulated metabolic pathways in all samples reflected that the bacterial community compositions of fresh alfalfa and silages at two growth stages always remained high metabolic activity. The gradually down-regulated pathways of ABC transporters, biosynthesis of secondary metabolites, biosynthesis of amino acids, carbon metabolism, two-component system, and microbial metabolism in diverse environments during ensiling indicated that the membrane transport, biosynthesis and metabolism of bacterial community in silages might be strongly influenced by the anaerobic environment, bacterial interaction and fermentation products (e.g., lactic acid, acetic acid and ethanol).
Predicted enzymes of bacterial community in fresh alfalfa and silages at initial and full flowering stages
The enzyme activity of different individual microorganisms may differ, leading to different functional contributions to certain reaction end-products . It is common knowledge that 1-phosphofructokinase, fructokinase and pyruvate kinase are mainly responsible for the Embden–Meyerhof pathway (EMP). Compared to AL2 group, the higher abundances of 1-phosphofructokinase and fructokinase in AL1 group on day 3 may be more related to the higher metabolic activity of undesirable bacteria due to the slower acidification at initial fermentation stage in AL1 group. The higher abundances of fructokinase, pyruvate kinase and 1-phosphofructokinase on day 60 in AL2 group than AL1 group were primarily related to the higher metabolic activity of beneficial LAB in AL2 group.
In anaerobic glycolysis, lactate dehydrogenase is the terminative enzyme in the sequence of reactions that promote the breakdown of glucose to lactate . Compared to AL1 group, the higher abundances of D-lactate dehydrogenase and lower abundances of L-lactate dehydrogenase in AL2 group on day 3 suggested D-lactate dehydrogenase was more critical than L-lactate dehydrogenase in stimulating lactic acid fermentation during the early stages. It is in accordance with the findings of Wang et al. , who reported D-lactate dehydrogenase was important in the production of LA at the beginning of ensiling. In the later research, more attention should be paid to investigating the role of D-lactate dehydrogenase in regulating the LA production during the initial stages of fermentation. After 60 days, higher proportions of D- and L-lactate dehydrogenase in AL2 than AL1 agreed with the large accumulation of LA in AL2 at the late stages of fermentation.
The ribulose-5-phosphate and glucose-6-phosphate dehydrogenase played important roles in pentose phosphate pathway . The higher proportions of L-ribulose-5-phosphate 3-epimerase and glucose-6-phosphate dehydrogenase in AL1 group than AL2 group on day 60 implied that the bacterial communities in well-preserved silage were less involved in pentose phosphate pathway. More research needs to be conducted to explore the role of pentose phosphate pathway during ensiling.
The nitrite reductase (nirB) and ornithine decarboxylase (odcA) in bacterial community were mainly responsible for the ammonia–nitrogen (NH3–N) and biogenic amine formation in alfalfa silages . In the current study, the higher NH3–N contents and lower abundances of Nitrite reductase (NADH) and Ornithine decarboxylase in AL1 group on day 3 seemed to be contradictory. However, it can be explained by the reports of Heinritz et al. , who concluded that the formation of ammonia during ensiling was related to plant enzyme and bacterial activity. During the initial stages of fermentation, higher NH3–N contents in AL1 group could be mainly ascribed to the plant proteolytic enzyme due to the less lactic acid production and slower acidification in AL1 group. After 60 days, the higher NH3–N concentrations in AL1 group may be more correlated with the bacterial activity because of the higher abundances of Nitrite reductase (NADH) and Ornithine decarboxylase in AL1 group.
The up-regulated protein–N(pi)–phosphohistidine–sugar phosphotransferase, histidine kinase, DNA-directed DNA polymerase and DNA helicase of bacterial community in silage samples indicated that these bacterial enzymes were positively involved in various biochemistry reactions during ensiling. The down-regulated NADH: ubiquinone reductase, glutathione transferase, iron–chelate-transporting ATPase and peptidylprolyl isomerase of bacterial community in silage samples suggested that these enzymes were inhibited by ensiling due to the acidic environment and fermentation products. However, the specific function of abovementioned enzymes requires further study.
Predicted modules of bacterial community in fresh alfalfa and silages at initial and full flowering stages
Genome-scale metabolic models including functional network modules, can provide important insights into cell function. These modules are defined as groups of reactions with correlated fluxes, and the identification of modules can enable researchers to know the internal “wiring” of genetic networks [39, 40]. On day 3, the higher proportions of glycolysis (Embden–Meyerhof pathway), glucose = > pyruvate, and Pentose phosphate pathway (pentose phosphate cycle) in AL1 group than AL2 group were in agreement with the variations of abovementioned key enzymes involved in Embden–Meyerhof pathway and pentose phosphate pathway, demonstrating the consistency of predicted enzymes and modules in bacterial communities. On day 60, lower proportions of citrate cycle (TCA cycle, Krebs cycle) in AL2 group than AL1 group confirmed that the bacterial community in well-preserved silage was less involved in TCA cycle.
Notably, the module of NADH: quinone oxidoreductase, prokaryotes was obviously down-regulated in well-fermented AL2 group on day 60, consistent with the changes of abovementioned NADH: ubiquinone reductase. Brandt  described that the NADH: quinone oxidoreductase can catalyze electron transfer from NADH to quinone, and it is by far the largest enzyme of respiratory chain. Hence, it was speculated that the downregulation of NADH: quinone oxidoreductase may reflect the inhibition of undesirable bacteria in well-fermented silage. However, the role of NADH: quinone oxidoreductase in bacterial community during ensiling needs to be further investigated.
Predicted phenotype of bacterial community in fresh alfalfa and silages at initial and full flowering stages
Nowadays, shotgun metagenomics and marker gene amplicon sequencing (16S) can be used to directly measure or predict the functional profiles of microbiota, but current methods do not readily estimate the functional capability of individual microorganisms. Hence, an algorithm named BugBase was used to predict biologically interpretable phenotypes, such as oxygen tolerance, Gram staining and pathogenic potential, within complex microbiome based on whole-genome shotgun or marker gene sequencing data . To our knowledge, this is the first investigation to predict the phenotype of bacterial community in the field of silage.
Herein, the higher proportions of facultatively anaerobic and lower proportions of anaerobic in AL2 than AL1 group on day 60 implied that most lactic acid bacteria strains in AL2-60 group belonged to facultatively anaerobic bacteria rather than obligate anaerobic bacteria. The higher proportions of potentially pathogenic and gram negative in AL1 group than AL2 group on day 60 could be explained by the less lactic acid accumulation and higher pH environment in AL1-60 group, promoting the growth of some pathogenic and/or gram negative bacteria. The higher proportions of gram positive in AL2 group than AL1 group on day 60 was probably because the bacterial community in AL2-60 group was mainly occupied by the lactic acid bacteria, which belonged to gram positive bacteria .