Dynamics in fermentation quality, bacterial community, and metabolic profile during silage fermentation of late-harvested elephant grass.

The present study aimed to evaluate the effects of delayed harvest and storage length on fermentation products, bacterial community, and metabolic shifts of elephant grass silage. The late-harvested elephant grass (LG) was naturally fermented (NLG) for 1, 3, 7, 15, 30, and 60 days, respectively. After 60-day ensiling, NLG displayed homolactic fermentation with low pH value, butyric acid, and ammonia nitrogen concentrations, and high lactic acid concentration, and ratio of lactic acid to acetic acid. Pseudomonas, Sphingomonas, and Pantoea dominated the bacterial community in LG, but Lactobacillus, Lactococcus, and Pediococcus were the advantageous genera in a 3-day and 60-day NLG. The correlation heatmap revealed that Acetobacter was positively related to acetic acid, ethanol, ammonia nitrogen, and butyric acid concentrations. There were distinct differences in the KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic profiles of fresh and ensiled LG. Ensiling suppressed the metabolism of amino acid, vitamins, and energy, while promoted the metabolism of carbohydrate. The LG can be well-fermented without additives, but its low crude protein content should not be ignored when applied in agricultural practice. The ensiling process remarkably affected the fermentation quality, bacterial community, and metabolic profiles of NLG.

The effects of epiphytic microbiota and chemical composition of Italian ryegrass harvested at different growth stages on silage fermentation.

BACKGROUND: The influence of epiphytic microbiota and chemical composition on fermentation quality and microbial community of Italian ryegrass silage was evaluated. Italian ryegrass harvested at the filling stage (FS) and the dough stage (DS) was sterilized by gamma-ray irradiation and inoculated as follows: (I) FS epiphytic microbiota + irradiated FS (FF); (II) FS epiphytic microbiota + irradiated DS (FD); (III) DS epiphytic microbiota + irradiated DS (DD); (IV) DS epiphytic microbiota + irradiated FS (DF). RESULTS: After 60 days of ensiling, silage made from irradiated FS had a lower pH and ammonia nitrogen (NH3 -N) content and a higher lactic acid (LA) content than that made from irradiated DS. Similarly, silage inoculated with the epiphytic microbiota of DS had a lower pH and NH3 -N content and a higher LA content than that inoculated with the epiphytic microbiota of FS. However, LA-type fermentation (lactic acid:acetic acid > 2:1) was presented at DF and DD. The principal coordinates analysis showed that the distance between FF and DF and FD and DD was closer than other treatments, suggesting that the microbial community of silages made from irradiated FS (or DS) was more similar. CONCLUSION: The epiphytic microbiota played a more important role in the fermentation type, whereas the chemical composition had a great influence on the contents of fermentation end-products. However, chemical composition had a stronger effect on the microbial community of silage than the epiphytic microbiota. © 2022 Society of Chemical Industry.

Assessing the impact of phyllosphere microbiota on dynamics of in-silo fermentation of Italian ryegrass harvested at heading and blooming stages.

BACKGROUND: The present study aimed to investigate the relationship between the phyllosphere microbiota of Italian ryegrass (Lolium multiflorum Lam.) harvested at heading (H) [> 50% earing rate or 216 g kg-1 fresh weight (FW)] and blooming (B) (> 50% bloom or 254 g kg-1 FW) stages and in-silo fermentation products, and the composition, abundance, diversity and activity of bacterial community. In total, 72 (4 treatments × 6 ensiling durations × 3 replicates) laboratory scale (400 g) silages of Italian ryegrass were prepared: (i) irradiated heading stage silages (IRH) (n = 36) were inoculated with phyllosphere microbiota inoculum (2 mL) eluted from fresh Italian ryegrass at either heading (IH) (n = 18) or blooming (IB) (n = 18) stages; (ii) irradiated blooming stage silages (IRB) (n = 36) were inoculated with either IH (n = 18) or IB (n = 18). Triplicate silos of each treatment were analyzed after 1, 3, 7, 15, 30 and 60 days of ensiling. RESULTS: In fresh forage, Enterobacter, Exiguobacterium and Pantoea were the three major genera at heading stage, and Rhizobium, Weissella and Lactococcus were the most abundant genera at blooming stage. Higher metabolic activity was found in IB. After 3 days of ensiling, the large amounts of lactic acid in IRH-IB and IRB-IB can be attributed to the higher abundances of Pediococcus and Lactobacillus, 1-phosphofructokinase, fructokinase, l-lactate dehydrogenase and glycolysis I, II and III. CONCLUSION: The composition, abundance, diversity and functionality of the phyllosphere microbiota of Italian ryegrass at different growth stages could remarkably affect silage fermentation characteristics. © 2023 Society of Chemical Industry.

The reversion of DNA methylation-induced miRNA silence via biomimetic nanoparticles-mediated gene delivery for efficient lung adenocarcinoma therapy.

BACKGROUND: Lung cancer is one of the fatal cancers worldwide, and over 60% of patients are lung adenocarcinoma (LUAD). Our clinical data demonstrated that DNA methylation of the promoter region of miR-126-3p was upregulated, which led to the decreased expression of miR-126-3p in 67 cases of lung cancer tissues, implying that miR-126-3p acted as a tumor suppressor. Transduction of miR-126-3p is a potential therapeutic strategy for treating LUAD, yet the physiological environment and properties of miRNA challenge current transduction approaches. METHODS: We evaluated the expression of miR-126-3p in 67 pairs of lung cancer tissues and the corresponding adjacent non-tumorous tissues by Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The relationship between the overall survival of lung cancer patients and miR-126-3p was analyzed by the Cancer Genome Atlas cohort database (Oncolnc, http://www.oncolnc.org ). We analyzed DNA methylation Methylation-specific PCR (MSP) analysis. To determine whether ADAM9 is the direct target of miR-126-3p, we performed the 3'-UTR luciferase reporter assay. The protein levels in the cells or tissues were evaluated with western blotting (WB) analysis. The biodistribution of nanoparticles were monitored by in vivo tracking system. RESULTS: We describe the development of novel stealth and matrix metalloproteinase 2 (MMP2)-activated biomimetic nanoparticles, which are constructed using MMP2-responsive peptides to bind the miR-126-3p (known as MAIN), and further camouflaged with red blood cell (RBC) membranes (hence named REMAIN). REMAIN was able to effectively transduce miRNA into lung cancer cells and release them via MMP2 responsiveness. Additionally, REMAIN possessed the advantages of the natural RBC membrane, including extended circulation time, lower toxicity, better biocompatibility, and immune escape. Moreover, in vitro and in vivo results demonstrated that REMAIN effectively induced apoptosis of lung cancer cells and inhibited LUAD development and progression by targeting ADAM9. CONCLUSION: The novel style of stealth and MMP2-activated biomimetic nanoparticles show great potential in miRNA delivery.

A survey of fermentation parameters, bacterial community compositions and their metabolic pathways during the ensiling of sorghum.

AIMS: To investigate the fermentative products, bacterial community compositions and their metabolic pathways of sorghum (Sorghum dochna Snowden) during ensiling. METHODS AND RESULTS: Sorghum was harvested at soft dough stage and ensiled in laboratory-scale silos (10 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through high-throughput sequencing technology, and 16S rRNA gene-predicted functional profiles were analysed according to the KEGG (Kyoto Encyclopedia of Genes and Genomes) using Tax4Fun. The sorghum silages had high lactic acid (~85.4 g kg-1 dry matter [DM]) contents, and low pH (~3.90), butyric acid (~1.27 g kg-1 DM) and ammonia nitrogen (~86.3 g kg-1 total nitrogen [TN]) contents. During the ensiling, glucose was the first fermentable substrate, and the contents of xylose and arabinose were increased. The dominant genus Lactococcus on day 3 was replaced by Lactobacillus on day 60. The metabolism of amino acid, energy, cofactors and vitamins was inhibited, whilst the metabolism of nucleotide and carbohydrate was promoted after ensiling. 1-Phosphofructokinase and pyruvate kinase of bacterial community played important roles in stimulating the lactic acid fermentation. CONCLUSIONS: Knowledge about bacterial community dynamics and their metabolic pathways during sorghum ensiling is important for understanding the fermentative profiles and may promote the production of nutritious and stable sorghum silage. SIGNIFICANCE AND IMPACT OF THE STUDY: The high-throughput sequencing technology combined with 16S rRNA gene-predicted functional analyses revealed the differences in silages not only for distinct bacterial community but also for specific functional metabolites. This could provide some new insights into bacterial community and functional profiles to further improve the silage quality.

Separating the chemical and microbial factors of oat harvested at two growth stages to determine the main factor on silage fermentation.

AIMS: This work evaluated the effects of epiphytic microbiota and chemical components on fermentation quality and microbial community of ensiled oat. METHODS AND RESULTS: Oat harvested at the heading stage (HS) and the milk stage (MS) was sterilized by gamma-ray irradiation and inoculated as the following: (1) HS epiphytic microbiota + sterilized HS (H-H); (2) MS epiphytic microbiota + sterilized HS (M-H); (3) MS epiphytic microbiota + sterilized MS (M-M); and (4) HS epiphytic microbiota + sterilized MS (H-M). After 60-day fermentation, silages inoculated with the epiphytic microbiota of HS had higher acetic acid content than those inoculated with MS. Silage made from sterilized MS had lower pH, ammonia nitrogen and butyric acid contents and higher dry matter, water-soluble carbohydrates and lactic acid contents than that made from sterilized HS. The microbial communities of oat silages were similar, and they were mainly Lactobacillus. CONCLUSIONS: The chemical component rather than the epiphytic microbiota at harvest exerted more effects on oat silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This work reveals the different effects of chemical and microbial factors on the fermentation of silage, which is instructive for us to produce quality silage.

Exploring the ensiling characteristics and bacterial community of red clover inoculated with the epiphytic bacteria from temperate gramineous grasses.

AIMS: To explore the microbiological factors that cause the difference in silage fermentation characteristics between temperate gramineous (Italian ryegrass and oat) grass and legume forage. METHODS AND RESULTS: Through γ-ray irradiation sterilization and bacterial transplantation technology, the sterilized red clover was inoculated as follows: (i) aseptic water (STRC); (ii) epiphytic bacteria on red clover (RCRC); (iii) epiphytic bacteria on oat (RCOT); and (iv) epiphytic bacteria on Italian ryegrass (RCIR). Red clover was ensiled in laboratory-scale silos made of vacuum-packed plastic bags for 1, 3, 7, 14, 30 and 60 days. STRC remained unfermented state based on similar chemical components with fresh red clover. Compared with RCRC and RCIR, higher lactic acid content and ratio of lactic acid to acetic acid, and lower pH, acetic acid and ammonia nitrogen contents were observed in RCOT after 60 days of ensiling. Using next-generation sequencing, higher abundances of Methylobacterium and Sphingomonas were observed in the epiphytic bacteria on red clover. Pediococcus was dominant in RCRC after 3 days of ensiling. Lactobacillus was the most predominant in each group after 60 days of fermentation. RCRC and RCIR had a more hetero-fermentative process, while RCOT possessed a more homo-fermentative pathway. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was accelerated by the epiphytic bacteria from red clover. CONCLUSIONS: Inoculating the epiphytic bacteria from oat could alter the fermentation characteristics and bacterial compositions of red clover silage. The relative abundance and activity of Methylobacterium, Sphingomonas, Enterobacteriaceae and hetero-fermentative Lactobacillus in red clover silage could be changed by the epiphytic bacteria on temperate gramineous grass. SIGNIFICANCE AND IMPACT OF THE STUDY: The exogenous micro-organisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.

Characterization and identification of a novel microbial consortium M2 and its effect on fermentation quality and enzymatic hydrolysis of sterile rice straw.

AIMS: To isolate and enrich lignocellulolytic microbial consortia from yak (Bos grunniens) rumen and evaluate their effects on the fermentation characteristics and enzymatic hydrolysis in rice straw silage. METHODS AND RESULTS: A novel microbial consortium M2 with high CMCase and xylanase activities was enriched and observed to be prone to use natural carbon sources. Its predominant genus was Enterococcus, and most carbohydrate-active enzyme (CAZyme) genes belonged to the glycosyl hydrolases class. The consortium M2 was introduced with or without combined lactic acid bacteria (XA) to rice straw silage for 60 days. Inoculating the consortium M2 notably decreased the structural carbohydrate contents and pH of rice straw silages. Treatment that combines consortium M2 and XA resulted in the highest levels of lactic acid and lignocellulose degradation. The consortium M2 alone or combined with XA significantly (p < 0.01) increased water-soluble carbohydrates (WSCs), mono- and disaccharides contents compared with the XA silage. Combined addition obviously improved the enzymatic conversion efficiency of rice straw silage with higher glucose and xylose yields (23.39 and 12.91 w/w% DM, respectively). CONCLUSIONS: Ensiling pretreatment with the microbial consortium M2 in sterile rice straw improved fermentation characteristics. The combined application of consortium M2 with XA had synergistic effects on promoting the degradation of structural carbohydrates and enzymatic hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Rice straw is difficult to ensile because of its low WSC and high structural carbohydrate contents. The microbial consortium M2 identified herein exhibits great potential for degrading fibrous substrates, and their combination with XA provides a faster and more effective synergistic strategy for biorefinery of lignocellulosic biomass.

Effect of epiphytic microbiota from napiergrass and Sudan grass on fermentation characteristics and bacterial community in oat silage.

AIMS: To investigate the effects of epiphytic microbiota from napiergrass and Sudan grass on ensiling characteristics and microbial community of oat silage. METHODS AND RESULTS: By γ-ray irradiation sterilization and microbiota transplantation technology, the sterilized oat was inoculated as follows: (a) aseptic water (STOT), (b) epiphytic bacteria on oat (OTOT), (c) epiphytic bacteria on napiergrass (OTNP) and (d) epiphytic bacteria on Sudan grass (OTSD). STOT remained in the unfermented state based on similar chemical components with fresh oat. Compared with OTOT and OTSD, higher lactic acid content and ratio of lactic acid to acetic acid, and lower pH, acetic acid and ammonia nitrogen contents were observed in OTNP after 60 days of ensiling. At the late stage, Lactobacillus was the most predominant in each group. Lactococcus was eventually replaced by Lactobacillus in OTSD, whereas Lactococcus was found throughout the whole ensiling process in OTNP. Higher abundance of Weissella was observed in OTSD at the early and late stages. The result of co-occurrence network analysis proved that Lactococcus was pivotal in determining the silage fermentation pattern during ensiling. According to the 16S rRNA gene-predicted functional profiles, the inoculation of epiphytic microbiota from oat enhanced the metabolism of amino acids, whereas the inoculation of epiphytic microbiota from napiergrass and Sudan grass accelerated the carbohydrate metabolism. CONCLUSIONS: The epiphytic microbiota on napiergrass promoted a homo-fermentative process, whereas the epiphytic microbiota on oat and Sudan grass facilitated a hetero-fermentative pattern in oat silage, which was closely related to the abundance and metabolism of Lactococcus, Weissella and Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: The exogenous microorganisms that promote the carbohydrate metabolism and inhibit the metabolism of amino acids could be a good potential source to improve the silage quality of temperate grass.

Dynamics of the bacterial communities and predicted functional profiles in wilted alfalfa silage.

AIMS: To investigate the fermentation characteristics, bacterial community and predicted functional profiles during the ensiling of wilted alfalfa (Medicago sativa L.). METHODS AND RESULTS: First-cutting alfalfa was harvested at the early bloom stage, wilted for 6 h, and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. The bacterial communities of wilted alfalfa and silages on day 3 and 60 were assessed through high throughput sequencing technology, and their functional characteristics were evaluated according to the Kyoto Encyclopedia of Genes and Genomes using Tax4Fun. After 60 days of ensiling, alfalfa silage showed a moderate fermentation quality, indicated by high lactic acid (56.7 g kg-1 dry matter [DM]) and acetic acid (39.4 g kg-1 DM) contents, and low concentrations of butyric acid (2.12 g kg-1 DM) and ammonia nitrogen (128 g kg-1 total nitrogen). Lactobacillus rapidly became predominant on day 3 and increased to 60.4% on day 60. Results of functional prediction analyses showed that the metabolism of amino acid, energy, cofactors and vitamins were reduced, while metabolism of nucleotide and carbohydrate were increased during ensiling. Fructokinase, 1-phosphofructokinase and pyruvate kinase played important roles in producing lactic acid. The production of acetic acid may be correlated with the enhancement of 6-phosphogluconate dehydrogenase and acetyl-CoA synthetase. CONCLUSIONS: Knowledge regarding bacterial dynamics and their metabolic pathways during alfalfa ensiling is important for understanding the fermentation process and may contribute to the production of nutritious and stable alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: High throughput sequencing technology combined with 16S rRNA gene-predicted functional analyses could provide a new and comprehensive insight into bacterial community dynamics and functional profiles to further improve the silage quality.

Using γ-ray irradiation and epiphytic microbiota inoculation to separate the effects of chemical and microbial factors on fermentation quality and bacterial community of ensiled Pennisetum giganteum.

AIMS: This study aimed to separate the effects of chemical and microbial factors on the fermentation quality and bacterial community of ensiled Pennisetum giganteum. METHODS AND RESULTS: Fresh P. giganteum of two vegetative stages (stage I, GI ; stage II, GII ) was treated as follows: GI epiphytic microbiota + Î³-ray presterilized GI (MI CI ), GII epiphytic microbiota + Î³-ray presterilized GI (MII CI ), GI epiphytic microbiota + Î³-ray presterilized GII (MI CII ), and GII epiphytic microbiota + Î³-ray presterilized GII (MII CII ). Triplicates per treatment were sampled after 30 days of ensiling for chemical and microbial analyses and high-throughput sequencing. Silages made from CII (MI CII and MII CII ) had higher lactic acid concentration and the ratio of lactic to acetic acid, and lower pH and ammonia nitrogen concentration than silages produced by CI (MI CI and MII CI ). Species differential analyses showed that the changes of chemical composition rather than epiphytic microbiota significantly affected the relative abundance of Lactobacillus, Pediococcus and Pantoea in P. giganteum silages. CONCLUSION: These above results manifested that chemical composition was the main factor influencing the fermentation quality and bacterial community of P. giganteum silage in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results may, therefore, be the first record to provide an in-depth understanding of the relative contributions of chemical and microbial parameters on fermentation quality and bacterial community, which is of great importance for modulating silage fermentation and improving silage quality.

Two novel screened microbial consortia and their application in combination with Lactobacillus plantarum for improving fermentation quality of high-moisture alfalfa.

AIMS: To enrich lignocellulolytic microbial consortia and evaluate whether a combination of these consortia and Lactobacillus plantarum can facilitate degradation of structural carbohydrates and improve fermentation quality of high-moisture alfalfa silage. METHODS AND RESULTS: Two novel microbial consortia (CL and YL) with high lignocellulolytic potential were enriched, and had higher enzyme activities at slightly acidic conditions (pH 3.5-6.5). Two consortia were inoculated with and without combined L. plantarum (LP) to alfalfa for up to 120 days of ensiling. The two consortia alone or combined with LP significantly (p < 0.05) increased lactic-to-acetic acid ratios and decreased contents of volatile organic acids and NH3 -N as compared to the control. Treatments that combining microbial consortia and LP further resulted in the higher contents of lactic acid (LA), water soluble carbohydrates (WSC) and crude protein, dry matter (DM) recovery, and lower neutral detergent fibre, acid detergent lignin and cellulose contents, with YLP silage showing the lowest pH (4.41) and highest LA content (76.72 g kg-1  DM) and the conversion of WSC into LA (184.03%). CONCLUSIONS: The addition of lignocellulolytic microbial consortia (CL or YL) to alfalfa silages as attractive silage inoculants could improve fermentation quality, and that their combination with L. plantarum appeared more effective on the degradation of structural carbohydrates and conversion of soluble carbohydrates into LA. SIGNIFICANCE AND IMPACT OF THE STUDY: High-moisture alfalfa is difficult to ensile due to its high buffering capacity and low readily fermentable carbohydrate contents. Microbial consortia (CL and YL) can encode a broad selection of multi-functional CAZymes, and their combination with LP could be promising for the degradation of structural carbohydrates simultaneously with improvement fermentation quality, with high performance in LA production.

Gamma-ray irradiation and microbiota transplantation to separate the effects of chemical and microbial diurnal variations on the fermentation characteristics and bacterial community of Napier grass silage.

BACKGROUND: To investigate the contributions of chemical and microbial diurnal variations in fermentation characteristics and bacterial community of Napier grass silage, gamma-ray irradiated Napier grass harvested at 07.00 h (AM), 12.00 h (M) and 17.00 h (PM) was inoculated with the microbiota derived from Napier grass harvested at AM, M and PM in a 3 (irradiated forage: AMG , MG and PMG ) × 3 (microbiota: AMM , MM and PMM ) design and then ensiled for 14 and 60 days. RESULTS: Napier grass harvested at various times had different chemical compositions and epiphytic microbiota prior to ensiling. For silages inoculated with the same microbiota, the pH values, residual water soluble carbohydrates and dry matter contents increased, and lactic acid, acetic acid, propionic acid, butyric acid, ethanol and volatile fatty acids contents decreased in PMG and MG silages compared to AMG silages. MM and PMM inoculum promoted lactic acid fermentation as indicated by higher lactic acid contents and lactic/acetic acid ratios in MM and PMM -inculated silages compared to those in AMM -inoculated silages after 60 days of ensiling. During ensiling, epiphytic microbiota affected the Chao1 index, operational taxonomic units (OTUs) number and Shannon index, as well as the abundances, of more than half of the top 10 abundant genera, whereas chemical composition did not affect any of the bacterial diversity and richness indices and only showed significant impacts on the abundances of two genera. CONCLUSION: The results indicated that chemical diurnal variation exerted an influence mainly on the extent of fermentation, whereas microbial diurnal variation affected more the bacterial community and fermentation types during Napier grass ensiling. © 2022 Society of Chemical Industry.

Assessment of organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration silage.

The work aimed to investigate the effects of four organic acid salts on fermentation quality, aerobic stability, and in vitro rumen digestibility of total mixed ration (TMR) silage prepared with citric acid residue, wet brewers' grains, and Napier grass. The TMR was ensiled with the following: (1) no additives (control), (2) 0.1% sodium benzoate (SB), (3) 0.1% potassium sorbate (PS), (4) 0.5% sodium diacetate (SDA), (5) 0.5% calcium propionate (CAP) on a fresh weight basis. All silos (10 L) were opened after 60 days of ensiling to determine fermentation profiles and in vitro rumen digestibility, and then were subjected to a 9-day aerobic stability test. Four organic acid salts significantly (p < 0.05) increased dry matter contents, lactic acid bacteria count, and decreased ethanol content and yeast count compared with the control. The SDA and CAP significantly (p < 0.05) increased water-soluble carbohydrates, lactic acid, and crude protein contents, and decreased pH, ammonia nitrogen, neutral detergent fiber, and hemicellulose contents compared with other TMR silages after 60 days of ensiling. Organic acid salts significantly (p < 0.05) prolonged the hours of aerobic stability and significantly (p < 0.05) increased cumulative gas production and potential gas production compared with the control. The treatments of SDA and CAP significantly (p < 0.05) improved aerobic stability as indicated by higher (p < 0.05) lactic acid and water-soluble carbohydrates contents, and lower (p < 0.05) pH, ammonia nitrogen, ethanol contents, and yeast count compared with the control. The treatments of SDA and CAP significantly (p < 0.05) increased in vitro rumen parameters, as indicated by higher (p < 0.05) in vitro digestibility of dry matter, crude protein, and neutral detergent fiber after 60 days of ensiling. Overall, these results indicated that the addition of SDA and CAP could ensure the good fermentation quality and improve aerobic stability of TMR silages. By comprehensive consideration, CAP was recommended for improving fermentation quality, aerobic stability, and in vitro rumen digestibility of TMR silages prepared with wet brewers' grains, citric acid residue, and Napier grass.

Fermentation profile and microbial diversity of temperate grass silage inoculated with epiphytic microbiota from tropical grasses.

This study was designed to evaluate the effects of epiphytic microbiota from Italian ryegrass (IRIR), Napier grass (IRNP) and Sudan grass (IRSD) on ensiling characteristics and microbial community of Italian ryegrass silage. Each treatment was prepared in triplicate and ensiled in plastic bag silos for 1, 3, 7, 14, 30 and 60 days. The γ-ray irradiation sterilization method, microbiota transplantation and next generation sequencing technology were used. Results indicated that significantly (P < 0.05) higher ratio of lactic acid to acetic acid, and lower acetic acid and ammonia nitrogen contents were observed in IRNP than IRIR and IRSD after 60 days of ensiling. Lactobacillus was the most predominant in each treatment at the late stage of fermentation. Lactococcus was eventually replaced by Lactobacillus in IRSD, whereas higher abundance of Lactococcus was continuously found in IRNP. Co-occurrence network analysis demonstrated Lactococcus was pivotal in determining the silage fermentation pattern of Italian ryegrass. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was enhanced by the epiphytic microbiota from Italian ryegrass and Sudan grass, while the carbohydrate metabolism was accelerated by the epiphytic microbiota from Napier grass. Overall, IRNP had a homo-fermentative process, whereas IRIR and IRSD possessed a hetero-fermentative pattern. The Lactococcus and heterofermentative Lactobacillus were mainly responsible for this. It also confirmed that the exogenous microorganisms that promote the carbohydrate metabolism and inhibit the metabolism of amino acids could be a good potential source to improve the silage quality of temperate grass.

Effect of storage time on the fermentation quality, bacterial community structure and metabolic profiles of napiergrass (Pennisetum purpureum Schum.) silage.

This study was aimed to investigate the effect of storage time on fermentation characteristics, bacterial community structure and predicted metabolic pathways of napiergrass (Pennisetum purpureum Schum.) silage. First-cutting napiergrass was harvested at the vegetative stage and ensiled in laboratory-scale silos (1 L capacity). Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. The bacterial communities on day 3 and 60 were assessed through the high throughput sequencing technology, and metabolic pathways of bacterial community were predicted according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) via Tax4Fun. Napiergrass silage exhibited an acetic acid-type fermentation, indicated by lower lactic acid contents and ratio of lactic acid to acetic acid, and higher pH, ethanol and acetic acid contents. Before ensiling, the predominant genera in fresh napiergrass mainly included Acinetobacter, Enterobacteriaceae, Enterobacter and Lactococcus. After 60 days of ensiling, high proportions of Enterobacteriaceae, Enterobacter and Lactobacillus were found in napiergrass silages. The metabolism of amino acid, energy, cofactors and vitamins were inhibited, whereas metabolism of nucleotide and carbohydrate were promoted during ensiling. Overall, the combination of high throughput sequencing technology and 16S rRNA gene-predicted functional analyses revealed the differences during the initial and late stages of napiergrass silages not only for distinct bacterial community but also for specific functional metabolites. It could provide a comprehensive insight into bacterial community and functional profiles to further improve the silage quality.

Using a high-throughput sequencing technology to evaluate the various forage source epiphytic microbiota and their effect on fermentation quality and microbial diversity of Napier grass.

Napier grass (Pennisetum purpureum) is well-known due to its high biomass production. The epiphytic microbiota was prepared from Napier, alfalfa, and red clover grass and served as an inoculum. The chopped sterilized Napier grass was inoculated with reconstituted epiphytic microbiota, and treatments were designed as: distilled water (N0); Napier grass epiphytic microbiota (NP); alfalfa epiphytic microbiota (AL); and red clover epiphytic microbiota (RC). The results reveal that the reconstituted epiphytic microbiota bacteria efficiently adapted in Napier grass silage, improved fermentation, and produced lactic acid. The alfalfa-grass inoculum rapidly dropped pH and enhanced the lactic acid (LA) and the ratio of lactic acid-to-acetic acid (LA/AA) during the entire ensiling process. However, red clover attains high lactic acid, while Napier grass produces high acetic acid-type fermentation at terminal silage. After day 60 of ensiling, Lactobacillus proportion was higher in AL (85.45%), and RC (59.44%), inocula as compared with NP (36.41%), inoculum. The NP inoculum terminal silage was diverse than AL and RC inocula and dominated by Enterobacter (16.32%) and Enterobacteriaceae (10.16%) and also significantly (p < 0.05) higher in acetic acid. The present study concluded that AL and RC epiphytic microbiota successfully develop and more efficient than Napier grass microbiota. It is suggested that abundant microbiota isolate from alfala and red clover and develop more economical and efficient inocula for quality fermentation of Napier grass silage in practice.

Partial substitution of whole-crop corn with bamboo shoot shell improves aerobic stability of total mixed ration silage without affecting in vitro digestibility.

To evaluate the feasibility of bamboo shoot shell (BSS) application in total mixed ration silage (TMR) production, the effects of BSS substitution for whole-crop corn on the fermentation characteristics, nutritive value, aerobic stability and in vitro parameters of TMR silage were studied. Four TMR formulations were designed based on dry matter: (1) 0% bamboo shoot shell + 23% whole-crop corn (BSS0 ); (2) 4% bamboo shoot shell + 19% whole-crop corn (BSS4 ); (3) 8% bamboo shoot shell + 15% whole-crop corn (BSS8 ); and (4) 12% bamboo shoot shell + 11% whole-crop corn (BSS12 ). After 90 days of ensiling, the silos were opened and sampled for fermentation characteristics, nutritive value and in vitro analyses, and subsequent 14-day aerobic stability test. All TMR silages were well preserved except BSS12 , characterized by high lactic acid content and V-score, low pH and NH3 -N and butyric acid content. With increasing proportion of BSS, crude protein increased (p <.05), and water soluble carbohydrate decreased (p < .05). Under aerobic exposure, BSS-substituted (BSS4 , BSS8 and BSS12 ) silages were more stable than BSS0 silage, as characterized by relatively low silage temperature and high water soluble carbohydrate content. No obvious (p> .05) difference of BSS substitution was observed on in vitro ruminal gas production, digestibility, metabolizable energy and net energy for lactation. The substitution of whole-crop corn with 4% BSS and 8% BSS had no undesired effect on the fermentation characteristics and in vitro digestibility, and efficiently improving the aerobic stability of TMR silages. The BSS8 substitution level is recommended to maximize the BSS utilization.

Silage fermentation characteristics and microbial diversity of alfalfa (Medicago sativa L.) in response to exogenous microbiota from temperate grasses.

The objective of this study was to explore the microbiological factors that cause the difference in silage fermentation characteristics between grass and legume. Specifically, the effects of epiphytic microbiota from alfalfa, oat and Italian ryegrass on ensiling characteristics and microbial community of alfalfa were assessed. By γ-ray irradiation sterilization and microbiota transplantation technology, the sterile alfalfa was inoculated as follows: (i) aseptic water (STAL); (ii) epiphytic bacteria from alfalfa (ALAL); (iii) epiphytic bacteria from oat (ALOT); (iv) epiphytic bacteria from Italian ryegrass (ALIR). Alfalfa at the initial flowering stage was ensiled in laboratory-scale silos for 1, 3, 7, 14, 30 and 60 days. Compared with ALAL and ALIR, higher lactic acid contents and ratio of lactic acid to acetic acid, and lower acetic acid, propionic acid, ethanol and ammonia nitrogen contents were observed in ALOT after 60 days of fermentation. In each treated group, Lactobacillus was the most dominant genus after 60 days of ensiling. Relatively higher abundance of Weissella, Hafnia-Obesumbacterium, Enterobacteriaceae or hetero-fermentative Lactobacillus was found in ALAL and ALIR after 60 days. Co-occurrence network analysis proved Pediococcus and Lactococcus were pivotal in deciding the fermentation pattern of alfalfa silage. According to the 16S rRNA gene-predicted functional profiles, the metabolism of amino acids was inhibited by the epiphytic microbiota from oat. Overall, ALOT showed a homo-fermentative process, whereas ALAL and ALIR exhibited a hetero-fermentative pattern. Furthermore, the exogenous microorganisms inhibiting the metabolism of amino acids can be a good potential source to improve the silage quality of legume forage.

Abundance and diversity of epiphytic microbiota on forage crops and their fermentation characteristic during the ensiling of sterile sudan grass.

This study aimed to evaluate the effects of exogenous epiphytic microbiota inoculation on the fermentation quality and microbial community of sudan grass silage. Gamma irradiated sudan grass was ensiled with distilled water (STR), epiphytic microbiota of sudan grass (SUDm), forage sorghum (FSm), napier grass (NAPm) and whole crop corn (WCCm). The FSm inoculated silage have significantly lower lactic acid (LA) concentration and higher pH during early ensiling, while LA concentration gradually and significantly increased with the progression of ensiling and have lower pH in relation to other treatments for terminal silage. Inoculation of NAPm resulted in lower LA and higher acetic acid (AA) concentrations, higher pH, ammonia-N and dry matter losses for terminal silage, followed by SUDm silage. Inoculations of WCCm significantly increased LA production and pH decline during early ensiling and have higher LA and pH then NAPm and SUDm silages during final ensiling. The early fermentation of SUDm silage was dominated by genus of Pediococcus. The genera of Lactobacillus were predominant in WCCm and NAPm silages during 3 days of ensiling, while Weissella dominated initial microbial community of FS silage. The terminal silage of NAPm was dominated by Enterobacter and Rosenbergiella, while Enterobacter and Lactobacillus dominated terminal SUDm silage. The final silage of FSm was dominated by Lactobacillus, Weissella and Pediococcus, while Lactobacillus and Acetobacter dominated terminal WCCm silages. The results demonstrated that among the four forages the epiphytic microbiota from forage sorghum positively influenced the microbial community and fermentability of sudan grass silage.