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Alfalfa ( Medicago sativa L.) is one of the most extensively cultivated forage crops globally, and its nutritional quality critically influences the productivity of dairy cows. Silage fermentation is recognized as a crucial technique for the preservation of fresh forage, ensuring the retention of its vital nutrients. However, the detailed microbial components and their functions in silage fermentation are not fully understood. This study integrated large-scale microbial culturing with high-throughput sequencing to thoroughly examine the microbial community structure in alfalfa silage and explored the potential pathways of nutritional degradation via metagenomic analysis. The findings revealed an enriched microbial diversity in silage, indicated by the identification of amplicon sequence variants. Significantly, the large-scale culturing approach recovered a considerable number of unique microbes undetectable by high-throughput sequencing. Predominant genera, such as Lactiplantibacillus , Leuconostoc , Lentilactobacillus , Weissella , and Liquorilactobacillus , were identified based on their abundance and prevalence. Additionally, genes associated with Enterobacteriaceae were discovered, which might be involved in pathways leading to the production of ammonia-N and butyric acid. Overall, this study offers a comprehensive insight into the microbial ecology of silage fermentation and provides valuable information for leveraging microbial consortia to enhance fermentation quality., Importance: Silage fermentation is a microbial-driven anaerobic process that efficiently converts various substrates into nutrients readily absorbable and metabolizable by ruminant animals. This study, integrating culturomics and metagenomics, has successfully identified core microorganisms involved in silage fermentation, including those at low abundance. This discovery is crucial for the targeted cultivation of specific microorganisms to optimize fermentation processes. Furthermore, our research has uncovered signature microorganisms that play pivotal roles in nutrient metabolism, significantly advancing our understanding of the intricate relationships between microbial communities and nutrient degradation during silage fermentation., Competing Interests: The authors declare no conflict of interest.

The study of antibiotic resistance in the silage microbiome has attracted initial attention. However, the influences of lactic acid bacteria inoculants and dry matter (DM) content on antibiotic resistance genes (ARGs) reduction in whole-plant corn silage remain poorly studied. This study accessed the ARGs' risk and transmission mechanism in whole-plant corn silage with different DM levels and treated with Lactiplantibacillus plantarum or Lentilactobacillus buchneri. The macrolide and tetracycline were the main ARGs in corn silage. The dominant species (Lent. buchneri and Lactobacillus acetotolerans) were the main ARGs carriers in whole-plant corn silage. The application of Lent. buchneri increased total ARGs abundance regardless of corn DM. Whole-plant corn silage with 30 % DM reduced the abundances of integrase and plasmid compared with 40 % DM. The correlation and structural equation model analysis demonstrated that bacterial community succession, resulting from changes in DM content, was the primary driving factor influencing the ARGs distribution in whole-plant corn silage. Interestingly, whole-plant corn silage inoculated with Lent. buchneri reduced abundances of high-risk ARGs (mdtG, mepA, tetM, mecA, vatE and tetW) by regulating pathogens (Escherichia coli), mobile genetic elements (MGEs) genes (IS3 and IS1182), and this effect was more pronounced at 30 % DM level. In summary, although whole-plant corn silage inoculated with Lent. buchneri increased the total ARGs abundance at both DM levels, it decreased the abundance of high-risk ARGs by reducing the abundances of the pathogens and MGEs, and this effect was more noticeable at 30 % DM level., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing for financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The physical attributes of corn silage are enhanced by shredlage (SHR), while there is a rising interest in boosting its biological performance. This study aimed to assess and compare the impact of both the chopping method and different yeast strains on ensilage quality including the in vitro evaluation of corn silage. Both types of corn, including chopped and shredded, were harvested on the same day from the same field where the same corn hybrid (Suwan 5) was grown. Subsequently, whole-corn plants were fermented with additives. A 2 × 5 Factorial completely randomized design was employed, where factor A represents corn chopped (CON) and corn shredded (SHR), and factor B represents the additives: no additives, molasses + urea (M + U), M + U + Candida tropicalis KKU20, M + U + Pichia kudriavzevii KKU20, and M + U + saccharomyces cerevisiae. The results demonstrated that SHR fermentation with M + U and yeast significantly increased in vitro dry matter degradability (IVDMD) and organic matter degradability (IVOMD). Specifically, at 4 h post-incubation, the addition of Crabtree-negative yeast led to a 5.8% increase in total volatile fatty acids (TVFA) compared to the Crabtree-positive yeast group (P < 0.01). The C2 (acetic acid) + C4 (normal butyric acid and isobutyric acid): C3 (propionic acid) ratio showed a significant decrease without additives, but P. kudriavzevii KKU20 led to the highest ratio and methane production (P < 0.01). Based on this study, it could be concluded that SHR harvesting led to higher digestion efficiency in the rumen. The use of M + U + yeast also demonstrated uncertain effects on rumen fermentation efficiency, and the inclusion of P. kudriazevii KKU20 may potentially reduce rumen fermentation efficiency when used with corn silage., (© 2024. The Author(s).)

Factors contributing to variations in the quality and microbiota of ensiled forages and in bulk tank microbiota in milk from cows fed different forages were investigated. Nutritional quality, fermentation parameters and hygiene quality of forage samples and corresponding bulk tank milk samples collected in 3 periods from 18 commercial farms located in northern Sweden were compared. Principal coordinates analysis revealed that the microbiota in forage and bulk milk, analyzed using 16S rRNA gene-based amplicon sequencing, were significantly different. The genera Lactobacillus, Weissella, and Leuconostoc dominated in forage samples, whereas Pseudomonas, Staphylococcus, and Streptococcus dominated in bulk milk samples. Forage quality and forage-associated microbiota were affected by ensiling method and by use of silage additive. Forages stored in bunker and tower silos (confounded with use of additive) were associated with higher levels of acetic and lactic acid and Lactobacillus. Forage ensiled as bales (confounded with no use of additive) was associated with higher DM content, water-soluble carbohydrate content, pH, yeast count, and the genera Weissella, Leuconostoc, and Enterococcus. For bulk tank milk samples, milking system was identified as the major factor affecting the microbiota and type of forage preservation had little effect. Analysis of common amplicon sequence variants (ASV) suggested that forage was not the major source of Lactobacillus found in bulk tank milk., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In this study, the effects on silage performance and microbial community of sorghum straw treated with the addition of enzymes (cellulase (CE), xylanase (XE)) and extrusion puffing technology, combined with SEM, XRD, and FTIR techniques, were thoroughly investigated. The results showed that the enzyme preparations, especially xylanase, significantly improved the nutritional value and fermentation efficiency of straw and enhanced the silage effect. Extruding significantly changes the surface structure of the straw, increasing the surface area and porosity, and promoting the attachment of microorganisms. This study not only optimized the sorghum straw silage performance but also provided technical support for the efficient use of straw resources, which is of great significance for the sustainable development of animal husbandry and the resource utilization of agricultural waste., (© 2024. The Author(s).)

Background: Sweet sorghum is used mainly as an energy crop and feed crop in arid and semiarid regions, and ensiling is a satisfactory method for preserving high-quality sweet sorghum. The aim of this study was to reveal the dynamics of the fermentation quality, bacterial communities, and fermentation weight loss (FWL) of sweet sorghum silage during fermentation., Methods: Sweet sorghum was harvested at the first inflorescence spikelet stage and ensiled without (CK) or with lactic acid bacterial (LAB) additives (L). After ensiling, samples were collected on days 0, 1, 3, 5, 15, 40, and 100 to assess the fermentation quality, bacterial communities, and FWL., Results: For CK and L, on day 1, the pH was 5.77 and 5.57, respectively, and the lactic acid (LA) was 1.30 and 2.81 g/kg dry matter (DM), respectively. Compared with CK, L had a lower pH and higher LA from days 1 to 5 (P < 0.05), a lower FWL from days 5 to 100 (P < 0.05), and a greater abundance of Lactiplantibacillus from days 1 to 15 (P < 0.05). The main bacterial genera were Leuconostoc and Weissella in CK and Lactiplantibacillus, Leuconostoc, and Weissella in L on day 1; Lactiplantibacillus in all silages from days 3 to 40; and Lactiplantibacillus and Lentilactobacillus in all silages on day 100., Conclusions: Sweet sorghum silage fermented relatively slowly during the first day. Moreover, inoculation with LAB accelerated fermentation and optimized bacterial communities during the initial fermentation phase. Inoculation with LAB also reduced the silage FWL, and the LAB succession relay occurred in the silage throughout the fermentation process., (© 2024. The Author(s).)

High-throughput metagenomic sequence technology was employed to evaluate changes in microbial community composition and carbohydrate-active enzymes encoding gene enrichment status in Elymus nutans silages to altitudinal gradients in the world's highest alpine region of Qinghai-Tibetan Plateau (QTP). E. nutans were collected from three different altitudes in QTP: 2,600 m (low altitude), 3600 m (moderate altitude), and 4,600 m [high (H) altitude], and ensiled for 7, 14, 30, and 60 d. Results indicated an improvement in silage quality with the increasing altitude, although the acetic acid concentration and dry matter loss were greater in H altitude silages after 30 d of ensiling. Harmful bacteria or potential pathogens predominated in the microbial community on d 7 and 14 of fermentation, while genera belonging to lactic acid bacteria gradually became the main microorganisms with the increasing altitude on d 30 and 60 of ensiling. The abundance of carbohydrate-active enzymes genes responsible for macromolecular carbohydrate degradation in silage increased with increasing altitude, and those genes were mainly carried by Lactiplantibacillus and Pediococcus at 30 and 60 d of ensiling. The abundance of key enzymatic genes associated with glycolysis and organic acid production in carbohydrate metabolism pathway was higher in H altitude silages, and Lactiplantibacillus and Pediococcus were also the main hosts after 30 d of silage fermentation, except for the fact that acetic acid production was also related to genera Leuconostoc , Latilactobacillus , and Levilactobacillus ., Importance: The fermentation quality of Elymus nutans silage was getting better with the increase of altitude in the Qinghai-Tibetan Plateau. The abundance of hosts carrying carbohydrate-active enzymes genes and key enzyme genes related to organic acid production increased with increasing altitude during the later stages of fermentation. Lactiplantibacillus and Pediococcus were the core microorganisms responsible for both polysaccharide hydrolysis and silage fermentation in the late stage of ensiling. This study provided insights on the influence of different altitudes on the composition and function of silage microbiome in the Qinghai-Tibetan Plateau, and provided a reference approach for improving the quality and controllability of silage production in high altitude areas of the Qinghai-Tibetan Plateau., Competing Interests: The authors declare no conflict of interest.

In this experiment, alfalfa silage with different packing densities (500 kg/m 3 、600 kg/m 3 and 700 kg/m 3 ) was prepared under the conditions of outdoor high temperature and indoor room temperature, respectively. At the same time, the same lactobacillus additive was used for fermentation in each density treatment group. The chemical composition, fermentation quality and microbial community of alfalfa silage were analyzed. The results showed that the contents of dry matter (DM) and water-soluble carbohydrate (WSC) decreased with the increase of density during fermentation at high temperature. At the same time, when the density is 600 kg/m³, CP (crude protein) content is the highest, ADF (acid detergent fiber) content is the lowest. The contents and pH values of neutral detergent fiber (NDF), lactic acid (LA) and lactic acid bacteria (LAB) were significantly affected by temperature (p < 0.05). Density had significant effects on DM, NDF, WSC and LA contents (p < 0.05). The interaction between temperature and density had significant effects on the content of ADF and LAB (p < 0.05). At the same time, the abundance of Lactiplantibacillus plantarum in high temperature fermented silage was lower than that in normal temperature fermented feed. The number of Lactiplantibacillus plantarum in room temperature treatment group decreased with the increase of density. In summary, this study clarified the effects of different temperature and density on alfalfa fermentation quality and microbial community, and clarified that the density should be reasonably controlled within 600 kg/m³ during alfalfa silage, providing theoretical support for production practice., (© 2024. The Author(s).)

Hybrid Pennisetum, a top biomass energy source, faces usage limitations because of its scarce lactic acid bacteria and high fiber content. This study assessed the influence of rumen fluid pretreatment on hybrid Pennisetum's silage, with focus on silage duration and rumen fluid effects on quality and fiber decomposition. Advanced third-generation sequencing was used to track microbial diversity changes and revealed that rumen fluid considerably enhanced dry matter, crude protein, and water-soluble carbohydrates, thus improving fermentation quality to satisfactory pH levels (3.40-3.67). Ideal results, including the highest fiber breakdown and enzymatic efficiency (47.23 %), were obtained with 5 % rumen fluid in 60 days. The addition of rumen fluid changed the dominant species, including Paucilactobacillus vaccinostercus (0.00 % vs. 18.21 %) and Lactiplantibacillus plantarum (21.03 % vs. 47.02 %), and no Enterobacter was detected in the high-concentration treatments. Moreover, strong correlations were found between specific lactic acid bacteria and fermentation indicators, revealing the potential of achieving efficient and economically beneficial hybrid Pennisetum production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ruminal microbes catabolise feed carbohydrates mainly into SCFA, methane (CH 4 ), and carbon dioxide (CO 2 ), with predictable relationships between fermentation end products and net microbial increase. We used a closed in vitro batch culture system, incubating grass and maize silages, and measured total gas production at 8 and 24 h, as well as the truly degraded substrate, the net production of SCFA, CH 4 , and microbial biomass at 24 h, and investigated the impact of silage type and inoculum microbial mass on fermentation direction. Net microbial yield was negatively correlated with total gas at 8 h (P < 0•001), but not at 24 h (P = 0•052), and negatively correlated with CH 4 production (P < 0•001). Higher initial inoculum microbial mass was related to a lower net microbial yield (P < 0•001) but a higher CH 4 production (P < 0•001). A significant difference between grass silage and maize silage was detected within the context of these relationships (P < 0•050). The metabolic hydrogen (2H) recovery was 102.8 ± 12.3 % for grass silages and 118.8 ± 13.3% for maize silages. Overall, grass silages favoured more substrate conversion to microbial biomass and less to fermentation end products than maize silage. Lower inoculum microbial mass facilitated more microbial growth and, because of the 2H sink by microbial synthesis, decreased CH 4 production., Competing Interests: The authors declare no conflict of interest., (© Published by Cambridge University Press on behalf of The Nutrition Society 2024.)

This study investigated the influence of different temperatures (35℃ High temperature and average indoor ambient temperature of 25℃) and lactic acid bacterial additives (Lactiplantibacillus plantarym, Lentilactobacillus buchneri, or a combination of Lactiplantibacillus plantarym and Lentilactobacillus buchneri) on the chemical composition, fermentation quality, and microbial community of alfalfa silage feed. After a 60-day ensiling period, a significant interaction between temperature and additives was observed, affecting the dry matter (DM), crude protein (CP), acid detergent fiber (ADF), and neutral detergent fiber (NDF) of the silage feed (p < 0.05). Temperature had a highly significant impact on the pH value of the silage feed (p < 0.0001). However, the effect of temperature on lactic acid, acetic acid, propionic acid, and butyric acid was not significant (p > 0.05), while the inoculation of additives had a significant effect on lactic acid, acetic acid, and butyric acid (p > 0.05). As for the dynamic changes of microbial community after silage, the addition of three kinds of bacteria increased the abundance of lactobacillus. Among all treatment groups, the treatment group using complex bacteria had the best fermentation effect, indicating that the effect of complex lactic acid bacteria was better than that of single bacteria in high temperature fermentation. In summary, this study explained the effects of different temperatures and lactic acid bacterial additives on alfalfa fermentation quality and microbial community, and improved our understanding of the mechanism of alfalfa related silage at high temperatures., (© 2024. The Author(s).)

This study was performed to evaluate the effects of rye silage treated with sodium formate (Na-Fa) and lactic acid bacteria (LAB) inoculants on the ruminal fermentation characteristics, methane yield and energy balance in Hanwoo steers. Forage rye was harvested in May 2019 and ensiled without additives (control) or with either a LAB inoculant or Na-Fa. The LAB ( Lactobacillus plantarum ) were inoculated at 1.5 × 10 10 CFU/g fresh matter, and the inoculant was sprayed onto the forage rye during wrapping at a rate of 4 L/ton of fresh rye forage. Sixteen percent of the Na-Fa solution was sprayed at a rate of approximately 6.6 L/ton. Hanwoo steers (body weight 275 ± 8.4 kg ( n = 3, group 1); average body weight 360 ± 32.1 kg ( n = 3, group 2)) were allocated into two pens equipped with individual feeding gates and used in duplicated 3 × 3 Latin square design. The experimental diet was fed twice daily (09:00 and 18:00) during the experimental period. Each period comprised 10 days for adaptation to the pen and 9 days for measurements in a direct respiratory chamber. The body weights of the steers were measured at the beginning and at the end of the experiment. Feces and urine were collected for 5 days after 1 day of adaptation to the chamber, methane production was measured for 2 days, and ruminal fluid was collected on the final day. In the LAB group, the ratio of acetic acid in the rumen fluid was significantly lower ( p = 0.044) and the ratio of propionic acid in the rumen fluid was significantly higher ( p = 0.017). Methane production per DDMI of the Na-FA treatment group was lower than that of the other groups ( p = 0.052), and methane production per DNDFI of the LAB treatment group was higher than that of the other groups ( p = 0.056). The use of an acid-based additive in silage production has a positive effect on net energy and has the potential to reduce enteric methane emissions in ruminants., Competing Interests: Jayeon Kim is employed by Cargill Agri Purina Inc. and Geumhwi Bang is employed by Farmsco Co., Ltd. The authors declare that they have no competing interests except to Jayeon Kim and Geumhwi Bang., (© 2024 Choi et al.)

Silage has been identified as a source of different microbial toxins, that may impair farm animal health and productivity as human health can also be compromised. In this sense, the aim of this study was to determine the impact of silage additives on the concentrations of deoxynivalenol (DON) and zearalenone (ZEN) mycotoxins and, eventually, to evaluate the hygienic quality of orchardgrass (Dactylis glomerata L.) silage based on the concentration of them compared to control silage. This study evaluated the influence of biological and chemical additives used in six different varieties of orchardgrass silage on DON and ZEN mycotoxin contents for the first time. The content of both fusariotoxins (DON and ZEN) in fresh matter and grass silage were below the threshold stipulated by the European Commission. The concentration of DON ranges from ~21.86 to 37.26 ng/kg, ~10.21 to 15 ng/kg, ~20.72 to 29.14 ng/kg; and ZEN range from ~3.42 to 7.87 ng/kg, ~3.85 to 8.62 ng/kg and ~2.15 to 5.08 ng/kg, in control, biological and chemical silages, respectively. In general, the biological additive was more efficient for preventing DON contamination, whereas the chemical additive was more efficient for preventing ZEN contamination in grass silage. In summary, the results obtained in this work demonstrate that biological and chemical additives can inhibit fungal growth and mycotoxin production on Dactylis glomerata L. silage and whose use could prevent animal and human diseases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alba-Mejía et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Feeding ruminants with high-quality forage can enhance digestibility and reduce methane production. Development of high-quality silage from leguminous plants with lactic acid bacteria can improve digestibility and it mitigate the greenhouse gas emissions. In this study, we developed a high-quality alfalfa silage with improved fermentation index and microbial dynamics using Levilactobacillus brevis-KCC-44 at low or high moisture (LM/HM) conditions and preserved it for 75 or 150 days. Alfalfa fermentation with L. brevis enhances acidification and fermentation characteristics primarily due to the dominance of lactic acid bacteria (LAB) L. brevis (>95%) compared to alfalfa fermented with epiphytic LAB. The inoculant L. brevis improved the anaerobic fermentation indexes resulting in a higher level of lactic acid in both high (10.0 ± 0.12 & 8.90 ± 0.31%DM) and low moisture (0.55 ± 0.08 & 0.39 ± 0.0 %DM) in 75 and 150 days respectively, compared to control silage. In addition, the marginal amount of acetic acid (range from 0.23 ± 0.07 to 2.04 ± 0.27 %DM) and a reduced level of butyric acid (range between 0.03 ± 0.0 to 0.13 ± 02 %DM) was noted in silage treated with LAB than the control. The LAB count and abundance of Levilactobacillus were higher in alfalfa silage fermented with L. brevis. Microbial richness and diversity were reduced in alfalfa silage treated with L. brevis which prompted lactic acid production at a higher level even for a prolonged period of time. Therefore, this L.brevis is an effective inoculant for producing high-quality alfalfa silage since it improves fermentation indexes and provides reproducible ensiling properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rice anaerobic fermentation is a significant source of greenhouse gas (GHG) emissions, and in order to efficiently utilize crop residue resources to reduce GHG emissions, rice straw anaerobic fermentation was regulated using lactic acid bacteria (LAB) inoculants (FG1 and TH14), grass medium (GM) to culture LAB, and Acremonim cellulolyticus (AC). Microbial community, GHG emission, dry matter (DM) loss, and anaerobic fermentation were analyzed using PacBio single-molecule real-time and anaerobic fermentation system. The epiphytic microbial diversity of fresh rice straw was extremely rich and contained certain nutrients and minerals. During ensiling, large amounts of GHG such as carbon dioxide are produced due to plant respiration, enzymatic hydrolysis reactions, and proliferation of aerobic bacteria, resulting in energy and DM loss. Addition of FG1, TH14, and AC alone improved anaerobic fermentation by decreasing pH and ammonia nitrogen content ( P < 0.05) and increased lactic acid content ( P < 0.05) when compared to the control, and GM showed the same additive effect as LAB inoculants. Microbial additives formed a co-occurrence microbial network system dominated by LAB, enhanced the biosynthesis of secondary metabolites, diversified the microbial metabolic environment and carbohydrate metabolic pathways, weakened the amino acid metabolic pathways, and made the anaerobic fermentation cleaner. This study is of great significance for the effective utilization of crop straw resources, the promotion of sustainable livestock production, and the reduction of GHG emissions.IMPORTANCETo effectively utilize crop by-product resources, we applied microbial additives to silage fermentation of fresh rice straw. Fresh rice straw is extremely rich in microbial diversity, which was significantly reduced after silage fermentation, and its nutrients were well preserved. Silage fermentation was improved by microbial additives, where the combination of cellulase and lactic acid bacteria acted as enzyme-bacteria synergists to promote lactic acid fermentation and inhibit the proliferation of harmful bacteria, such as protein degradation and gas production, thereby reducing GHG emissions and DM losses. The microbial additives accelerated the formation of a symbiotic microbial network system dominated by lactic acid bacteria, which regulated silage fermentation and improved microbial metabolic pathways for carbohydrates and amino acids, as well as biosynthesis of secondary metabolites., Competing Interests: The authors declare no conflict of interest.

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058 T , SCR0080, LD0937 T and SCR0063 T , were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058 T and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058 T and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178 T , together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178 T , revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058 T and SCR0080 represent a new subspecies of L. zeae . The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058 T (=DSM 116376 T =NCIMB 15474 T ). According to the results of 16S rRNA gene sequencing, LD0937 T and SCR0063 T are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937 T and SCR0063 T was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 11542 5 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011 T (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937 T (=DSM 116105 T =NCIMB 15471 T ) and SCR0063 T (=DSM 116297 T =NCIMB 15473 T ), respectively.

The microbial hosts of antibiotic resistance genes (ARGs) found epiphytically on plant materials could grow and flourish during silage fermentation. This study employed metagenomic analysis and elucidated the occurrence and transmission mechanisms of ARGs and their microbial hosts in whole-crop corn silage inoculated with homofermentative strain Lactiplantibacillus plantarum or heterofermentative strain Lentilactobacillus buchneri ensiled under different temperature (20 and 30 °C). The results revealed that the corn silage was dominated by Lactobacillus, Leuconostoc, Lentilactobacillus, and Latilactobacillus. Both the ensiling temperature and inoculation had greatly modified the silage microbiota. However, regardless of the ensiling temperature, L. buchneri had significantly higher ARGs, while it only exhibited significantly higher mobile genetic elements (MGEs) in low temperature treatments. The microbial community of the corn silage hosted highly diverse form of ARGs, which were primarily MacB, RanA, bcrA, msbA, TetA (58), and TetT and mainly corresponded to macrolides and tetracyclines drug classes. Plasmids were identified as the most abundant MGEs with significant correlation with some high-risk ARGs (tetM, TolC, mdtH, and NorA), and their abundances have been reduced by ensiling process. Furthermore, higher temperature and L. buchneri reduced abundances of high-risk ARGs by modifying their hosts and reduced their transmission in the silage. Therefore, ensiling, L. buchneri inoculation and higher storage temperature could improve the biosafety of corn silage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study investigates the effectiveness of an exopolysaccharide (EPS)-producing strain (Lactiplantibacillus plantarum L75) alone or in combination with Saccharomyces cerevisiae on the fermentation characteristics, antioxidant capacities and microbial community successions of oat silage stored at various temperatures. A rapid decrease in pH and lactic acid accumulation was observed in silages treated with L. plantarum and S. cerevisiae (LS) as early as 3 days of ensiling (p < 0.05). Over the ensiling period of 7-60 days, L. plantarum (L)-inoculated groups showed the lowest pH, lowest ammonia nitrogen and the highest amount of lactic acid regardless of the storage temperatures. When the oat silage was stored at 15°C, LS-inoculated group exhibited a higher superoxide dismutase (SOD) activity than control and L-inoculated group. Furthermore, the proportion of Lactiplantibacillus in the combined inoculation group increased by 65.42% compared to the L-inoculated group (33.26%). Fungal community data revealed abundant Penicillium carneum in the control and L-inoculated groups stored at 15°C. Conclusively, these results showed that combined inoculation of L. plantarum L75 and S. cerevisiae improved the fermentation quality of oat silage at 15°C, thus proposing a technique for enhancing the fermentation quality of silage in regions with low temperatures during harvest season., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

Sweet sorghum, as a seasonal energy crop, is rich in cellulose and hemicellulose that can be converted into biofuels. This work aims at investigating the effects of synergistic regulation of Pichia anomala and cellulase on ensiling quality and microbial community of sweet sorghum silages as a storage and pretreatment method. Furthermore, the combined pretreatment effects of ensiling and ball milling on sweet sorghum were evaluated by microstructure change and enzymatic hydrolysis. Based on membership function analysis, the combination of P. anomala and cellulase (PA + CE) significantly improved the silage quality by preserving organic components and promoting fermentation characteristics. The bioaugmented ensiling with PA + CE restructured the bacterial community by facilitating Lactobacillus and inhibiting undesired microorganisms by killer activity of P. anomala. The combined bioaugmented ensiling pretreatment with ball milling significantly increased the enzymatic hydrolysis efficiency (EHE) to 71%, accompanied by the increased specific surface area and decreased pore size/crystallinity of sweet sorghum. Moreover, the EHE after combined pretreatment was increased by 1.37 times compared with raw material. Hence, the combined pretreatment was demonstrated as a novel strategy to effectively enhance enzymatic hydrolysis of sweet sorghum., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The successful ensiling of lucerne (Medicago sativa L.) depends on a rapid acidification in the silo and consequently relies on a sufficient proliferation of, particularly homofermentative, lactic acid bacteria. Similarly, growth of spoilage bacteria, such as enterobacteria and clostridia, must be suppressed and silage additives are therefore frequently applied to promote favourable conditions during ensiling. Three silage additives or soil were applied during lucerne ensiling and investigated for their effects on silage quality characteristics and abundances of total bacteria as well as the bacterial key players Lactobacillus spp., homofermentative Lact. plantarum, heterofermentative Lact. buchneri, Clostridium spp. and Enterobacteriaceae after 30 days of storage. Inoculation with viable Lact. plantarum resulted in highest concentration of this species and excellent silage quality, i.e. high lactic acid concentration coupled with low acetic acid and ammonia-nitrogen concentrations. A sodium nitrite and hexamine-based additive did not support growth of lactic acid bacteria, which was also apparent by higher pH and low lactic acid concentration. No effect of treatments was found on spoilage-related enterobacteria and clostridia, even not when adding soil to lucerne to increase initial clostridial contamination. However, soil treatment resulted in increased ammonia-nitrogen and acetic acid concentrations. Consequently, among the bacterial key players, lactic acid bacteria concentrations were related to silage quality. Regarding spoilage bacteria, however, alterations in silage quality characteristics were not reflected in the abundances of enterobacteria and clostridia. Future investigations should underpin the present findings and help to understand how silage additives affect microbial key players and silage fermentation. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the effects of spraying oregano essential oil (OEO) onto sugar beet pulp silage (SBPS) on silage quality and aerobic stability after opening. A factorial experiment with three replicates of three treatments and four time periods was conducted using laboratory-type plastic silos. The treatments were an untreated control, silage sprayed with 10 ml/75 cm2 OEO, and silage sprayed with 20 ml/75 cm2 OEO. The silages were sampled at 0, 72, 120, and 168 hours after spraying. Temperature, L*, a*, and b* colour values, pH, water-soluble carbohydrates, crude nutrient contents, Fleig score, metabolizable energy (ME) value, lactic acid bacteria (LAB), total live bacteria (TLB), yeast, and mould formation were assessed. Spraying OEO onto SBPS did not affect L*, a*, and b* values, pH, water-soluble carbohydrates, and Fleig score values, but decreased temperature. Spraying OEO onto the silage increased organic matter, ether extract, acid detergent fibre, neutral detergent fibre and acid detergent lignin contents without affecting crude protein, crude fibre, nitrogen free extract, and ME contents. Irrespective of treatment, crude protein, ether extract, acid detergent fibre, acid detergent lignin, nitrogen free extract, Fleig score, and ME contents of silages increased with time after spraying. The OEO spraying reduced LAB, TLB, and yeast contents in silages. In conclusion, OEO spraying onto opened SBPS reduced LAB, TLB, and yeast formation and stopped mould growth up to 72 hours without affecting their nutritional properties, suggesting that OEO could be used to ensure the stability of SBPS. [ABSTRACT FROM AUTHOR]

Importance: Feed shortage in the tropics is a major constraint to the production of livestock products such as milk and meat. In order to effectively utilize of local feed resources, the selected lactic acid bacteria (LAB) strain was used to prepare Napier grass and sugarcane top silage. The results showed that the two silages inoculated with LAB formed a co-occurrence microbial network dominated by Lactiplantibacillus during the fermentation process, regulated the microbial community structure and metabolic pathways, and improved the silage fermentation quality. This is of great significance for alleviating feed shortage and promoting sustainable production of livestock., Competing Interests: The authors declare no conflict of interest.

Importance: Ensiled whole-plant oats are an important feedstuff for ruminants in large parts of the world. Oat silage is rich in dietary fibers, minerals, vitamins, and phytochemicals beneficial to animal health. The fermentation of oat silage is a complex biochemical process that includes interactions between various microorganisms. The activity of many microbes in silage may cause an extensive breakdown of nutrition and lead to undesirable fermentation. Moreover, it is difficult to make high-quality oat silage because the number of epiphytic lactic acid bacterium microflora was lower than the requirement. Understanding the complex microbial community during the fermentation process and its relationship with community functions is therefore important in the context of developing improved fermentation biotechnology systems. These results suggested that the addition of Lactobacillus plantarum or Lactobacillus buchneri regulated the ensiling performance and microbial community in oat silage by shaping the metabolic pathways., Competing Interests: The authors declare no conflict of interest.

Silage as the main forage for ruminants could be a reservoir for antibiotic resistance genes (ARGs) through which these genes got access into the animals' system causing a latent health risk. This study employed metagenomics and investigated the ARGs' fate and transmission mechanism in high-moisture alfalfa silage treated with formic acid bactericide. The results showed that there were 22 ARGs types, in which multidrug, macrolide-lincosamide-streptogramine, bacitracin, beta-lactam, fosmidomycin, kasugamycin, and polymycin resistance genes were the most prevalent ARGs types in the ensiled alfalfa. The natural ensiling process increased ARGs enrichment. Intriguingly, after 5 days of ensiling, formic acid-treated silage reduced ARGs abundances by inhibiting host bacterial and plasmids. Although formic acid bactericide enhanced the fermentation characteristics of the high-moisture alfalfa by lowering silage pH, butyric acid concentration, dry matter losses and proteolysis, it increased ARGs abundances in alfalfa silage owing to increases in abundances of ARGs carriers and transposase after 90 days of ensiling. Notably, several pathogens like Staphylococcus, Clostridium, and Pseudomonas were inferred as potential ARGs hosts in high-moisture alfalfa silage, and high-moisture alfalfa silage may harbor a portion of the clinical ARGs. Fundamentally, microbes were distinguished as the foremost driving factor of ARGs propagation in ensiling microecosystem. In conclusion, although formic acid bactericide improved the fermentation characteristics of high-moisture alfalfa during ensiling and reduced ARGs enrichment at the initial ensiling stage, it increased ARGs enrichment at the end of ensiling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Highlights • Factors impacting spoilage in corn silage at the periphery of the exposed face in nine silage piles were investigated. • A feed-out speed of 0.29 m/day was sufficient to prevent spoilage of exposed face peripheral silage. • Decreasing days from pile opening to feedout from 110 to 61 reduced spoilage of exposed face peripheral silage. • Surface density in the range of 312–360 kg/m3 had little impact on spoilage of exposed face peripheral silage. Abstract Preserving silage in large piles is popular due to its flexibility and affordability that allows storage of large amounts of silage with low facility investment. Management of silage piles during feed-out impacts silage spoilage since, once the plastic covers are removed and silage near the exposed silage 'face' is exposed to air, undesirable microbes can proliferate to negatively impact its hygienic quality and nutritional value. Pile silage also has characteristics, such as a long feed-out phase and a large exposed face peripheral area, which makes it prone to spoilage during feed-out, especially in the exposed face area. Silage samples were collected from nine corn silage piles in the San Joaquin Valley of California (USA) in order to evaluate impacts of feed-out speed, days after opening and surface silage density on degree of silage spoilage at the periphery of the exposed face. Faster feed-out speed (i.e., 0.59 versus 0.29 m/day) suppressed the degree of spoilage, as indicated by lower temperature and pH, and higher lactic levels, as well as higher abundance of lactic acid bacteria (LAB). However, because surface spoilage in both fast- and slow-speed piles was judged to be in the spoilage initiation stage, the feed-out speed of 0.29 m/day was judged sufficient to prevent substantive spoilage. Fewer days after opening (i.e., 69 versus 110 days) impacted degree of spoilage as indicated by lower mold counts and higher lactic and acetic acid levels. As spoilage in long-open piles had reached mid-stage, while spoilage in short-open piles was still in the initiation stage, days after opening of 110 was judged too long to prevent surface spoilage. Higher surface density (i.e., 360 versus 312 kg/m3 wet weight) had little impact on spoilage, due to its low impact on all response parameters. Overall, faster feed-out speed and fewer days after pile opening had the most positive impacts on suppressing spoilage in corn silage at the periphery of the exposed face, while increasing surface silage density was less impactful. [ABSTRACT FROM AUTHOR]

AIMS: To determine the suitability of diets containing either approximately 85% fodder beet (Beta vulgaris L.) with barley straw or 65% fodder beet with pasture silage when fed to non-lactating dairy cows, by measuring intakes, digestibility, rumen function including microbial growth, and N excretion. METHODS: Holstein-Friesian cows fitted with permanent rumen fistulae were fed either 65% fodder beet with pasture silage (Silage; n=8) or 85% fodder beet with barley (Hordeum vulgare L.) straw (Straw; n=8) in an indoor facility over a 9-day period, for measurement of intakes, digestibility, rumen function and urine production. The cows were adapted to the diets over 2 weeks before the indoor measurements. Feed was available for about 6 hours/day, as practiced commercially for wintering non-lactating cows. RESULTS: Five cows fed the Straw diet had to be removed from the trial because of acute acidosis; four on Day 1 of the measurement period and one on Day 7. One cow allocated to the Silage diet refused to eat fodder beet bulbs and was also removed from the trial. Two cows fed the Silage diet were also treated for acidosis. DM intakes were lower with the Straw than Silage diets (6.4 (SE 0.4) vs. 8.3 (SE 0.5) kg/day) and organic matter (OM) digestibility was lower with the Straw than Silage diets (77 (SE 1) vs. 83 (SE 1) g/100g). The N content of the two diets was 1.14 and 1.75 g/100 g DM and there was a net loss of N by cows fed the Straw diet (−22.7 (SE 7) g/day). Rumen microbial N production was much lower in cows fed the Straw than the Silage diet (6.6 (SE 1.3) vs. 15.8 (SE 0.7) g microbial N/kg digestible OM intake). Concentrations of ammonia in rumen liquid collected on Days 5-6 were below detection limits (<0.1 mmol/L) in 36/48 (75%) samples collected from cows fed the Straw diet and in 27/48 (56%) cows fed the Silage diet. Mean urinary N excretion was lower in cows fed the Straw than the Silage diet (52.0 (SE 5.8) vs. 87.7 (SE 5.9) g/day). CONCLUSION AND CLINICAL RELEVENCE: An over-wintering diet for dry cows comprising about 65% fodder beet with 35% pasture silage provided adequate nutrition, although there was some risk of acidosis. In contrast, the diet containing about 85% fodder beet with barley straw resulted in lower DM intakes, poor rumen function, negative N balance so that both nutrition and welfare were compromised. [ABSTRACT FROM AUTHOR]

Ensiling of forages was recognized as a microbialdriven process as early as the late 1800s, when it was associated with the production of “sweet” or “sour” silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage. [ABSTRACT FROM AUTHOR]

Silage making can be conveniently divided into field, ensiling, storage, and feed-out phases. In all of these stages, controllable and uncontrollable components can affect silage quality. For instance, silages produced in hot or cold regions are strongly influenced by uncontrollable climate-related factors. In hot regions, crops for silage are influenced by (1) high temperatures negatively affecting corn yield (whole-crop and grain) and nutritive value, (2) butyric and alcoholic fermentations in warm-season grasses (Panicum, Brachiaria, and Pennisetum genera) and sugarcane, respectively, and (3) accelerated aerobic deterioration of silages. Ensiling expertise and economic factors that limit mechanization also impair silage production and utilization in hot environments. In cold regions, a short and cool growing season often limits the use of crops sensitive to cool temperature, such as corn. The fermentation triggered by epiphytic and inoculated microorganisms can also be functionally impaired at lower temperature. Although the use of silage inoculants has increased in Northern Europe, acid-based additives are still a good option in difficult weather conditions to ensure good fermentation quality, nutritive value, and high intake potential of silages. Acid-based additives have enhanced the quality of round bale silage, which has become a common method of forage preservation in Northern Europe. Although all abiotic factors can affect silage quality, the ambient temperature is a factor that influences all stages of silage making from production in the field to utilization at the feed bunk. This review identifies challenges and obstacles to producing silages under hot and cold conditions and discusses strategies for addressing these challenges. [ABSTRACT FROM AUTHOR]

The study investigated the influence of the target pH-values 4.5, 5, 5.5 and 6 in the acidification reactor on process parameters, such as substrate-specific methane yield and the intermediates, in the two-stage anaerobic digestion of sugar beet silage. The total specific methane yield (Nl kg −1 COD d −1 ) increased with an increase in the pH (pH 4.5: 140.58 ± 70.08, pH 5: 181.21 ± 55.71, pH 5.5: 218.32 ± 51.01, pH 6: 256.47 ± 28.78). The pH-value also had an effect on the dominant intermediate in hydrolysate. At the pH-value of 4.5, almost no acidification and microbial activity was observed. At pH 5 and 5.5, butyric acid production dominated, guided by H 2 production. At pH 6 acetic acid was the main product. The absence of H 2 and the highest SMY makes it favorable under practical aspects. [ABSTRACT FROM AUTHOR]

This study reports characterization of fibrolytic enzymes from rumen bacteria of migratory Gaddi sheep in North- West Himalayan Region (NWHR). A total of 70 cellulolytic bacteria were isolated. Two isolates viz., CDB1 and CDB5 due to higher secretion of fibrolytic enzymes, namely endoglucanase and β-glucosidase were studied. The endoglucanase and βglucosidase activities in isolate CDB1 and isolate CDB5 were found to be 0.720 and 0.696 U/mL and 0.820 and 0.758 U/mL, respectively. The isolate CDB1showed optimal enzyme production using yeast extract, while isolate CDB5 used peptone as nitrogen source for production of the enzyme. The endoglucanase (0.720 and 0.750 U/mL) and βglucosidase (0.710 and 0.722 U/mL) activities in CDB1 and CDB5 were optimal at 40°C and 50°C, respectively. Highest level of endoglucanase (0.742 and 0.850 U/mL) and βglucosidase activities (0.724 and 0.810 U/mL) produced by CDB1 and CDB5 were detected at pH 6.0 to 7.0, and 6.0 to 8.0, respectively. In view of ability of the isolates to inhibit some undesirable microorganisms, and activity of enzymes produced at 40°C and 50°C, it is envisaged that these isolates may have applications as probiotics and additives in silage making. [ABSTRACT FROM AUTHOR]

The aim of this study was to study the agronomic traits of different Sorghum bicolor (L.) Moench hybrids for silage productionin semiarid conditions. It was a 1-year evaluation conducted in a randomized block design with 24 treatments and three replicates. The treatments were sorghumhybrids developed by the breeding programof "EmbrapaMilho e Sorgo" (Brazilian Agricultural Research Corporation). The fresh matter yield (FMY) in the first cut varied from 22,643.56 to 44,033.15 kg/ha, with an average of 32,607.37 kg/ha, leading to the formation of two groups. Similar results were observed for the dry matter yield (DMY), in which the highest group yielded from 9,471.32 to 14,540.23 kg/ha dry matter (DM). For plant regrowth, there was an increase in the number of stems and a decrease in the amount of panicles. Two groups were formed for the accumulated dry matter yield (ADMY), averaging 14,217.91 kg/ha; the highest group showed mean values of 18,003.00 to 14,221.94 kg/ha. The evaluated sorghum hybrids exhibited satisfactory accumulated forage yields due to their high yield in regrowth, which indicates that they are suitable for use in animal production systems in semi-arid regions. [ABSTRACT FROM AUTHOR]

Background: Describing the microbial populations present in small grain silage and understanding their changes during ensiling is of interest for improving the nutrient value of these important forage crops. Barley, oat and triticale forages as well as an intercropped mixture of the 3 crops were harvested and ensiled in mini silos for a period of 90 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics and nutritive value were assessed in terminal silages and bacterial and fungal communities during ensiling and aerobic exposure were described using 16S and 18S rDNA sequencing, respectively. Results: All small grain silages exhibited chemical traits that were associated with well ensiled forages, such as low pH value (4.09 ± 0.28) and high levels of lactic acid (59.8 ± 14.59 mg/g DM). The number of microbial core genome operational taxonomic units (OTUs) decreased with time of ensiling. Taxonomic bacterial community profiles were dominated by the Lactobacillales after fermentation, with a notable increase in Bacillales as a result of aerobic exposure. Diversity of the fungal core microbiome was shown to also be reduced during ensiling. Operational taxonomic units assigned to filamentous fungi were found in the core microbiome at ensiling and after aerobic exposure, whereas the Saccharomycetales were the dominate yeast population after 90 days of ensiling and aerobic exposure. Bacterial and fungal orders typically associated with silage spoilage were identified in the core microbiome after aerobic exposure. Conclusion: Next Generation Sequencing was successfully used to describe bacterial communities and the first record of fungal communities throughout the process of ensiling and utilization. Adequately describing the microbial ecology of silages could lead to improved ensiling practices and the selection of silage inoculants that act synergistically with the natural forage microbiome. [ABSTRACT FROM AUTHOR]

In the winter time in Serbia, maize silage is the main ruminant feed. Therefore, managing maize silage is an important contributor to maintain the silage quality for livestock feed. In the study were evaluated the chemical composition, energetic and fermentation characteristics in whole-crop maize silage inoculated with different bacterial inoculants under field conditions in the commercial dairy farm, during the 2015. Three treatments were tested: negative control (untreated silage), a positive control (competitor inoculant) and Silko treatment (contains a mixture of 4 strains of Lactobacillus plantarum (LP1, LP2, LP3 and LP4). Maize is ensiled in the milk-wax grain maturity. After 90 days of ensiling, the maize silages were analyzed. The application of bacterial inoculants improved the chemical composition and energetic characteristics of silage. The inoculant Silko was more effective at improving the fermentation characteristics than competitor inoculant. Ash, cellulose, soluble N/TN, NH3-N/TN, ADF, NDF, acetic acid and pH were significantly lower in Silko treatment than positive control. There were no differences in crude fat, crude protein, ME, NEL, lactic acid and butyric acid between the treated silages. Generally, the new product bacterial inoculant Silko proved in field trials its ability to support the ensiling process in maize. The main action of the bacterial inoculant Silko is performed in two ways: the reduced degradation of protein in silage and the improvement of the aerobic stability due to the lower pH, higher content of acetic acid than negative control. [ABSTRACT FROM AUTHOR]

Macroalgal water content is an on-going problem for the use of readily accessible seaweeds in sustainable biorefining, including fuel production. Silage is a reduced-water, compactable, easily stored, transportable material. Ensiling could establish a non-seasonal supply of preserved algal biomass, but requires high initial dry matter content to mitigate environmental pollution risks from effluent. This study investigated potential dewatering methods for kelp harvested throughout the year. Treatments included air-drying, osmotic media and acids. Significant interactions between treatment and harvest-time were observed for traits of interest. Fresh weight loss during treatment was composed of changes in water and dry matter content. Air-drying gave reliable increase in final dry matter content; in summer and autumn 30% dry matter content was reached after 24 h. Dilute hydrochloric acid reduced stickiness and rendered material suitable for dewatering by screw-pressing; it may be possible to use the consequent pH reduction to promote efficient preservation. [ABSTRACT FROM AUTHOR]

Making silage is a green process to use the fast-growing water hyacinth (Eichhornia crassipes) biomass. However, the high moisture (∼95%) of the water hyacinth is the biggest challenge to making silage while its effects on fermentation processes are less studied. In this study, water hyacinths silage with different initial moistures were conducted to investigate the fermentation microbial communities and their roles on the silage qualities. Results show that both silages with 70% (S70) and 90% (S90) of initial moistures achieved the target of silage fermentation, however, their microbial processes were significantly different. Their succession directions of microbial communities were different: Plant cells in S70 were destroyed by the air-dry treatment, thus there were more soluble carbohydrates, which helped the inoculated fermentative bacteria become dominant (Lactobacillus spp. > 69%) and produce abundant lactic acid; In contrast, stochastic succession became dominant over time in S90 (NST = 0.79), in which Lactobacillus spp. and Clostridium spp. produced butyric that also obviously decreased the pH and promoted the fermentation process. Different microbial succession led to different metabolic patterns: S70 had stronger starch and sucrose metabolisms while S90 had stronger amino acid and nitrogen metabolisms. Consequently, S70 had higher lactic acid, crude protein and lower ammonia nitrogen and S90 had higher in vitro digestibility of dry matter and higher relative feeding value. Moreover, the variance partitioning analysis indicated that moisture could only explain less information (5.9%) of the microbial assemblage than pH value (41.4%). Therefore, the colonization of acid-producing bacteria and establishment of acidic environment were suggested as the key on the silage fermentation no matter how much is the initial moisture. This work can provide a basis for the future preparation of high-moisture raw biomasses for silage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

To investigate community synergy of lactic acid bacteria (LAB) and cleaner fermentation of oat silage, oat silages were prepared with or without (control) commercial LAB inoculants LI1 (containing Lactiplantibacillus plantarum, Lentilactobacillus buchneri, Lacticaseibacillus paracasei, and Pediococcus acidilactici) and LI2 (containing Lactiplantibacillus plantarum and Lentilactobacillus buchneri). The microbial community, LAB synergy, and cleaner fermentation were analyzed at 1, 3, 6, 15, 35, and 90 days of ensiling. The LAB inoculant improved fermentation quality, with significantly ( P <  0.05) lower pH, ammonia nitrogen content, and gas production and higher lactic acid and acetic acid contents than those of the control. Enterobacteriaceae was the main bacterial community in early stage of fermentation, which utilizes sugar to produce CO 2 gas, causing dry matter (DM) and energy loss. As fermentation progressed, the microbial diversity decreased, and the microbial community shifted from Gram-negative to Gram-positive bacteria. The inoculation of multispecies LAB displayed community synergy; Pediococcus acidilactici formed a dominant community in the early stage of fermentation, which produced an acid and anaerobic environment for the subsequent growth of Lentilactobacillus and Lacticaseibacillus species, thus forming a LAB-dominated microbial community. The predicted functional profile indicated that the silage inoculated with LI1 enhanced the carbohydrate metabolism pathway but inhibited the amino acid metabolism pathway, which played a role in promoting faster lactic acid production, reducing the decomposition of protein to ammonia nitrogen, and improving the fermentation quality of silage. Therefore, oat silage can be processed to high-quality and cleaner fermented feed by using an LAB inoculant, and LI1 showed better efficiency than LI2. IMPORTANCE Oat natural silage is rich in Enterobacteriaceae , increasing gas production and fermentation loss. Lactic acid bacteria interact synergistically to form a dominant community during ensiling. Pediococci grow vigorously in the early stage of fermentation and create an anaerobic environment. Lactobacilli inhibit the harmful microorganisms and result in cleaner fermentation of oat silage., Competing Interests: The authors declare no conflict of interest.

Alfalfa silage fermentation quality, metabolome, bacterial interactions, and successions as well as their predicted metabolic pathways were explored under different dry matter contents (DM) and lactic acid bacteria (LAB) inoculations. Silages were prepared from alfalfa with DM contents of 304 (LDM) and 433 (HDM) g/kg fresh weight and inoculated with Lactiplantibacillus plantarum (L. plantarum, LP), Pediococcus pentosaceus (P. pentosaceus, PP), or sterile water (control). The silages were stored at a simulated hot climate condition (35°C) and sampled at 0, 7, 14, 30, and 60 days of fermentation. The results revealed that HDM significantly improved the alfalfa silage quality and altered microbial community composition. The GC-TOF-MS analysis discovered 200 metabolites in both LDM and HDM alfalfa silage, mainly consisting of amino acids, carbohydrates, fatty acids, and alcohols. Compared with LP and control, PP-inoculated silages had increased concentrations of lactic acid (P < 0.05) and essential amino acids (threonine and tryptophan) as well as decreased pH, putrescine content, and amino acid metabolism. However, alfalfa silage inoculated with LP had higher proteolytic activities than control and PP-inoculated silage, as revealed by a higher concentration of ammonia nitrogen (NH 3 -N), and also upregulated amino acid and energy metabolism. HDM content and P. pentosaceus inoculation significantly altered the composition of alfalfa silage microbiota from 7 to 60 days of ensiling. Conclusively, these results indicated that inoculation with PP exhibited great potential in enhancing the fermentation of silage with LDM and HDM via altering the microbiome and metabolome of the ensiled alfalfa, which could help in understanding and improving the ensiling practices under hot climate conditions. KEY POINTS: • HDM improved fermentation quality and declined putrescine content of alfalfa silage • P. pentosaceus inoculation enhanced the fermentation quality of alfalfa silage • P. pentosaceus is an ideal inoculant for alfalfa silage under high temperature., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The effects of Lactobacillus buchneri, Lactobacillus hilgardii and citric acid on organic acid production, substrate consumption, protein degradation and microbial community were investigated in this study. The results indicated that combined inoculants induced a significant increase in levels of lactic acid (43 g/kg dry matter), acetic acid (14 g/kg dry matter), butyric acid (5 g/kg dry matter), total organic acid (60 g/kg dry matter) and ammonia nitrogen (20 g/kg total nitrogen). Furthermore, citric acid addition into the combined inoculants caused a significant increase in levels of acetic acid (12 g/kg dry matter), water-soluble carbohydrate (12 g/kg dry matter) and a reduction in ammonia nitrogen formation (22 g/kg total nitrogen). Microbiologically, combining inoculants and citric acid enriched Lactobacillus buchneri and Lactobacillus hilgardii and upregulated the functional pathways related to acid production and resistance. Collectively, combining citric acid and heterofermentative inoculants was beneficial to recycle Chinese cabbage waste in producing organic acids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Natural lactic acid bacteria (LAB) populations in tropical grasses and their fermentation characteristics on silage prepared with cellulase enzyme and LAB inoculants were studied. A commercial inoculant Lactobacillus plantarum Chikuso 1 (CH), a local selected strain Lactobacillus casei TH14 (TH14), and 2 cellulases, Acremonium cellulase (AC) and Maicelase (MC; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), were used as additives to silage preparation with fresh and wilted (6 h) Guinea grass and Napier grass. Silage was prepared using a laboratory-scale fermentation system. Treatments were CH, TH14, AC at 0.01% fresh matter, AC 0.1%, MC 0.01%, MC 0.1%, CH+AC 0.01%, CH+AC 0.1%, CH+MC 0.01%, CH+MC 0.1%, TH14+AC 0.1%, TH14+AC 0.01%, TH14+MC 0.1%, and TH14+MC 0.01%. Microorganism counts of Guinea grass and Napier grass before ensiling were 102 LAB and 106 aerobic bacteria; these increased during wilting. Based on morphological and biochemical characteristics, and 16S rRNA gene sequence analysis, natural strains from both grasses were identified as L. plantarum, L. casei, Lactobacillus acidipiscis, Leuconostoc pseudomesenteroides, Leuconostoc garlicum, Weissella confusa, and Lactococcus lactis. Lactobacillus plantarum and L. casei are the dominant species and could grow at lower pH and produce more lactic acid than the other isolates. Crude protein and neutral detergent fiber were 5.8 and 83.7% of dry matter (DM) for Guinea grass, and 7.5 and 77.1% of DM for Napier grass. Guinea grass had a low level of water-soluble carbohydrates (0.39% of DM). Guinea grass silage treated with cellulase had a lower pH and higher lactic acid content than control and LAB treatments. The 0.1% AC and MC treatments had the best result for fermentation quality. All high water-soluble carbohydrate (2.38% DM) Napier grass silages showed good fermentation quality. Compared with control and LAB-inoculated silage, the cellulasetreated silages had significantly higher crude protein content and lower neutral detergent fiber and acid detergent fiber contents. The results confirmed that cellulase could improve tropical silage quality, inhibiting protein degradation and promoting fiber degradation. [ABSTRACT FROM AUTHOR]

The study objective was to evaluate the effects of the addition of exogenous protease on the fermentation and nutritive value of rehydrated corn and sorghum grain silages during various storage periods. Treatments were applied using a 2 × 6 × 3 factorial combination, with 2 types of rehydrated grains (corn and sorghum), 6 doses of the enzyme (0, 0.3, 0.6, 0.9, 1.2, and 1.5%, based on natural matter) and 3 fermentation periods (0, 60, and 90 days) in a completely randomized design, with 4 replications. The protease aspergilopepsin I, of fungal origin, produced by Aspergillus niger, was used. The lactic acid concentration increased linearly as the enzyme dose increased in corn (CG) and sorghum (SG) grain silages, at 60 and 90 days of fermentation. There was an increase in the concentrations of ammonia nitrogen and soluble protein, as well as the in situ starch digestibility in rehydrated CG and SG silages, compared to the treatment without the addition of protease. The addition of 0.3% exogenous protease at the moment of CG ensiling and 0.5% in rehydrated SG increased the proteolytic activity during fermentation, providing an increase in in situ starch digestibility in a shorter storage time., (© 2023. The Author(s).)

Ensilaging is one of the most common feed preservation processes using lactic acid bacteria to stabilize feed and save feed quality. The silage bacterial community is well known but the role of the virome and its relationship with the bacterial community is scarce. In the present study, metagenomics and amplicon sequencing were used to describe the composition of the bacterial and viral community during a 40-day grass silage preservation. During the first two days, we observed a rapid decrease in the pH and a shift in the bacterial and viral composition. The diversity of the dominant virus operational taxonomic units (vOTUs) decreased throughout the preservation. The changes in the bacterial community resembled the predicted putative host of the recovered vOTUs during each sampling time. Only 10% of the total recovered vOTUs clustered with a reference genome. Different antiviral defense mechanisms were found across the recovered metagenome-assembled genomes (MAGs); however, only a history of bacteriophage infection with Lentilactobacillus and Levilactobacillus was observed. In addition, vOTUs harbored potential auxiliary metabolic genes related to carbohydrate metabolism, organic nitrogen, stress tolerance, and transport. Our data suggest that vOTUs are enriched during grass silage preservation, and they could have a role in the establishment of the bacterial community.

Aims: The aim of this study was to investigate the dynamics of bacterial communities and natural fermentation quality in three steppe types [meadow steppe (MS); typical steppe (TS); and desert steppe (DS)] on the Mongolian Plateau., Methods and Results: PacBio single molecule with real-time sequencing technology was applied to provide insights into the dynamics of the physicochemical characteristics and the complex microbiome of native grass after 1, 7, 15, and 30 days of fermentation process. The dry matter, crude protein, and water soluble carbohydrate (WSC) contents of the three groups slowly decreased after 1 day of fermentation process, and the lowest WSC concentration after 30 days of ensiling was detected in the DS group compared to that in the MS and TS groups. There was no significant effect of steppe types on lactic acid and butyric acid content (P > 0.05). The pH was higher in the early stages of fermentation. After 30 days of fermentation, the pH of MS and DS dropped to ∼5.60, while TS was as high as 5.94. At different ensiling days, the pH of TS was significantly higher than that of MS (P < 0.05). The ammonia nitrogen content of MS was significantly higher than TS and DS (P < 0.05). During the whole fermentation process, Leuconostoc mesenteroides and Pseudocitrobacter faecalis were the main species of DS, while Enterobacter roggenkampii and Faecalibacterium prausnitzii dominated the fermentation process in MS and TS, respectively., Conclusions: The fermentation quality of native grass silage of different steppe types was less satisfactory, with the silage quality ranging from DS, MS, and TS in descending order. The epiphytic bacteria dominating the fermentation process differed between steppe types of silage. Leuconostoc mesenteroides as the main strain of DS had a modulating effect on pH and LA content, while the main strains of MS and TS (Enterobacter roggenkampii and Faecalibacterium prausnitzii) dominated the silage without significant effect on improving fermentation characteristics and nutritional quality., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

In this research, we evaluated the effect of exogenous lactic acid bacteria and Amomum villosum essential oil (AVEO) on the chemical composition, microbial community composition, microbial functional diversity, and fermentation quality of Broussonetia papyrifera (BP) and Pennisetum sinese (PS) mixed silages. The BP:PS mixing ratios were 100:0, 70:30, 50:50, 30:70, and 0:100. After 3 and 30 days of ensiling at 22°C-25°C, microbial diversity and function, and fermentation quality, were assessed. Increasing PS content resulted in decreased ammoniacal nitrogen and pH, increased water-soluble carbohydrate content, increased relative abundance of Lactococcus and Acinetobacter, and reduced relative abundance of Caproiciproducens and Pseudomonas. A 50:50 BP:PS ratio effectively improved the fermentation quality compared to anaerobic fermentation with BP or PS alone, while AVEO treatment further improved fermentation quality by increasing Lactococcus relative abundance. Moreover, as fermentation proceeded, ensiling enhanced the 'Human diseases', 'Environmental information processing', and 'Cellular processes' functions at the first level, as well as the 'Two-component system' and 'ABC transporters' functions at the third level. Different additives affected the fermentation of BP and PS mixed silage by regulating microbial community succession and metabolic pathways during ensiling., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims This study investigated the influence of two microbial inoculants; Lactobacillus formosensis and Lactobacillus buchneri on fermentation quality, chemical composition, aerobic stability and in vitro ruminal biological activity of condensed tannins in sweet potato vines silage. Methods and Results Sweet potato vines were ensiled for 28 and 60 days; without inoculant ( CON), with Lact. buchneri ( LB) or with Lact. formosensis ( LF), both inoculants applied to achieve 1 × 106 CFU g−1 fresh forage. Lactobacillus formosensis silage had lower pH and higher lactic acid than all treatments. Yeasts and moulds were not detected in LB silage after ensiling. Lactobacillus buchneri silage was more aerobically stable than all treatments, whereas LF was more stable than CON silage. In vitro ruminal biological activity of condensed tannins was lower in microbial-inoculated silages than CON after ensiling. Conclusion Lactobacillus formosensis improved fermentability by reducing silage pH and improved aerobic stability by producing more propionate, which inhibited yeast activity. Lactobacillus buchneri improved aerobic stability of the silage by producing more acetate. Both strains effectively reduced the antinutritional effect of condensed tannins after ensiling. Significance and Impact of the Study Lactobacillus formosensis has the potential to be used as a silage inoculant because of its ability to improve fermentability and aerobic stability in sweet potato vines silage. [ABSTRACT FROM AUTHOR]

The current study was undertaken to investigate the effects of acid-based preservative (AP) on fermentation, hygienic and post-storage quality as well as digestibility of low dry matter (DM) direct cut wheat silages. Wheat was harvested at boot stage of maturity (265 g DM kg-1). Silages with no additive served as control (AP0). The AP was applied at 2 mL kg-1 (AP2), 3 mL kg-1 (AP3) and 4 mL kg-1 (AP4) of fresh forage weight. After AP treatment, the chopped forages were ensiled in 1.5 L anaerobic jars. Four jars per treatment were sampled on d 90 after ensiling, for chemical and microbiological analysis. Overall, AP treatment did not affect (P>0.05) the concentrations of lactic and acetic acid of the wheat silages. In contrast, inclusion of AP increased the concentrations of formic and propionic acid whereas decreased the concentrations of butyric acid, ethanol, ammonia-N and fermantation losses when compared to the control silage (P<0.05). Under aerobic conditions, AP treated silages had lower pH, CO2 production and the numbers of yeast and mold than AP0 (control) silage (P<0.05). Fibrous fractions were decreased (P<0.05) with the application of AP, but 96 h in vitro gas production and in vitro organic matter digestibility of the silages were not affected (P>0.05) by AP inclusion. In conclusion, the AP administration in to direct-cut wheat silage apparently improved fermentation properties and post storage quality. Its antimicrobial properties caused a reduction in proteolysis. [ABSTRACT FROM AUTHOR]

Ensiling is a feed preservation method of moist forage crops that generally depends on naturally developing lactic acid bacteria to convert water-soluble carbohydrates into organic acids. While bacterial community dynamics have been previously assessed in bench-scale and pilot ensiling facilities, almost no studies have assessed the microbiomes of large-scale silage facilities. This study analyzed bacterial community composition in mature silage from bunker silos in three commercial production centers as related to pH, organic matter, volatile fatty acid composition, and spatial distribution within the ensiling bunker. It revealed significant physicochemical differences between 'preserved' regions situated in the center and along the walls of the silage bunkers that were characterized by high concentrations of lactic acid and other volatiles and pH values below 5, and 'spoiled' regions in the corners (shoulders) of the bunkers that had low lactic acid concentrations and high pH values. Preserved silage was dominated (>90 %) by lactic acid bacteria and characterized by high similarity and low taxonomic diversity, whereas spoiled silage had highly diverse microbiomes with low abundances of lactic acid bacteria (<5 %) that were sometimes characterized by high levels of Enterobacteriaceae. Spatial position had a much stronger impact on the microbial community composition than feedstock type, sampling date, or production center location supporting previous studies demonstrating that ecology and not geography is a major driver of environmental microbiomes. [ABSTRACT FROM AUTHOR]

The aim of this research was to evaluate vitreousness in commercial corn hybrids with flint and dent grains and to study the effect of this characteristic on agronomic behavior, nutritional value, and ruminal degradability of grains harvested in silage maturity. Twelve commercial corn hybrids were evaluated. They were divided in two groups (six with flint grains and six with dent grains). The experiments were conducted in two municipalities represented by Guarapuava and Laranjeiras do Sul, both in Paraná State. The harvest for the grain quality analysis was performed at ? of the milk line stage in the grain. The following characteristics were evaluated: grain yield, grain yield in silage maturity, vitreousness, ruminal digestibility of grain, neutral detergent fiber, acid detergent fiber and crude protein. The grains in the group of dent hybrids exhibited better degradability associated with lower vitreousness than the group of flint hybrids. The group of dent hybrids exhibited better ruminal digestibility of the grains associated with lower vitreousness versus the group of flint hybrids. There is a negative correlation between vitreousness and the digestibility of the grain; thus, vitreousness can be a criterion for selecting genotypes for forage production. [ABSTRACT FROM AUTHOR]

Previous studies have shown that silages treated with lactic acid bacteria (LAB) inoculants enhance ruminants' performance. The objective of the current experiments was to develop an in vitro model to study interactions between LAB silage inoculants and inoculated silages and Escherichia coli (EC) in rumen fluid (RF). Our hypothesis was that some inoculants inhibit EC in RF. For that purpose buffered RF was incubated under anaerobic conditions at 39°C with commercial strains of LAB silage inoculants or with laboratory corn and wheat silages treated with these LAB, an EC strain and with various ruminant feed ingredients. The EC strain was originally isolated from cattle manure and tagged with a plasmid expressing the green fluorescence protein and kanamycin and streptomycin resistance. Results indicate that the LAB or the treated silages did not suppress EC numbers in the RF. When the pH of the RF decreased below 5·0 the EC disappeared. We conclude that both LAB inoculants for silage and EC survived in RF for several days; however, the inoculants and silages treated with such inoculants did not inhibit EC in RF in vitro. Significance and Impact of the Study Forage crops, silage and hay are initial stages of the food chain for humans. Cattle harbours and sheds enterobacteria regularly, some strains of which are pathogens. These can contaminate forage crops through field fertilization with cattle manure. The objective of this study was to develop an in vitro model to test whether lactic acid bacteria, which are used in silage inoculants, alone or in treated silages can inhibit Escherichia coli in rumen fluid. This study presents safety aspects and it is also part of a broad research effort aimed at finding out how LAB silage inoculants and inoculated silages enhance ruminant performance or exert probiotic effects in ruminants. [ABSTRACT FROM AUTHOR]