Effect of Regulation of Whole-Plant Corn Silage Inoculated with Lactobacillus buchneri or Bacillus licheniformis Regarding the Dynamics of Bacterial and Fungal Communities on Aerobic Stability.

Enhancing the aerobic stability of whole-plant corn silage is essential for producing high-quality silage. Our research assessed the effect of inoculation with Lactobacillus buchneri or Bacillus licheniformis and its modulation of the bacterial and fungal microbial community structure in an aerobic stage of whole-plant corn silage. Following treatment with a distilled sterile water control, Lactobacillus buchneri, and Bacillus licheniformis (2 × 105 cfu/g), whole-plant corn was ensiled for 60 days. Samples were taken on days 0, 3, and 7 of aerobic exposure, and the results showed that inoculation with Lactobacillus buchneri or Bacillus licheniformis improved the aerobic stability of silage when compared to the effect of the control (p < 0.05). Inoculation with Bacillus licheniformis attenuated the increase in pH value and the decrease in lactic acid in the aerobic stage (p < 0.05), reducing the filamentous fungal counts. On the other hand, inoculation with Lactobacillus buchneri or Bacillus licheniformis increased the diversity of the fungal communities (p < 0.05), complicating the correlation between bacteria or fungi, reducing the relative abundance of Acetobacter and Paenibacillus in bacterial communities, and inhibiting the tendency of Monascus to replace Issatchenkia in fungal communities, thus delaying the aerobic spoilage process. Due to the prevention of the development of aerobic spoilage microorganisms, the silage injected with Lactobacillus buchneri or Bacillus licheniformis exhibited improved aerobic stability.

Effects of Bacillus subtilis or Lentilactobacillus buchneri on aerobic stability, and the microbial community in aerobic exposure of whole plant corn silage.

This study aimed to evaluate the effects of Bacillus subtilis or Lentilactobacillus buchneri on the fermentation quality, aerobic stability, and bacterial and fungal communities of whole plant corn silage during aerobic exposure. Whole plant corn was harvested at the wax maturity stage, which chopped to a length of approximately 1 cm, and treated with the following: distilled sterile water control, 2.0 × 105 CFU/g of Lentilactobacillus buchneri (LB) or 2.0 × 105 CFU/g of Bacillus subtilis (BS) for 42 days silage. Then, the samples were exposed to air (23-28°C) after opening and sampled at 0, 18 and 60 h, to investigate fermentation quality, bacterial and fungal communities, and aerobic stability. Inoculation with LB or BS increased the pH value, acetic acid, and ammonia nitrogen content of silage (P < 0.05), but it was still far below the threshold of inferior silage, the yield of ethanol was reduced (P < 0.05), and satisfactory fermentation quality was achieved. With the extension of the aerobic exposure time, inoculation with LB or BS prolonged the aerobic stabilization time of silage, attenuated the trend of pH increase during aerobic exposure, and increased the residues of lactic acid and acetic acid. The bacterial and fungal alpha diversity indices gradually declined, and the relative abundance of Basidiomycota and Kazachstania gradually increased. The relative abundance of Weissella and unclassified_f_Enterobacteria was higher and the relative abundance of Kazachstania was lower after inoculation with BS compared to the CK group. According to the correlation analysis, Bacillus and Kazachstania are bacteria and fungi that are more closely related to aerobic spoilage and inoculation with LB or BS could inhibit spoilage. The FUNGuild predictive analysis indicated that the higher relative abundance of fungal parasite-undefined saprotroph in the LB or BS groups at AS2, may account for its good aerobic stability. In conclusion, silage inoculated with LB or BS had better fermentation quality and improved aerobic stability by effectively inhibiting the microorganisms that induce aerobic spoilage.

Natural fermentation quality, bacteria, and functional profiles of three cuttings of alfalfa silage in a year in Inner Mongolia, China.

Alfalfa is harvested two or three times a year in central and western Inner Mongolia, China. However, the variations in bacterial communities as affected by wilting and ensiling, and the ensiling characteristics of alfalfa among the different cuttings, are not fully understood. To enable a more complete evaluation, alfalfa was harvested three times a year. At each time of cutting, alfalfa was harvested at early bloom, wilted for 6 h, and then ensiled in polyethylene bags for 60 days. The bacterial communities and nutritional components of fresh alfalfa(F), wilted alfalfa(W) and ensiled alfalfa(S), and the fermentation quality and functional profile of bacterial communities of the three cuttings alfalfa silage, were then analyzed. Functional characteristics of silage bacterial communities were evaluated according to the Kyoto Encyclopedia of Genes and Genomes. The results showed that all nutritional components, fermentation quality, bacterial communities, carbohydrate, amino acid metabolism and key enzymes of bacterial communities were influenced by cutting time. The species richness of F increased from the first cutting to the third cutting; it was not changed by wilting, but was decreased by ensiling. At phylum level, Proteobacteria were more predominant than other bacteria, followed by Firmicutes (0.063-21.39%) in F and W in the first and second cuttings. Firmicutes (96.66-99.79%) were more predominant than other bacteria, followed by Proteobacteria (0.13-3.19%) in S in the first and second cuttings. Proteobacteria, however, predominated over all other bacteria in F, W, or S in the third cutting. The third-cutting silage showed the highest levels of dry matter, pH and butyric acid (p < 0.05). Higher levels of pH and butyric acid were positively correlated with the most predominant genus in silage, and with Rosenbergiella and Pantoea. The third-cutting silage had the lowest fermentation quality as Proteobacteria were more predominant. This suggested that, compared with the first and second cutting, the third cutting is more likely to result in poorly preserved silage in the region studied.

Effect of exogenous microorganisms on the fermentation quality, nitrate degradation and bacterial community of sorghum-sudangrass silage.

This study aims to investigate the effects of adding Lactobacillus buchneri (LB), Lactobacillus brevis (LBR) and Bacillus subtilis (BS) on the fermentation quality, nitrate degradation and bacterial community of sorghum-sudangrass silage. The results showed that the addition of LB significantly increased the pH and acetic acid content (p < 0.05), but high-quality silage was obtained. The addition of LBR and BS improved the fermentation quality of sorghum-sudangrass silage. The use of additives reduced the nitrate content in sorghum-sudangrass silage. The LB group increased the release of N2O at 3-7 days of ensiling (p < 0.05), and LBR and BS increased the release of N2O at 1-40 days of ensiling (p < 0.05). On the first day of ensiling, all silages were dominated by Weisslla, over 3 days of ensiling all silages were dominated by Lactobacillus. Acinetobacter, Serratia, Aquabacterium, and unclassified_f_enterobacteriaceae showed significant negative correlations with nitrate degradation during sorghum-sudangrass ensiling (p < 0.05). The BS and LBR groups increased the metabolic abundance of denitrification, dissimilatory nitrate reduction, and assimilatory nitrate reduction (p < 0.05). Overall, the additive ensures the fermentation quality of sorghum-sudangrass silage and promotes the degradation of nitrate by altering the bacterial community.

Effect of Ensiling Density and Storage Temperature on Fermentation Quality, Bacterial Community, and Nitrate Concentration of Sorghum-Sudangrass Silage.

This study aimed to evaluate the fermentation quality, bacterial community, and nitrate content of sorghum-sudangrass silage with two ensiling densities [550 kg fresh weight (FW)/m3 (low density, LD) and 650 kg FW/m3 (high density, HD)] stored at two temperatures [10°C (low temperature, LT) and 25°C (normal temperature, NT)] for 60 days. The fermentation parameters, microbial counts, bacterial community, nutritional composition, and nitrate and nitrite levels were assessed. The pH and ammonia nitrogen (N) in all silages were below 4.0 and 80 g/kg total N, respectively. Compared with LT treatments, NT treatments had lower pH and lactic acid (LA) bacteria and yeasts counts and contained higher LA and LA/acetic acid (LA/AA) (p < 0.05). The LT-LD contained more ammonia-N than LT-HD (p < 0.05) and had higher nitrate and lower nitrate degradation than other treatments (p < 0.05). Lactobacillus was the most dominant genus with all treatments (57.2-66.9%). The LA, LA/AA, and abundances of Pantoea, Pseudomonas, and Enterobacter in the silage negatively correlated with nitrate concentration and positively correlated with nitrate degradation (p < 0.05). Moreover, pH and ammonia-N were positively correlated with nitrate concentration and negatively correlated with nitrate degradation (p < 0.05). Overall, all silage had satisfactory fermentation quality, and the silage with HD and NT had better fermentation quality and higher nitrate degradation. The bacterial communities in all silages were dominated by Lactobacillus. The nitrate degradation during the fermentation process might be related to the fermentation quality and the activity of Pantoea, Pseudomonas, and Enterobacter in silage.

iTRAQ-based quantitative proteome analysis insights into cold stress of Winter Rapeseed (Brassica rapa L.) grown in the field.

Winter rapeseed (Brassica rapa L.) is a major oilseed crop in Northern China, where its production was severely affected by chilling and freezing stress. However, not much is known about the role of differentially accumulated proteins (DAPs) during the chilling and freezing stress. In this study, isobaric tag for relative and absolute quantification (iTRAQ) technology was performed to identify DAPs under freezing stress. To explore the molecular mechanisms of cold stress tolerance at the cellular and protein levels, the morphological and physiological differences in the shoot apical meristem (SAM) of two winter rapeseed varieties, Longyou 7 (cold-tolerant) and Lenox (cold-sensitive), were explored in field-grown plants. Compared to Lenox, Longyou 7 had a lower SAM height and higher collar diameter. The level of malondialdehyde (MDA) and indole-3-acetic acid (IAA) content was also decreased. Simultaneously, the soluble sugars (SS) content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, soluble protein (SP) content, and collar diameter were increased in Longyou 7 as compared to Lenox. A total of 6330 proteins were identified. Among this, 98, 107, 183 and 111 DAPs were expressed in L7 CK/Le CK, L7 d/Le d, Le d/Le CK and L7 d/L7 CK, respectively. Quantitative real-time PCR (RT-qPCR) analysis of the coding genes for seventeen randomly selected DAPs was performed for validation. These DAPs were identified based on gene ontology enrichment analysis, which revealed that glutathione transferase activity, carbohydrate-binding, glutathione binding, metabolic process, and IAA response were closely associated with the cold stress response. In addition, some cold-induced proteins, such as glutathione S-transferase phi 2(GSTF2), might play an essential role during cold acclimation in the SAM of Brassica rapa. The present study provides valuable information on the involvement of DAPs during cold stress responses in Brassica rapa L, and hence could be used for breeding experiments.

Dynamics of Bacterial and Fungal Communities and Metabolites During Aerobic Exposure in Whole-Plant Corn Silages With Two Different Moisture Levels.

The study was aimed to investigate the effect of moisture content on microbial communities, metabolites, fermentation quality, and aerobic stability during aerobic exposure in whole-plant corn silages preserved long time to improve the quality and aerobic stability of the silage during feed-out. Corn plants with two different moisture levels (high-moisture content, 680 g/kg; low-moisture content, 620 g/kg) were harvested at one-third and two-thirds milk-line stages, respectively, ensiled in laboratory-scale silos, and then sampled at 350 day after ensiling and at 2 and 5 day after opening to investigate bacterial and fungal communities, metabolites, and aerobic stability. High-moisture content increased aerobic stability and pH and decreased lactic acid and microbial counts in silages (P < 0.05). During aerobic exposure, the low-moisture silages had higher pH and lactic acid bacterial count and lower lactic acid than the high-moisture silages (P < 0.05); Acinetobacter sp. was the most main bacterial species in the silages; Candida glabrata and unclassified Candida had an increasing abundance and negatively correlation with aerobic stability of high-moisture silages (P < 0.05), while C. glabrata, Candida xylopsoci, unclassified Saccharomycetaceae, and unclassified Saccharomycetales negative correlated with aerobic stability of low-moisture silages (P < 0.05) with a rising Saccharomycetaceae; the silages had a reducing concentration of total metabolites (P < 0.05). Moreover, the high-moisture silages contained greater total metabolites, saturated fatty acids (palmitic and stearic acid), essential fatty acids (linoleic acid), essential amino acids (phenylalanine), and non-essential amino acids (alanine, beta-alanine, and asparagine) than the low-moisture silages at 5 day of opening (P < 0.05). Thus, the high-moisture content improved the aerobic stability. Acinetobacter sp. and Candida sp. dominated the bacterial and fungal communities, respectively; Candida sp. resulted in the aerobic deterioration in high-moisture silages, while the combined activities of Candida sp. and Saccharomycetaceae sp. caused the aerobic deterioration in low-moisture silages. The greater aerobic stability contributed to preserve the palmitic acid, stearic acid, linoleic acid, phenylalanine, alanine, beta-alanine, and asparagine during aerobic exposure.

Succession of Bacterial Community During the Initial Aerobic, Intense Fermentation, and Stable Phases of Whole-Plant Corn Silages Treated With Lactic Acid Bacteria Suspensions Prepared From Other Silages.

The present study was aimed at investigating the bacterial community in lactic acid bacteria (LAB) suspensions prepared from whole-plant corn silage (LAB suspension-CS) and Elymus sibiricus silage (LAB suspension-ES) and the bacterial community succession of whole-plant corn silages inoculated with LAB suspension-CS or LAB suspension-ES during initial aerobic phase, intense fermentation phase, and stable phase. The LAB suspensions were cultured in sterile Man, Rogosa, Sharpe broth at 37°C for 24 h and used as inoculants for ensiling. The chopped whole-plant corn was treated with distilled water (CK), LAB suspension-CS (CSL), or LAB suspension-ES (ESL) and then ensiled in vacuum-sealed plastic bags containing 500 g of fresh forage. Silages were sampled at 0 h, anaerobic state (A), 3 h, 5 h, 10 h, 24 h, 2 days, 3 days, 10 days, 30 days, and 60 days of ensiling with four replicates for each treatment. The results showed that Lactobacillus, Weissella, and Lachnoclostridium_5 dominated the bacterial community in LAB suspension-CS; Lactobacillus was the most predominant bacterial genus in LAB suspension-ES. During the initial aerobic phase (from 0 h to A) of whole-plant corn silage, the pH and the abundances of Pantoea, Klebsiella, Rahnella, Erwinia, and Serratia increased. During the intense fermentation phase (from A to 3 days), the pH decreased rapidly, and the microbial counts increased exponentially; the most predominant bacterial genus shifted from Pantoea to Weissella, and then to Lactobacillus; inoculating LAB suspensions promoted the bacterial succession and the fermentation process, and LAB suspension-CS was more effective than LAB suspension-ES. During the stable phase (from 3 to 60 days), the pH and the microbial counts decreased, and Lactobacillus dominated the bacterial community with a little decrease. The results also confirmed the existence of LAB fermentation relay during fermentation process, which was reflected by Weissella, Lactococcus, and Leuconostoc in the first 5 h; Weissella, Lactococcus, Leuconostoc, Lactobacillus, and Pediococcus between 5 and 24 h; and Lactobacillus from 24 h to 60 days.

An identification method of the weak link of stiffness for cantilever beam structure.

To find the weak link of the structural stiffness is important to improve machine tool stiffness. However, how to overcome the static deformation with difficulty acquisition is a difficult problem in machine tool structure. The article takes the cantilever beam structure as a numerical example, the weak link is modeled as EA reduction in stiffness. Thorough finite element simulations are performed to assess the robustness and limitations of the method in several scenarios with single and multiple weaknesses. The sensors are used to acquire the acceleration data, the structural modal parameters are obtained by the singular value decomposition technique, and the dynamic characteristics are systematically reconstructed by using the modal state-space method to obtain static stiffness. Then, an identification method proposed by measured data and reconstructed data to identify the weak link of stiffness of the cantilever structure. Furthermore, the comparison of numerical and experimental results validate the correctness and effectiveness of this method. The research has certain practical engineering value and provides an accurate guidance for the optimization of machine tool stiffness.

Research on the elastic-plastic external contact mechanical properties of cylinder.

Based on Hertz contact theory, an elastic-plastic contact mechanics model of outer cylinder under different contact angles of axis is proposed. The relationship among contact angle, load and contact deformation, contact stiffness and contact area is established. The finite element method is used to simulate the elastic-plastic contact process of the cylinder. The influence of the load and radius of the cylinder model on the contact deformation and the contact stiffness is compared and analyzed under different contact angles. The error of the analysis results of the finite element and the mechanical model is within 9%. On this basis, the influence of contact deformation, contact area and contact angle on the contact stiffness of the outer cylinder in elastic and plastic stage is explored. The results show that in the stage of elastic and plastic deformation, the amount of contact deformation and contact area increase with the increase of load. The contact stiffness decreases with the increase of contact angle and increases with the increase of cylinder radius. The amount of contact deformation decreases with the increase of cylinder radius, and tends to constant gradually. In the elastic stage, the contact stiffness increases with the increase of load. The contact area decreases with the increase of contact angle and increases with the increase of cylinder radius. In the plastic stage, the contact stiffness is constant with the increase of load, and the contact area is independent of contact angle and cylinder radius.

Protein Immobilization on Gold Nanoparticles: Quantitative Analysis.

Conjugation of gold nanoparticles (AuNPs) with biologically relevant molecules underpins many applications in medicine and biochemistry. Immobilization of functional proteins on AuNPs often affects protein structure and function. Such effects are protein dependent and require thorough investigation using suitable quantitative tests. Good experimental design and the use of a comprehensive set of control samples are essential when characterizing the consequences of protein immobilization and its effect on protein structure and function. However, traditional approaches to making control samples, that is, immobilized protein versus protein in solution in absence of any nanoparticles, do not provide sufficiently identical reaction conditions and complicate interpretation of the results. Accurate quantification of protein conjugation to AuNPs and ensuring complete removal of unconjugated protein remain the two key challenges in such functional assays. This report describes a simple and straightforward procedure allowing for quantitative analysis of protein conjugation to AuNPs. The principles are illustrated using fluorescence and circular dichroism measurements, and can be applied to other analytical techniques or be adapted with minor modifications for use with other proteins.

Fermentation quality and nutritive value of total mixed ration silages based on desert wormwood (Artemisia desertorum Spreng.) combining with early stage corn.

This study aimed to investigate the fermentation quality and nutritive value of total mixed ration (TMR) silages based on desert wormwood (DW) combined with early stage corn (ESC) as forage and determine an optimum formula. Desert wormwood and ESC were harvested, chopped, and mixed with other ingredients according to a formula, packed into laboratory silos at densities of 500-550 g/L, and stored in the dark for 60 days. The DW proportions in the forage of TMR were 1, 0.75, 0.50, 0.25 and 0, based on fresh weight. As the proportion of DW decreased, the pH also decreased (P < 0.05), while lactic acid, lactic acid/acetic acid, crude protein, starch, and the in vitro digestibility of dry matter and neutral detergent fiber increased (P < 0.05). Ammonia nitrogen/total nitrogen in the TMR silages with DW proportions of 0.75, 0.25 and 0 in the forage was more than 10%. These results indicated that the quality of the TMR silage containing DW alone as forage was poor, TMR silages containing DW proportions of 0.75 and 0.25, and ESC alone, in the forage were not well preserved. The optimum TMR silage formula contained a DW proportion of 0.5 in the forage.