Effects of steam explosion pretreatment and Lactobacillus buchneri inoculation on fungal community of unensiled and ensiled total mixed ration containing wheat straw during air exposure.

AIM: To compare the effects of steam explosion and Lactobacillus buchneri inoculation on fungal community in ensiled total mixed ration (TMR) during aerobic exposure. METHODS AND RESULTS: The TMRs were prepared using wheat straw with or without steam explosion, sweet potato residue, lucerne hay, maize meal and soybean meal, and ensiled with or without L. buchneri inoculation. Fungal communities were detected by high-throughput sequencing. All ensiled TMRs were well ensiled and steam explosion has a major effect on improving aerobic stability. The fungal species, such as Xeromyces bisporus and Cryptococcus victoriae, that dominated in the TMR decreased after ensiling, with a concomitant increase in Candida humilis, Pichia kudriavzevii, Aspergillus flavus and Phanerochaete chrysosporium. Most mould species decreased, with C. humilis and P. kudriavzevii dominating during aerobic exposure. CONCLUSION: Steam explosion could improve the aerobic stability in ensiled TMR by inhibition of C. humilis. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput sequencing used in this study provides insight into the fungal community in ensiled TMR during aerobic exposure, which could contribute towards elucidating the mechanism by which aerobic deterioration develops.

Dynamics of microbial community during ensiling direct-cut alfalfa with and without LAB inoculant and sugar.

AIM: To gain deeper insights into the clostridial community dynamics and chemical transformations during the ensiling of alfalfa. METHODS AND RESULTS: Direct-cut alfalfa silage (with the dry matter content of 240 g kg-1 ) was prepared with or without the addition of a lactic acid bacterial inoculant and sucrose. Silages were sampled at 0, 3, 7, 14, 28 and 56 days after ensiling and their bacterial community was determined using high-throughput sequencing with a special focus on the clostridial community. A clostridial fermentation occurred in the control silage, with high contents of acetic acid, butyric acid and ammonia nitrogen and Clostridia counts; while the inoculated silage was well preserved, with low pH and high lactic acid content. Lactic acid bacteria dominated the bacterial community during the ensiling process. In the control silage, Weissella confusa, Lactobacillus brevis, Enterococcus mundtii and Pediococcus acidilactici were identified at the beginning of the fermentation. Thereafter, W. confusa, Lactobacillus helsingborgensis and Bifidobacterium asteroides appeared and quickly prevailed. In the inoculated silage, Lactobacillus plantarum dominated the whole ensiling process. The genus Clostridium dominated the clostridial community, and was depressed with the inoculated treatment. Clostridium perfringens, Garciella sp. and Clostridium baratii were the main initiators of the clostridial fermentation of the control silage, while Clostridium tyrobutyricum became the most abundant Clostridia with prolonged ensiling. Overall in the inoculated silage, little changes in the clostridial community were observed throughout the ensiling period. Treating alfalfa silage with a homolactic acid-based bacterial inoculant prevented a clostridial fermentation resulting in more efficient fermentation. CONCLUSION: Distinct changes in the bacterial community with a special focus on the clostridial community were associated with the development of the clostridial fermentation during the ensiling of alfalfa. SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput sequencing based on a novel Clostridia-specific primer set proved a potentially useful tool to study the clostridial community dynamics, and could aid to elucidate the mechanism by which the clostridial fermentation develops during the ensiling of alfalfa.

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.