Recently, returning straw to the fields has been proved as a direct and effective method to tackle soil nutrient loss and agricultural pollution. Meanwhile, the slow decomposition of straw may harm the growth of the next crop. This study aimed to determine the effects of rumen microorganisms (RMs) on straw decomposition, bacterial microbial community structure, soil properties, and soil enzyme activity. The results showed that RMs significantly enhanced the degradation rate of straw in the soil, reaching 39.52%, which was 41.37% higher than that of the control on the 30th day after straw return. After 30 d, straw degradation showed a significant slower trend in both the control and the experimental groups. According to the soil physicochemical parameters, the application of rumen fluid expedited soil matter transformation and nutrient buildup, and increased the urease, sucrase, and cellulase activity by 10%‒20%. The qualitative analysis of straw showed that the hydroxyl functional group structure of cellulose in straw was greatly damaged after the application of rumen fluid. The analysis of soil microbial community structure revealed that the addition of rumen fluid led to the proliferation of Actinobacteria with strong cellulose degradation ability, which was the main reason for the accelerated straw decomposition. Our study highlights that returning rice straw to the fields with rumen fluid inoculation can be used as an effective measure to enhance the biological value of recycled rice straw, proposing a viable solution to the problem of sluggish straw decomposition.
Animals ; Rumen/metabolism* ; Agriculture/methods* ; Soil/chemistry* ; Microbiota ; Bacteria/metabolism* ; Oryza/metabolism* ; Soil Microbiology ; Cellulose

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.
Animals ; Cyclization ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Industrial Microbiology ; methods ; Microbiota ; Protein Engineering ; Rumen ; enzymology ; microbiology ; Temperature

Using lipase segregation agar containing trioleoylglycerol, we obtained 18 lipase positive clones by screening from a metagenome library of dairy rumen microflora containing 15,360 clones. The average insert size of lipase positive clones was about 60 kb. Lipase enzyme activity analysis by p-NPP method indicated that Lipase6, Lipase7 and Lipase8 had higher lipolytic activities to substrates of p-nitrophenyl palmitate (C16), p-nitrophenyl alaurate (C12) and p-nitrophenyl palmitate (C16) respectively. The optimum pH of Lipase 6, Lipase 7 and Lipase 8 were 7.5. The halflife period of Lipase 8 with the value of 15 min in 70 degrees C decreased with the increase of temperature. In conclusion, the lipases screened in this study had different substrates specificity and good thermo stability, which laid a basis for large-scale industrial application.
Animals ; Bacteria ; genetics ; Cattle ; Cloning, Molecular ; Female ; Gene Library ; Lipase ; genetics ; metabolism ; Metagenome ; genetics ; Rumen ; microbiology ; Substrate Specificity ; Temperature

Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the hottest issues in the field of microbial diversity research. In this study, the soil and sheep rumen chyme samples were used to extract DNA, respectively. Then the 25 ng total DNA was used to amplify the 16S rRNA V3 region with 20, 25, 30 PCR cycles, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the operational taxonomic unit (OUT) amount, rarefaction curve, microbial number and species were compared through data analysis. It was found that at the same amount of DNA template, the proportion of the community composition was not the best with more numbers of PCR cycle, although the species number was much more. In all, when the PCR cycle number is 25, the number of species and proportion of the community composition were the most optimal both in soil or chyme samples.
Animals ; Bacteria ; classification ; DNA, Bacterial ; genetics ; Gene Library ; High-Throughput Nucleotide Sequencing ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; Rumen ; microbiology ; Sheep ; Soil Microbiology

In previous study we isolated a gram-positive bacterial strain, designated Niu-O16, from bovine rumen gastric juice. The growing cells of bacterial strain Niu-O16 is capable of biotransforming isoflavone daidzein into dihydrodaidzein efficiently under anaerobic conditions. In this study we investigated the optimal bioconversion conditions for the resting cells of bacterial strain Niu-O16 to convert daidzein into dihydrodaidzein. Single factor test showed that the optimal conditions for the initial pH of phosphate buffer, the concentration of the resting cell and the concentration of the substrate daidzein were 6.0-8.0, 32-64 mg/mL (wet weight) and 0.8-1.2 mmol/L, respectively. Orthogonal experiments were used to determine the optimal combination of the resting cell concentration, substrate concentration and biotransformation time. The results showed that the optimal combination included resting cell concentration 32 mg/mL, substrate concentration 0.8 mmol/L and the biotransformation time 24 h. Furthermore, the biotransformation kinetics under optimal conditions were studied, under which conditions the highest bioconversion rate was 63.9% in the resting cell system. The results might provide information for resting cell biotransforming of anaerobes as well as its industrial application.
Anaerobiosis ; Animals ; Biotransformation ; Cattle ; Culture Techniques ; methods ; Gastric Juice ; microbiology ; Gram-Positive Bacteria ; growth & development ; isolation & purification ; physiology ; Isoflavones ; biosynthesis ; chemistry ; metabolism ; Kinetics ; Rumen ; microbiology

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.
Animals ; Cattle ; Neocallimastigales/metabolism* ; Anaerobiosis ; Rumen/microbiology* ; Propionates/metabolism* ; Isobutyrates/metabolism* ; Cellulose/metabolism* ; Fungi ; Starch/metabolism* ; Glucose/metabolism* ; Acetates ; Sucrose/metabolism* ; Cellulases ; Cellulase

The effect of tea saponins (TS) on rumen fermentation and methane emission was examined using an in vitro gas production technique named Reading Pressure Technique. Three levels of TS addition (0, 0.2, 0.4 mg/ml) were evaluated in the faunated and defaunated rumen fluid. Compared to the control, TS addition decreased the 24 h gas production in the faunated rumen fluid, but had a minor effect on gas yield in the defaunated rumen fluid. The TS significantly reduced methane production in vitro. In the faunated rumen fluid, 0.2 or 0.4 mg/ml TS decreased the 24 h methane emission by 12.7% or 14.0%, respectively. Rumen fluid pH value was affected neither by TS addition nor by defaunation. The TS addition had only minor effects on volatile fatty acids, but the yield and pattern of volatile fatty acids were greatly affected by defaunation. While the molar proportion of acetate was not affected by defaunation, the propionate was significantly increased and the butyrate significantly decreased. Ammonia-N concentration and microbial protein yield were influenced by TS inclusion and defaunation. Inclusion of 0.4 mg/ml TS increased the microbial protein mass by 18.4% and 13.8% and decreased the ammonia-N concentration by 8.3% and 19.6% in the faunated and defaunated rumen fluid, respectively. Protozoa counts were significantly reduced by TS inclusion. The current study demonstrated the beneficial effect of TS on methane production and rumen fermentation, and indicated that this may be due to the effect of the associated depression on protozoa counts.
Animals ; Camellia sinensis ; metabolism ; Eukaryota ; drug effects ; physiology ; Fermentation ; drug effects ; Gastrointestinal Contents ; drug effects ; microbiology ; In Vitro Techniques ; Methane ; metabolism ; Plant Extracts ; pharmacology ; Rumen ; metabolism ; microbiology ; Saponins ; pharmacology ; Seeds ; metabolism ; Sheep ; Tea ; chemistry

Metagenomic cosmid libraries containing 1.26 x 10(5) clones, covering about 4.8 x 10(6) kb metagenomic DNA of uncultured microorganisms from the contents of buffalo rumens were constructed, and 118 independent clones expressing beta-glucosidase activity were isolated from the libraries. Screening of these clones showed that eight clones expressed relatively higher beta-glucosidase activity at pH 5.0 and 37 degrees C. One out of the eight clones was subcloned. Sequencing analysis showed that an open reading frame (ORF) of 2223 bp, termed umcel3G, potentially encodes a beta-glucosidase. The encoded product shared highest homology with a beta-glucosidase from Bacillus sp. at 60% identity and 73% similarity. The umcel3G was over-expressed in Escherichia coli and the size of the translated product Umcel3G on SDS-PAGE was in agreement with the predicted molecular mass. Zymogram analysis showed that Umcel3G exhibited beta-glucosidase activity, confirming that this ORF encodes a beta-glucosidase. The Umcel3G, purified with Ni-NTA column, exhibited optimal activity at pH 6.0-6.5 and 45 degrees C. Certain ions such as Ca2+, Zn2+ had significant positive effect on the activity of Umcel3G. However, some ions such as Fe3+, Cu2+ gave significant inhibitory effect on the enzyme. The Ni-NTA purified recombinant beta-glucosidase Umcel3G had a specific activity of 22.8 IU/mg at pH4.5, 35 degrees C and at the presence of 5 mmol/L Ca2+, indicating that this enzyme has potential applications in the fermentative production of ethanol by simultaneous saccharification and cofermentation (SSCF) of lignocelluloses.
Animals ; Bacteria ; enzymology ; genetics ; Buffaloes ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Open Reading Frames ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Rumen ; microbiology ; beta-Glucosidase ; biosynthesis ; genetics ; isolation & purification

Aeroto-Niu-O16, an oxygen-tolerant bovine rumen bacterium, is capable of aerobically reducing isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein through catalytic hydrogenation. In this study, it was found that bacterium strain Aeroto-Niu-O16 was able to cleavage the C-ring of liquiritigenin (LG), which is one of the main biologically active components of licorice roots, in the presence of atmospheric oxygen. LG was prepared by acid hydrolysis of the crude extract of licorice roots. The metabolite of LG obtained in strain Aeroto-Niu-O16 was identified as davidigenin (DG) based on the data of UV, MS, 1H and 13C NMR. The maximal concentration of LG that the strain Aeroto-Niu-O16 was able to transform effectively was 0.8 mmol x L(-1) and the average productivity of the metabolite DG was 71.7%. Furthermore, when 0.1% (m/v) of L-cysteine or sodium thiosulfate was added in the cultural medium, the average bioconversion rate of LG was increased from 71.7% to 78.3% and 77.2%, respectively. The in vitro antioxidant investigation showed that 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of DG was significantly or extremely significantly higher than that of LG at the concentrations from 0.2 mmol x L(-1) to 1.6 mmol x L(-1). We discoverd for the first time that LG can be converted to DG, which has stronger and wider biological activities, through microbial biotransformation method.
Animals ; Antioxidants ; isolation & purification ; metabolism ; pharmacology ; Bacteria, Anaerobic ; isolation & purification ; metabolism ; Biotransformation ; Biphenyl Compounds ; metabolism ; Cattle ; Chalcone ; analogs & derivatives ; metabolism ; pharmacology ; Cysteine ; pharmacology ; Flavanones ; isolation & purification ; metabolism ; pharmacology ; Glycyrrhiza ; chemistry ; Picrates ; metabolism ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Rumen ; microbiology ; Thiosulfates ; pharmacology