Screening, characterization, and optimization of the fermentation conditions of a novel cellulase-producing microorganism from soil of Qinghai-Tibet Plateau.

Cellulases play an important role in the bioconversion of lignocellulose. Microorganisms found in extreme environments are a potentially rich source of cellulases with unique properties. Due to the uniqueness of the environment, the abundant microbial resources in the Qinghai-Tibet Plateau (QTP) are worth being explored. The aim of this study was to isolate and characterize an acidic, mesophilic cellulase-producing microorganism from QTP. Moreover, the fermentation conditions for cellulase production were optimized for future application of cellulase in the development of lignocellulose biomass. A novel cellulase-producing strain, Penicillium oxalicum XC10, was isolated from the soil of QTP. The cellulase produced by XC10 was a mesophilic cellulase that exhibited good acid resistance and some cold-adaptation properties, with maximum activity at pH 4.0 and 40°C. Cellulase activity was significantly enhanced by Na+ (p < 0.05) and inhibited by Mg2+, Ca2+, Cu2+, and Fe3+ (p < 0.05). After optimization, maximum cellulase activity (8.56 U/mL) was increased nearly 10-fold. Optimal fermentation conditions included an inoculum size of 3% (v/v) in a mixture of corn straw (40 g/L), peptone (5 g/L), and Mg2+ (4 g/L), at pH 4.0, 33°C, and shaking at 200 rpm. The specific properties of the P. oxalicum XC10 cellulase suggest the enzyme may serve as an excellent candidate for the bioconversion and utilization of lignocellulose biomass generated as agricultural and food-processing wastes.

Acclimation of copper absorption ability of Candida utilis.

The use of inorganic copper in feed is hazardous. As probiotics of animals, Candida utilis can absorb copper ions and transform them to organic copper. This study aimed to domesticate the ability of C. utilis (CICC 32211) to absorb and transform copper ions. So, C. utilis (CICC 32211) was cultured in media with different concentrations of copper ions for 24, 48 and 72 h to identify the optimum copper ion concentration. C. utilis (CICC 32211) strains were domesticated for three generations, then the absorption and distribution of copper ions in the yeast cells were studied. We found that the optimum concentration of copper ions was 110 µg/mL. After 48 h, the number of yeast cells was low, but copper ion absorption efficiency was maximized (43.83%). Most of the enriched copper ions were distributed in the yeast cell wall (up to 30.58% when grown in the medium with 110 µg/mL copper ions), while the intracellular copper ion content was low (2.095%). High concentrations of copper ions affected the morphological structure, element content and distribution of yeast cells.

Bacterial community diversity associated with different levels of dietary nutrition in the rumen of sheep.

The sheep rumen microbial community plays an important role in animal performance and the environment. Few studies have paid close attention to the impact of different levels of dietary nutrition on rumen microbial populations. A total of 112 healthy Tan sheep were selected and randomly allotted to one of four dietary treatments (groups I, II, III, and IV). Each treatment included four replicated pens with seven sheep each for a total of 28 sheep per treatment. The sheep were fed four diets with nutrient levels that were 84, 96, 108, or 120% of the recommendation. In this study, a next-generation sequencing strategy and quantitative real-time PCR analysis were applied to investigate changes in whole ruminal bacteria with increased dietary energy and protein levels. The study observed 133 genera belonging to 16 phyla distributed throughout the rumen samples, with Firmicutes and Bacteroidetes predominating. Additionally, the higher nutritional dietary level linearly increased (P < 0.05) the number of Bacteroidetes and Proteobacteria but linearly decreased (P < 0.05) the Firmicutes richness. At the species level, the abundance of Prevotella ruminicola, Ruminococcus flavefaciens, and Succinivibrio dextrinosolvens linearly increased (P < 0.05), whereas the abundance of Selenomonas ruminantium and Veillonella parvula did not (P > 0.05). Furthermore, we predicted the potential functions of rumen bacteria. In particular, the relative abundances of the genes related to carbohydrates were overrepresented, and the genes involved in amino acid metabolism linearly increased (P < 0.05). These findings provide the first deep insights into the rumen microbial composition and the targeted improvement of dietary protein and energy use efficiency in Tan sheep.

Rapid assembly of multiple DNA fragments through direct transformation of PCR products into E. coli and Lactobacillus.

This article describes a rapid, highly efficient and versatile method for seamlessly assembling multiple DNA fragments into a vector at any desired position. The inserted fragments and vector backbone were amplified by high-fidelity PCR containing 20 bp to 50 bp overlapping regions at 3' and/or 5' termini. These linearised fragments were equimolarly mixed, and then cyclised in a prolonged overlap extension PCR without adding primers. The resulting PCR products were DNA multimers that could be directly transformed into host strains, yielding the desired chimeric plasmid. The proposed method was illustrated by constructing an Escherichia coli co-expression vector. The feasibility of the method in Lactobacillus was further validated by assembling an E. coli-Lactobacillus shuttle vector. Results showed that three to four fragments could be simultaneously and precisely inserted in a vector in only 2-3 days using the proposed method. The acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, the proposed method is sufficiently competent for high-efficiency insertion of multiple DNA fragments into a plasmid and has theoretically good application potential for gene cloning and protein expression because it is simple, easy to implement, flexible and yields highly positive clones.

Coexpression and secretion of endoglucanase and phytase genes in Lactobacillus reuteri.

A multifunctional transgenic Lactobacillus with probiotic characteristics and an ability to degrade ß-glucan and phytic acid (phytate) was engineered to improve nutrient utilization, increase production performance and decrease digestive diseases in broiler chickens. The Bacillus subtilis WL001 endoglucanase gene (celW) and Aspergillus fumigatus WL002 phytase gene (phyW) mature peptide (phyWM) were cloned into an expression vector with the lactate dehydrogenase promoter of Lactobacillus casei and the secretion signal peptide of the Lactococcus lactis usp45 gene. This construct was then transformed into Lactobacillus reuteri XC1 that had been isolated from the gastrointestinal tract of broilers. Heterologous enzyme production and feed effectiveness of this genetically modified L. reuteri strain were investigated and evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the molecular mass of phyWM and celW was approximately 48.2 and 55 kDa, respectively, consistent with their predicted molecular weights. Endoglucanase and phytase activities in the extracellular fraction of the transformed L. reuteri culture were 0.68 and 0.42 U/mL, respectively. Transformed L. reuteri improved the feed conversion ratio of broilers from 21 to 42 days of age and over the whole feeding period. However, there was no effect on body weight gain and feed intake of chicks. Transformed L. reuteri supplementation improved levels of ash, calcium and phosphorus in tibiae at day 21 and of phosphorus at day 42. In addition, populations of Escherichia coli, Veillonella spp. and Bacteroides vulgatus were decreased, while populations of Bifidobacterium genus and Lactobacillus spp. were increased in the cecum at day 21.

Characterization and Assessment of Native Lactic Acid Bacteria from Broiler Intestines for Potential Probiotic Properties.

Probiotics are the most promising alternative to antibiotics for improving animal production and controlling pathogenic infections, while strains derived from natural hosts are considered highly desirable due to their good adaptation to the gastrointestinal tract. The aim of this study was to screen Lactobacillus with broad-spectrum antibacterial activity from broilers fed an antibiotic-free diet and evaluate their potential as poultry probiotics. A total of 44 lactic acid bacteria (LAB) strains were isolated from the intestines of healthy broilers, among which 3 strains exhibited outstanding antimicrobial activity and were subsequently identified through 16S rRNA sequencing as Enterococcus faecium L8, Lactiplantibacillus plantarum L10, and Limosilactobacillus reuteri H11. These three isolates demonstrated potent bacteriostatic activity against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella cholerae, with inhibition zones ranging from 15.67 ± 1.53 to 21.33 ± 0.58 mm. The selected LAB strains exhibited high tolerance to acid and bile salts, with L. reuteri H11 displaying the highest survival rate (ranging from 34.68% to 110.28%) after exposure to 0.3% (w/v) bile salts for 6 h or a low pH environment (pH 2, 2.5, and 3) for 3 h. Notably, L. reuteri H11 outperformed other strains in terms of hydrophobicity (84.31%), auto-aggregation (53.12%), and co-aggregation with E. coli ATCC 25922 (36.81%) and S. aureus ATCC 6538 (40.20%). In addition, the three LAB isolates were either fully or moderately susceptible to the tested antibiotics, except for strain L8, which resisted gentamycin and vancomycin. Consequently, these three LAB strains, especially L. reuteri H11, isolated from the intestines of broiler chickens, represent promising probiotic candidates that can be employed as feed additives to enhance production performance and control poultry pathogens.

Probiotic Properties of Chicken-Derived Highly Adherent Lactic Acid Bacteria and Inhibition of Enteropathogenic Bacteria in Caco-2 Cells.

Lactic acid bacteria (LAB) as probiotic candidates have various beneficial functions, such as regulating gut microbiota, inhibiting intestinal pathogens, and improving gut immunity. The colonization of the intestine is a prerequisite for probiotic function. Therefore, it is necessary to screen the highly adherent LAB. In this study, the cell surface properties, such as hydrophobicity, auto-aggregation, co-aggregation, and adhesion abilities of the six chicken-derived LAB to Caco-2 cells were investigated. All six strains showed different hydrophobicity (21.18-95.27%), auto-aggregation (13.61-30.17%), co-aggregation with Escherichia coli ATCC 25922 (10.23-36.23%), and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 (11.71-39.35%), and adhesion to Caco-2 cells (8.57-26.37%). Pediococcus pentosaceus 2-5 and Lactobacillus reuteri L-3 were identified as the strains with strong adhesion abilities (26.37% and 21.57%, respectively). Moreover, these strains could survive in a gastric acid environment at pH 2, 3, and 4 for 3 h and in a bile salt environment at 0.1%, 0.2%, and 0.3% (w/v) concentration for 6 h. Furthermore, the cell-free supernatant of P. pentosaceus 2-5 and L. reuteri L-3 inhibited the growth of enteropathogenic bacteria and the strains inhibited the adhesion of these pathogens to Caco-2 cells. In this study, these results suggested that P. pentosaceus 2-5 and L. reuteri L-3, isolated from chicken intestines might be good probiotic candidates to be used as feed additives or delivery vehicles of biologically active substances.

Non-invasive metabolomic profiling of culture media of ICSI- and IVF-derived early developmental cattle embryos via Raman spectroscopy.

The aim of the present study was to compare differences in composition between in vitro cultured early developmental embryos resulting from either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Non-invasive metabolomic profiling of culture media was conducted with laser tweezer Raman spectroscopy (LTRS), providing molecular information that was used to aid the diagnosis or treatment of embryos that were adversely affected by ICSI treatment, ultimately improving the ICSI embryonic developmental potential. Cattle embryos were generated via ICSI and IVF with development to the 2-, 4-, 8-, 16-,32-cell, and blastocyst stages with individual in vitro culturing occurring for 4 h. The culture media for embryos in different developmental stages were separately analyzed using LTRS. The resulting composition of culture media used for culturing IVF- and ICSI-derived embryos was mainly altered in contents of carbohydrates, lipids, DNA, and proteins. Bands at 1004 cm-1 (phenylalanine) and 1529 cm-1 (-C = C-carotenoid) had specific patterns related to the metabolicactivity of embryos; using LTRS, and these may be considered as biomarkers for embryonic development. Furthermore, the vibrations of lipids at different stages increased more with assessment of ICSI culture media than in IVF media. Discriminant function analysis can be utilized for the classification of culture media used for culture of ICSI- and IVF-derived embryos. In conclusion, LTRS can be used for development of an independent assay to assess embryo status during both ICSI and IVF procedures, which provides novel insights into differences in structure and components of single cells.

Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system.

Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest ß-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained P(luxS) and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of P(luxS) containing a putative promoter. This putative promoter was a hybrid promoter and its ß-Gal activity was higher than P(luxS). Reconstructing the hybrid promoter from pShuttle-09 to P(lapS) further improved the ß-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.