Responses of microbial community composition and CAZymes encoding gene enrichment in ensiled Elymus nutans to altitudinal gradients in alpine region.

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.

Hybrid substrate-based pH autobuffering GABA fermentation by Levilactobacillus brevis CD0817.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (L-glutamic acid or L-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium L-glutamate plus acidic L-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved L-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10 L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery L-glutamic acid (750 g/L), monosodium L-glutamate (34 g/L [0.2 mol/L]), glucose (5 g/L), yeast extract (35 g/L), MnSO4·H2O (50 mg/L [0.3 mmol/L]), and Tween 80 (1.0 g/L). The appropriate fermentation temperature was 30 °C. The fermenter trial results revealed that GABA was slowly synthesized from 0-4 h, rapidly synthesized until 32 h, and finally reached 353.1 ± 8.3 g/L at 48 h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA fermentations.

Genomic and proteomic analyses reveal the reduction mechanism of hexavalent chromium by the culturing supernatant of strain Pediococcus acidilactici 13-7.

This study used lactic acid bacteria with high antioxidative properties to screen for strains capable of reducing hexavalent chromium [Cr (VI)] in their culturing supernatants. The strain Pediococcus acidilactici 13-7 exhibited potent Cr (VI)-reducing capability and remarkable resistance to Cr (VI) even at concentration as high as 24 mM. Comparative genomics analysis revealed a unique gene, ChrR, associated with Cr (VI) reduction in this strain, distinguishing it from four reference strains of P. acidilactici. The proteomic investigation identified proteins linked to the ChrR gene, such as nqo1, frdA, and gshR, indicating significant enrichment in redox-related functions and oxidative phosphorylation pathways. These findings suggest that P. acidilactici 13-7 possesses superior electron transfer capacity compared to other strains, making it more adaptable under highly oxidative conditions by modulating the external environment to mitigate oxidative stress. Collectively, the results demonstrated the potential application of this lactic acid bacterial strain for bioremediation of heavy metals by its ability to reduce Cr (VI), and shed light on the molecular mechanisms underlying Cr (VI) reduction of the strain P. acidilactici 13-7.

Responses of microbial community dynamics, co-occurrences, functional shifts, and natural fermentation profiles of Elymus nutans silage to altitudinal gradients.

IMPORTANCE: On the Qinghai-Tibet Plateau (QTP), feed shortages are common due to cold environmental conditions and the short growing season of crops. Therefore, effective preservation, such as the ensiling of local forage, is becoming increasingly important to balance the seasonal imbalance between the forage supply and the nutritional needs of domestic animals in this area. However, the structure of the microbial community of the forage, which is influenced by climatic conditions such as altitude differences, has a major impact on the fermentation quality and microbial succession of the ensiled forage. Therefore, we investigated microbial community dynamics, co-occurrence, functional shifts, and natural fermentation profiles of Elymus nutans silage as a function of altitudinal gradients. Results show that silage from Chenduo at higher elevations has better fermentation quality and higher abundance of Lacticaseibacillus and Levilactobacillus than ensiled forage from other regions. This work may contribute to guiding for silage production in QTP.

Effects of bacteriocin-producing Lactiplantibacillus plantarum on bacterial community and fermentation profile of whole-plant corn silage and its in vitro ruminal fermentation, microbiota, and CH4 emissions.

BACKGROUND: Silage is widely used to formulate dairy cattle rations, and the utilization of antibiotics and methane emissions are 2 major problems for a sustainable and environmentally beneficial ruminant production systems. Bacteriocin has received considerable attention because of its potential as an alternative to antibiotics in animal husbandry. However, the impact of bacteriocin-producing lactic acid bacteria on the microbiological conversion process of whole-plant corn silage and rumen fermentation remains limited. The purpose of this study was to assess the effect of 2 class IIa bacteriocin-producing strains Lactiplantibacillus plantarum ATCC14917 and CICC24194 on bacterial community composition and ensiling profiles of whole-plant corn silage and its in vitro rumen fermentation, microbiota, and CH4 emissions. RESULTS: Both bacteriocin-producing strains increased the lactic acid concentration in silage fermented for 7 d, whereas the lowest lactic acid was observed in the ATCC14917 inoculated silage fermented for 90 d (P < 0.05). The highest DM content was observed in the CICC24194 treatment (P < 0.05), and the silages treated with both strains had the lowest DM loss (P < 0.05). Bacteriocin-producing strains promoted the growth of Levilactobacillus brevis on d 60 of ensiling. In addition, treatment with bacteriocin-producing strains increased the in vitro DM digestibility (P < 0.05) and decreased the CH4 production (P < 0.05). The results of random forest and clustering analyses at the genus level showed that ATCC14917 increased the relative abundance of the influential variable Bacillus compared to that in the control group, whereas CICC24194 decreased the relative abundance of the influential variable Ruminococcaceae UCG-005. The CICC24194 treatment had the lowest total bacterial, fungal, protozoan, and methanogen populations (P < 0.05). CONCLUSIONS: Both class IIa bacteriocin-producing L. plantarum strains improved the fermentation quality of whole-plant corn silage by regulating the bacterial community composition during ensiling, with CICC24194 being the most effective. Both bacteriocin-producing strains mitigated CH4 production and improved digestibility by modulating the interactions among rumen bacteria, protozoa, methanogens, and the composition of fibrolytic bacteria.

Investigating the efficacy of an exopolysaccharide (EPS)-producing strain Lactiplantibacillus plantarum L75 on oat silage fermentation at different temperatures.

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.

Effects of inoculating feruloyl esterase-producing Lactiplantibacillus plantarum A1 on ensiling characteristics, in vitro ruminal fermentation and microbiota of alfalfa silage.

BACKGROUND: Ferulic acid esterase (FAE)-secreting Lactiplantibacillus plantarum A1 (Lp A1) is a promising silage inoculant due to the FAE's ability to alter the plant cell wall structure during ensiling, an action that is expected to improve forage digestibility. However, little is known regarding the impacts of Lp A1 on rumen microbiota. Our research assessed the influences of Lp A1 in comparison to a widely adopted commercial inoculant Lp MTD/1 on alfalfa's ensilage, in vitro rumen incubation and microbiota. RESULTS: Samples of fresh and ensiled alfalfa treated with (either Lp A1 or Lp MTD/1) or without additives (as control; CON) and ensiled for 30, 60 and 90 d were used for fermentation quality, in vitro digestibility and batch culture study. Inoculants treated silage had lower (P < 0.001) pH, acetic acid concentration and dry matter (DM) loss, but higher (P = 0.001) lactic acid concentration than the CON during ensiling. Compared to the CON and Lp MTD/1, silage treated with Lp A1 had lower (P < 0.001) aNDF, ADF, ADL, hemicellulose, and cellulose contents and higher (P < 0.001) free ferulic acid concentration. Compared silage treated with Lp MTD/1, silage treated with Lp A1 had significantly (P < 0.01) improved ruminal gas production and digestibility, which were equivalent to those of fresh alfalfa. Real-time PCR analysis indicated that Lp A1 inoculation improved the relative abundances of rumen's total bacteria, fungi, Ruminococcus albus and Ruminococcus flavefaciens, while the relative abundance of methanogens was reduced by Lp MTD/1 compared with CON. Principal component analysis of rumen bacterial 16S rRNA gene amplicons showed a clear distinction between CON and inoculated treatments without noticeable distinction between Lp A1 and Lp MTD/1 treatments. Comparison analysis revealed differences in the relative abundance of some bacteria in different taxa between Lp A1 and Lp MTD/1 treatments. Silage treated with Lp A1 exhibited improved rumen fermentation characteristics due to the inoculant effects on the rumen microbial populations and bacterial community. CONCLUSIONS: Our findings suggest that silage inoculation of the FAE-producing Lp A1 could be effective in improving silage quality and digestibility, and modulating the rumen fermentation to improve feed utilization.

Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking.

Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).1.

DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA.

Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5' root homologous to the known sequence and a heterologous 3' bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy.

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5'- and 3'-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3'-end and the WWP at the 5'-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.

Disassociation of glutamate from γ-aminobutyric acid by zinc acetate-assisted differential precipitation/dissolution: Application to the quantification of γ-aminobutyric acid.

γ-aminobutyric acid (GABA) is a key physiologically active molecule in organisms. Separation of glutamate from its decarboxylated product GABA has been vigorously pursued. The interaction between these two compounds severely hindered their disassociation. Herein, we present a new strategy, termed zinc acetate-assisted differential precipitation/dissolution (ZA-DPD), for the removal of glutamate by step by step recovering pure GABA solution and discarding pure glutamate pellet, essentially attributed to the use of two core reagents (zinc acetate-assisted glutamate-precipitating reagent, and glutamate-rejecting reagent). In each precipitation, the zinc acetate-assisted glutamate-precipitating reagent guaranteed most GABA still soluble although the rest co-precipitated with glutamate; in the coupled dissolution, the co-precipitated GABA was fully dissolved with or without (in the case of glutamate-rejecting reagent used in the final dissolution) co-dissolution of glutamate. The process was repeated twice until glutamate was thoroughly removed. An accurate quantitative method coupling ZA-DPD with colorimetry was thereafter established for the determination of GABA. This study may facilitate the areas associated with GABA or glutamate.

Genomic insights into a robust gamma-aminobutyric acid-producer Lactobacillus brevis CD0817.

Lactobacillus brevis CD0817, a strain isolated from a healthy adult gut, was currently the most efficient lactic acid bacterial cell factory for gamma-aminobutyric acid. In this study, the complete genome sequence of CD0817 was determined and compared with some related L. brevis genomes. The CD0817 genome consists of one 2,990,570-bp chromosome and four plasmids. The comparative genomic and phylogenetic analysis revealed that L. brevis CD0817 was not very conserved with low GABA-producing L. brevis strains. A significant divergence was that CD0817 harbors only the gadCA operon whereas the low GABA-producing L. brevis strains contain the operon and gadB. The gadB seemed to only marginally contribute to the accumulation of GABA. The high GABA production ability of CD0817 may be associated with its extraordinary genome.