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Live Bacillus subtilis natto Promotes Rumen Fermentation by Modulating Rumen Microbiota In Vitro.

ABSTRACT: Previous studies have shown that Bacillus subtilis natto affects rumen fermentation and rumen microbial community structure, which are limited to detect a few microbial abundances using traditional methods. However, the regulation of B. subtilis natto on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear. This study explored the effects of live and autoclaved B. subtilis natto on ruminal microbial composition and diversity in vitro using 16S rRNA gene sequencing and the underlying mechanisms. Rumen fluid was collected, allocated to thirty-six bottles, and divided into three treatments: CTR, blank control group without B. subtilis natto; LBS, CTR with 10⁹ cfu of live B. subtilis natto; and ABS, CTR with 10⁹ cfu of autoclaved B. subtilis natto. The rumen fluid was collected after 0, 6, 12, and 24 h of fermentation, and pH, ammonia nitrogen (NH₃-N), microbial protein (MCP), and volatile fatty acids (VFAs) were determined. The diversity and composition of rumen microbiota were assessed by 16S rRNA gene sequencing. The results revealed LBS affected the concentrations of NH₃-N, MCP, and VFAs (p < 0.05), especially after 12 h, which might be attributed to changes in 18 genera. Whereas ABS only enhanced pH and NH₃-N concentration compared with the CTR group (p < 0.05), which might be associated with changes in six genera. Supplementation with live B. subtilis natto improved ruminal NH₃-N and propionate concentrations, indicating that live bacteria were better than autoclaved ones. This study advances our understanding of B. subtilis natto in promoting ruminal fermentation, providing a new perspective for the precise utilization of B. subtilis natto in dairy rations.

SUBMITTER: Chang M

PROVIDER: S-EPMC8225115 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length.ResultsWe applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for gamma-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases.ConclusionsThe determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http://natto-genome.org/.

Project description:Rumen microbiota transplantation (RMT) can improve rumen fermentation and ruminant performance. However, due to the microbial distinction in the fluid and solid phases, the current understanding of their specific roles in RMT is insufficient. Thus, this study was conducted to determine the effects of the microbiota from the recombination of the rumen fluid and solid phases on in vitro fermentation. The rumen fresh fluid (FF) and fresh solid (FS) phases were collected, and FS was washed for the fresh solid washing solution (FW). The fractions of FF, FS, and FW were autoclaved to obtain autoclaved fluid (AF), solid (AS), and washing solution (AW). Then, these phases were recombined to form eight treatments: FFFS, FFAS, FFFW, FFAW, AFFS, AFAS, AFFW, and AFAW. After 24 h of fermentation, the gas production in AFFS, FFFS, and FFAS was significantly higher than that of other groups. AFAS and AFAW had significantly lower alpha diversity than did other groups. The solid phase was enriched with fiber-degrading bacteria, including Treponema, Succinivibrio, and Ruminococcus. The fluid phase was dominated by Prevotella, Christensenellaceae R-7 group, and Rikenellaceae RC9 gut group. The washing solution had more Ruminobacter, Lachnospiraceae, and Fibrobacter. Moreover, the double-autoclaved phases displayed increased abundances of harmful bacteria, as AFAS and AFAW had higher Streptococcus and Prevotellaceae YAB2003 group abundances. A network analysis showed that the signature microbiota in AFAS and AFAW were negatively associated with the keystone microbiota in the other groups. In summary, the recombination of the solid phase and the autoclaved fluid phase had the best in vitro fermentation result, which provided certain references for RMT. IMPORTANCE This is the first study to systematically evaluate the in vitro fermentation efficiency of diets by bacteria harvested and recombined from the fluid and solid phases of rumen contents, and it took into account the effect of washing the rumen solid phase. Using "reconstituted rumen content", this study confirmed that bacteria from different fractions of the rumen digesta resulted in different fermentation production of diets and found the characteristic bacteria in each phase of rumen contents. Our data reveal that the bacteria in the solid phase have more positive effects on the in vitro fermentation parameters, that the combination of the autoclaved fluid phase and the fresh solid phase have the most ideal fermentation effect, and that the autoclave process significantly influenced the microbial composition and increased the abundance of harmful bacteria. This study provides a landmark reference for the future use of rumen microbiota transplantation to improve animal feed utilization and growth performance.

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Live Bacillus subtilis natto Promotes Rumen Fermentation by Modulating Rumen Microbiota In Vitro.

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