Toward the augmentation of rumen fermentation: Deciphering microbial dynamics

Tondini, Sara Maria

Permalink

https://hdl.handle.net/2142/121484 Copy

Description

Title

Toward the augmentation of rumen fermentation: Deciphering microbial dynamics

Author(s)

Tondini, Sara Maria

Issue Date

2023-07-11

Director of Research (if dissertation) or Advisor (if thesis)

McCann, Joshua C

Doctoral Committee Chair(s)

McCann, Joshua C

Committee Member(s)

• Shike, Daniel W
• Mackie, Roderick I
• Smith, Alexandra H

Department of Study

Animal Sciences

Discipline

Animal Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• rumen
• microbiome
• modeling
• bacterial interactions
• transcriptomics

Abstract

The rumen microbiome has been extensively studied to elucidate mechanisms and unravel complex interactions among rumen microorganisms. Pioneering culture-based research has revealed fundamental characterizations of specific model organisms but has limited capacity to describe community interactions. In contrast, modern culture-independent methods have allowed deeper insight into community composition but lack utility in defining microbial functions. Therefore, a combination of experimental and computational approaches is needed to further describe rumen microorganisms and identify potential targets for practical nutritional applications. The research in this dissertation combines multiple methodological approaches to gain a deeper comprehension of rumen microbial functions, interactions and community dynamics. Feed additives are important nutritional tools used to augment rumen microbial populations to increase substrate degradation and enhance fermentation. To investigate an alternative approach to modulating the rumen microbial population, polyclonal antibodies were developed against pure cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85) and validated for their ability to inhibit growth of each bacteria. Inoculation time (0 h and 4 h) and dose response (CON-0; LO-1.3 × 10-4; MD-0.013; HI-1.3 mg antibody per ml of medium) were evaluated to determine antibody efficacy. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density after a 52 h growth period when compared with CON or LO. Addition of anti-FS85 to non-cellulolytic strains did not affect (P ≥ 0.89) optical density, substrate disappearance or total volatile fatty acid concentrations providing further evidence of specificity against targeted bacteria. Western blotting with anti-FS85 indicated selective binding to F. succinogenes S85 proteins, and 7 of the 8 selected proteins were associated with the outer-membrane. Overall, polyclonal antibodies were more efficacious at inhibiting growth of targeted cellulolytic bacteria than non-targeted bacteria. Validated polyclonal antibodies could serve as an effective approach to modify rumen bacterial populations Understanding the microbial interactions that allow for enhanced substrate degradation and subsequent metabolite production is essential to improve rumen fermentation. To identify potential mechanisms for competitive or cooperative growth, rumen bacteria were grown in mono- and co-culture and their abundance, cellobiose utilization, metabolite production, and transcriptomic profiles were compared. Experiment 1 evaluated the interaction between R. albus 7 and F. succinogenes S85, and experiment 2 evaluated the interaction between R. albus 7 and R. flavefaciens FD-1. In Exp. 1, R. albus 7 and F. succinogenes S85 had greater (P < 0.05) abundance in co-culture compared with monoculture. Additionally, succinate and formate concentrations increased (P < 0.05) in co-culture. Transcriptional pathway analysis showed that F. succinogenes S85 had upregulated (≥ 1 log(fold-change); P < 0.05) genes involved with glycolysis, fermentation, and amino acid biosynthesis when co-cultured with R. albus 7. Additionally, R. albus 7 had upregulated genes involved with ammonia assimilation, amino acid synthesis, thiamin biosynthesis, and iron-sulfur cluster biosynthesis when in co-culture with F. succinogenes S85. Increased expression of metabolic pathways and fermentation end-products suggests a benefit from F. succinogenes S85 and R. albus 7 being co-cultured. In Exp. 2, R. albus 7 outcompeted R. flavefaciens FD-1 in co-culture according to qPCR abundance results. Transcriptional pathway analysis showed that R. flavefaciens FD-1 had upregulated genes involved with ammonia assimilation and downregulated genes involved with nucleotide synthesis, fermentation, and carbohydrate biosynthesis. R. albus 7 had upregulated genes involved in nucleotide synthesis and downregulated genes involved in glycolysis and cofactor synthesis. Decreased expression of metabolic pathways suggests a negative interaction between R. albus 7 and R. flavefaciens FD-1. All bacteria displayed changes in transcriptional response when co-cultured demonstrating that cooperative or competitive microbial interactions influence gene expression. In the rumen, substrate degradation is a critical task required for breakdown of various feedstuffs, and rumen bacteria use specialized functions to cooperatively access the energy stored in plant cell walls. The ability to predict interactions of rumen bacteria through computational modeling would enable characterization of complex dynamics to enhance fermentation. Therefore, a model was generated to describe pairwise interactions and substrate utilization of four rumen bacteria. R. albus 7, R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown in mono- and pairwise cultures in a cellobiose-containing medium for 18 h and sampled for optical density, abundance, and cellobiose utilization. The hybrid-model utilized a combination of interaction-focused population data and substrate-focused consumer resource data. Modeled interactions indicated four of the six co-cultures resulted in one-species dominant cultures and only two co-cultures displayed co-existence. Overall, the interaction- and substrate-based model developed in this study accurately described experimental population and substrate dynamics in mono- and pairwise cultures. Additionally, the model was able to describe intrinsic growth rates and substrate requirements for all four strains. This proposed framework can serve as a pipeline for understanding more complex, mixed-microbial communities. The approaches validated in this work can be used to further assess the interaction dynamics of rumen and gut microorganisms to develop practical nutritional solutions for improved animal health and performance.

Graduation Semester

2023-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2023 Sara Tondini

Owning Collections