A Comparison of Three Artificial Rumen Systems for Rumen Microbiome Modeling
Abstract
:1. Introduction
2. Materials and Methods
2.1. Rumen Fluid Collection
2.2. Feed Collection and Preparation
2.3. In Vitro Rumen Systems
2.4. Experimental Design
2.5. Ankom Gas Production System
2.6. RUSITEC, Polypropylene (PP) Vessels
2.7. RUSITEC Prime, Stainless Steel Vessels
2.8. Sample Collection
2.9. Volatile Fatty Acid and Greenhouse Gas Analysis
2.10. Statistics
2.11. DNA Extractions
2.12. PCR Amplification, Library Preparation, and Sequencing
2.13. Microbiome Analysis
3. Results
3.1. Gas Production
3.2. VFA Comparison
3.3. 16S rRNA Gene Sequencing Results
3.4. ITS Sequencing Results
4. Discussion
4.1. In Vitro Rumen Modeling
4.2. Platform Comparison
4.3. Selecting Semi-Continuous Modeling Platforms
4.4. Primed for Rumen Modeling
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Firmicutes | Bacteroidota | Proteobacteria | Verrucomicrobiota | Euryarchaeota | Thermoplasmatota | Spirochaetota | Fibrobacterota | Patescibacteria | Cyanobacteria | Actinobacteriota | Chloroflexi | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
Cow-24 h 1 | 34.34238 | 52.09 | 0.81 | 4.18 | 1.95 | 0.27 | 1.34 | 1.32 | 0.76 | 1.45 | 0.27 | 0.53 |
Cow-48 h 1 | 30.95768 | 58.68 | 0.52 | 2.33 | 1.01 | 0.32 | 2.02 | 0.89 | 0.90 | 1.28 | 0.23 | 0.28 |
Cow-72 h 1 | 37.34452 | 50.87 | 0.70 | 2.27 | 1.34 | 0.41 | 2.68 | 1.20 | 0.88 | 0.84 | 0.24 | 0.41 |
Cow-96 h 1 | 36.784 | 49.67 | 0.76 | 2.99 | 1.71 | 0.36 | 2.27 | 1.58 | 0.97 | 1.34 | 0.23 | 0.38 |
Cow-120 h 1 | 28.51376 | 53.59 | 1.37 | 4.42 | 1.63 | 0.55 | 3.08 | 1.98 | 0.86 | 2.69 | 0.13 | 0.35 |
Cow-Total 2 | 33.59 ± 3.79 | 52.98 ± 3.5 | 0.83 ± 0.32 | 3.24 ± 1.01 | 1.53 ± 0.36 | 0.38 ± 0.11 | 2.28 ± 0.66 | 1.39 ± 0.41 | 0.87 ± 0.08 | 1.52 ± 0.7 | 0.22 ± 0.05 | 0.39 ± 0.09 |
Ankom-24 h | 18.39 ± 1.68 | 62.39 ± 2.14 | 2.78 ± 0.79 | 9.96 ± 0.94 | 1.09 ± 0.33 | 0.54 ± 0.11 | 0.67 ± 0.11 | 0.07 ± 0.04 | 0.59 ± 0.09 | 2.44 ± 0.19 | 0.15 ± 0.06 | 0.28 ± 0.05 |
Ankom-48 h | 41.87 ± 3.64 | 37.1 ± 2.45 | 1.71 ± 0.22 | 10.69 ± 1.02 | 2.61 ± 0.27 | 0.24 ± 0.09 | 0.95 ± 0.27 | 0.04 ± 0.01 | 0.28 ± 0.04 | 1.81 ± 0.48 | 0.71 ± 0.03 | 1.16 ± 0.05 |
Ankom-72 h | 70.25 ± 6.23 | 13.67 ± 5.14 | 1.13 ± 0.41 | 7.18 ± 0.97 | 2.47 ± 0.38 | 0.12 ± 0.04 | 0.66 ± 0.29 | 0.02 ± 0 | 0.23 ± 0.06 | 1.08 ± 0.49 | 1.2 ± 0.2 | 1.26 ± 0.1 |
Ankom-96 h | 82.51 ± 0.77 | 6.47 ± 1.11 | 0.68 ± 0.08 | 3.86 ± 0.43 | 2.2 ± 0.23 | 0.11 ± 0.07 | 0.47 ± 0.14 | 0.01 ± 0.01 | 0.17 ± 0.06 | 0.9 ± 0.43 | 1.12 ± 0.22 | 0.96 ± 0.2 |
Ankom-120 h | 91.82 ± 2.01 | 2.71 ± 0.78 | 0.17 ± 0.03 | 1.61 ± 0.42 | 1.47 ± 0.54 | 0.05 ± 0.04 | 0.18 ± 0.11 | 0 ± 0 | 0.07 ± 0.04 | 0.44 ± 0.12 | 0.85 ± 0.11 | 0.44 ± 0.07 |
Ankom-Total 2 | 81.52 ± 9.93 | 7.62 ± 5.51 | 0.66 ± 0.47 | 4.22 ± 2.49 | 2.05 ± 0.57 | 0.09 ± 0.06 | 0.44 ± 0.27 | 0.01 ± 0.01 | 0.16 ± 0.08 | 0.8 ± 0.44 | 1.05 ± 0.23 | 0.88 ± 0.38 |
RUSITEC PP-24 h | 30.26 ± 4.35 | 54.37 ± 2.54 | 2.23 ± 0.78 | 5.53 ± 0.85 | 1.46 ± 0.68 | 1.37 ± 0.39 | 0.61 ± 0.13 | 0.04 ± 0.03 | 0.84 ± 0.08 | 1.27 ± 0.73 | 0.11 ± 0.05 | 0.56 ± 0.22 |
RUSITEC PP-48 h | 35.55 ± 1.12 | 41.99 ± 0.68 | 3.64 ± 0.28 | 5.16 ± 0.51 | 1.26 ± 0.01 | 2.43 ± 0.39 | 3.27 ± 0.68 | 0.82 ± 0.46 | 0.7 ± 0.05 | 1.84 ± 0.38 | 0.18 ± 0.07 | 0.98 ± 0.12 |
RUSITEC PP-72 h | 45.06 ± 8.38 | 35.72 ± 3.82 | 5.23 ± 1.86 | 3.47 ± 2.25 | 0.97 ± 0.4 | 1.9 ± 0.9 | 2.47 ± 0.96 | 0.78 ± 0.33 | 0.23 ± 0.12 | 1.65 ± 1.2 | 0.21 ± 0.13 | 0.88 ± 0.36 |
RUSITEC PP-96 h | 48.26 ± 9.7 | 34.75 ± 6.67 | 5.29 ± 1.39 | 2.01 ± 0.14 | 0.53 ± 0.15 | 1.56 ± 0.61 | 2.5 ± 1.07 | 1.14 ± 0.66 | 0.06 ± 0.03 | 2.16 ± 1.73 | 0.32 ± 0.08 | 0.56 ± 0.12 |
RUSITEC PP-120 h | 42.13 ± 1.28 | 40.43 ± 1.29 | 3.64 ± 0.49 | 1.5 ± 0.05 | 0.56 ± 0.08 | 1.48 ± 0.08 | 2.7 ± 0.23 | 1.1 ± 0.01 | 0.03 ± 0.01 | 4.07 ± 0.51 | 0.58 ± 0.07 | 0.68 ± 0.03 |
RUSITEC PP-Total 2 | 40.12 ± 8.79 | 41.53 ± 8.18 | 4.03 ± 1.57 | 3.68 ± 1.9 | 0.98 ± 0.5 | 1.77 ± 0.63 | 2.28 ± 1.15 | 0.75 ± 0.54 | 0.4 ± 0.35 | 2.06 ± 1.27 | 0.26 ± 0.17 | 0.74 ± 0.25 |
RUSITEC Prime-24 h | 23.89 ± 3.29 | 57.27 ± 2.73 | 3.47 ± 0.44 | 6.62 ± 0.29 | 1.41 ± 0.74 | 1.59 ± 0.56 | 0.69 ± 0.16 | 0.19 ± 0.05 | 1 ± 0.1 | 2.12 ± 0.2 | 0.06 ± 0.04 | 0.42 ± 0.1 |
RUSITEC Prime-48 h | 32.5 ± 2.3 | 42.9 ± 2 | 4.74 ± 0.23 | 8.32 ± 0.65 | 1.55 ± 0.4 | 1.83 ± 0.18 | 2.09 ± 0.19 | 0.5 ± 0.22 | 0.91 ± 0.04 | 1.95 ± 0.08 | 0.19 ± 0.06 | 0.68 ± 0.1 |
RUSITEC Prime-72 h | 39.5 ± 3.89 | 36.33 ± 1.2 | 4.76 ± 1.18 | 7.4 ± 2.32 | 1.34 ± 0.3 | 1.93 ± 0.54 | 2.6 ± 0.61 | 1.32 ± 1.5 | 0.43 ± 0.14 | 1.65 ± 0.45 | 0.22 ± 0.07 | 1.14 ± 0.22 |
RUSITEC Prime-96 h | 44.16 ± 1.49 | 33.73 ± 2.43 | 4.2 ± 0.11 | 6.2 ± 1.2 | 1.61 ± 0.51 | 2.22 ± 0.73 | 2.46 ± 0.13 | 0.66 ± 0.24 | 0.25 ± 0.06 | 1.12 ± 0.25 | 0.21 ± 0.08 | 1.5 ± 0.46 |
RUSITEC Prime-120 h | 47.1 ± 2.37 | 32.75 ± 1.08 | 4.97 ± 0.82 | 4.48 ± 0.25 | 1.14 ± 0.27 | 2.38 ± 0.45 | 2.86 ± 0.19 | 0.66 ± 0.09 | 0.09 ± 0.02 | 0.85 ± 0.57 | 0.27 ± 0.01 | 0.97 ± 0.34 |
RUSITEC Prime-Total 2 | 37.43 ± 8.99 | 40.6 ± 9.53 | 4.43 ± 0.81 | 6.6 ± 1.68 | 1.41 ± 0.44 | 1.99 ± 0.53 | 2.14 ± 0.84 | 0.67 ± 0.7 | 0.54 ± 0.38 | 1.54 ± 0.58 | 0.19 ± 0.09 | 0.94 ± 0.45 |
Desulfobacterota | Elusimicrobiota | Synergistota | Planctomycetota | Bdellovibrionota | Unassigned | WPS-2 | SAR324_clade(Marine_group_B) | Campilobacterota | Armatimonadota | <0.1% | ||
Cow-24 h 1 | 0.18 | 0.21 | 0.01 | 0.08 | 0.01 | 0.18 | 0.00 | 0.00 | 0.01 | 0.00 | 0.01 | |
Cow-48 h 1 | 0.25 | 0.12 | 0.02 | 0.06 | 0.01 | 0.09 | 0.01 | 0.00 | 0.00 | 0.00 | 0.00 | |
Cow-72 h 1 | 0.37 | 0.16 | 0.03 | 0.08 | 0.02 | 0.13 | 0.00 | 0.01 | 0.00 | 0.01 | 0.00 | |
Cow-96 h 1 | 0.41 | 0.17 | 0.03 | 0.10 | 0.01 | 0.17 | 0.00 | 0.02 | 0.00 | 0.00 | 0.00 | |
Cow-120 h 1 | 0.22 | 0.32 | 0.04 | 0.07 | 0.01 | 0.14 | 0.00 | 0.01 | 0.01 | 0.00 | 0.00 | |
Cow-Total 2 | 0.29 ± 0.1 | 0.19 ± 0.08 | 0.03 ± 0.01 | 0.08 ± 0.02 | 0.01 ± 0 | 0.14 ± 0.04 | 0 ± 0 | 0.01 ± 0.01 | 0 ± 0 | 0 ± 0 | 0 ± 0 | |
Ankom-24 h | 0.13 ± 0.03 | 0.28 ± 0.03 | 0.03 ± 0.01 | 0.05 ± 0.03 | 0.02 ± 0.02 | 0.11 ± 0.02 | 0 ± 0.01 | 0.02 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 1 | |
Ankom-48 h | 0.29 ± 0.12 | 0.24 ± 0.06 | 0.05 ± 0.01 | 0.11 ± 0.02 | 0.01 ± 0 | 0.08 ± 0.01 | 0 ± 0 | 0.04 ± 0.01 | 0 ± 0 | 0.01 ± 0 | 0 ± 0 | |
Ankom-72 h | 0.37 ± 0.17 | 0.14 ± 0.01 | 0.08 ± 0.03 | 0.05 ± 0.02 | 0 ± 0 | 0.04 ± 0.02 | 0 ± 0 | 0.02 ± 0 | 0 ± 0 | 0.01 ± 0 | 0 ± 1 | |
Ankom-96 h | 0.29 ± 0.22 | 0.06 ± 0.03 | 0.07 ± 0.04 | 0.06 ± 0.01 | 0 ± 0 | 0.04 ± 0.01 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0.01 ± 0.01 | 0 ± 0 | |
Ankom-120 h | 0.12 ± 0.05 | 0.03 ± 0.01 | 0.01 ± 0.01 | 0.02 ± 0.01 | 0 ± 0 | 0.02 ± 0.02 | 0 ± 0 | 0 ± 0.01 | 0 ± 0 | 0 ± 0 | 0 ± 0 | |
Ankom-Total 2 | 0.26 ± 0.18 | 0.08 ± 0.05 | 0.06 ± 0.04 | 0.04 ± 0.02 | 0 ± 0 | 0.03 ± 0.02 | 0 ± 0 | 0.01 ± 0.01 | 0 ± 0 | 0.01 ± 0.01 | 0 ± 0 | |
RUSITEC PP-24 h | 0.4 ± 0.12 | 0.27 ± 0.21 | 0.06 ± 0.01 | 0.16 ± 0.04 | 0.17 ± 0.07 | 0.18 ± 0.03 | 0 ± 0.01 | 0.06 ± 0.02 | 0.01 ± 0 | 0.01 ± 0.01 | 0.01 ± 0.01 | |
RUSITEC PP-48 h | 0.57 ± 0.28 | 0.65 ± 0.07 | 0.23 ± 0.07 | 0.18 ± 0.05 | 0.14 ± 0.05 | 0.25 ± 0.03 | 0.01 ± 0.01 | 0.06 ± 0.02 | 0.02 ± 0 | 0.01 ± 0.01 | 0.01 ± 0.01 | |
RUSITEC PP-72 h | 0.27 ± 0.1 | 0.35 ± 0.22 | 0.16 ± 0.1 | 0.16 ± 0.1 | 0.04 ± 0.03 | 0.25 ± 0.24 | 0.06 ± 0.07 | 0.04 ± 0.01 | 0.02 ± 0 | 0 ± 0.01 | 0.01 ± 0.01 | |
RUSITEC PP-96 h | 0.16 ± 0.05 | 0.21 ± 0.1 | 0.07 ± 0.03 | 0.11 ± 0.04 | 0.01 ± 0 | 0.15 ± 0.15 | 0.09 ± 0.05 | 0.02 ± 0.02 | 0.01 ± 0.01 | 0 ± 0 | 0 ± 0 | |
RUSITEC PP-120 h | 0.23 ± 0.08 | 0.32 ± 0.18 | 0.08 ± 0.02 | 0.13 ± 0.02 | 0.01 ± 0 | 0.16 ± 0.06 | 0.12 ± 0.04 | 0.02 ± 0 | 0.02 ± 0 | 0 ± 0 | 0 ± 0 | |
RUSITEC PP-Total 2 | 0.33 ± 0.2 | 0.36 ± 0.21 | 0.12 ± 0.09 | 0.15 ± 0.06 | 0.08 ± 0.08 | 0.2 ± 0.12 | 0.05 ± 0.06 | 0.04 ± 0.02 | 0.01 ± 0.01 | 0.01 ± 0.01 | 0.01 ± 0.01 | |
RUSITEC Prime-24 h | 0.25 ± 0.07 | 0.4 ± 0.07 | 0.05 ± 0 | 0.13 ± 0.01 | 0.21 ± 0.08 | 0.15 ± 0.02 | 0.01 ± 0.01 | 0.05 ± 0.01 | 0.01 ± 0 | 0.01 ± 0.01 | 0 ± 0 | |
RUSITEC Prime-48 h | 0.35 ± 0.12 | 0.58 ± 0.05 | 0.17 ± 0.04 | 0.18 ± 0.03 | 0.19 ± 0.06 | 0.19 ± 0.03 | 0.01 ± 0 | 0.06 ± 0.01 | 0.05 ± 0.01 | 0.01 ± 0 | 0 ± 0 | |
RUSITEC Prime-72 h | 0.23 ± 0.16 | 0.42 ± 0.13 | 0.17 ± 0.09 | 0.22 ± 0.08 | 0.07 ± 0.06 | 0.13 ± 0.05 | 0.01 ± 0.02 | 0.04 ± 0.02 | 0.04 ± 0.01 | 0.01 ± 0 | 0 ± 0 | |
RUSITEC Prime-96 h | 0.39 ± 0.15 | 0.23 ± 0.1 | 0.18 ± 0.06 | 0.32 ± 0.11 | 0.07 ± 0.01 | 0.14 ± 0.02 | 0.11 ± 0.07 | 0.05 ± 0.01 | 0.09 ± 0.02 | 0.01 ± 0.01 | 0 ± 0 | |
RUSITEC Prime-120 h | 0.42 ± 0.2 | 0.25 ± 0.1 | 0.12 ± 0.06 | 0.24 ± 0.08 | 0.02 ± 0.02 | 0.1 ± 0.02 | 0.11 ± 0.02 | 0.05 ± 0.03 | 0.11 ± 0.03 | 0 ± 0 | 0 ± 0 | |
RUSITEC Prime-Total 2 | 0.33 ± 0.15 | 0.38 ± 0.15 | 0.14 ± 0.07 | 0.22 ± 0.09 | 0.11 ± 0.09 | 0.14 ± 0.04 | 0.05 ± 0.06 | 0.05 ± 0.02 | 0.06 ± 0.04 | 0.01 ± 0 | 0 ± 0 |
Neocallimastigomycota | Basidiomycota | Ascomycota | Mucoromycota | Rozellomycota | Mortierellomycota | Unassigned | |
---|---|---|---|---|---|---|---|
Cow-24 h 1 | 96.11250628 | 0.772045634 | 2.648585301 | 0.150678051 | 0.004305087 | 0.203774126 | 0.108105522 |
Cow-48 h 1 | 96.84371793 | 0.84187354 | 2.098238412 | 0.106597651 | 0.007437045 | 0.046605485 | 0.055529939 |
Cow-72 h 1 | 98.86820007 | 0.245738198 | 0.742363393 | 0.092678406 | 0.031828947 | 0 | 0.019190983 |
Cow-96 h 1 | 97.73148245 | 0.797102957 | 1.307831589 | 0.104892922 | 0 | 0 | 0.058690087 |
Cow-120 h 1 | 84.96034062 | 10.95354187 | 3.323833396 | 0.683308725 | 0.020602273 | 0 | 0.058373107 |
Cow-Total 2 | 94.9 ± 5.65 | 2.72 ± 4.61 | 2.02 ± 1.03 | 0.23 ± 0.26 | 0.01 ± 0.01 | 0.05 ± 0.09 | 0.06 ± 0.03 |
Ankom-24 h | 89.12 ± 2.78 | 2.56 ± 0.35 | 7.23 ± 2.1 | 0.61 ± 0.35 | 0.08 ± 0.08 | 0 ± 0 | 0.4 ± 0.1 |
Ankom-48 h | 68.48 ± 13.45 | 6.19 ± 1.4 | 18.2 ± 8.58 | 5.85 ± 4.33 | 0.16 ± 0.2 | 0 ± 0 | 1.12 ± 0.49 |
Ankom-72 h | 37.14 ± 7.59 | 16.63 ± 1.76 | 36.24 ± 3.78 | 7 ± 0.35 | 1.18 ± 2.03 | 0.5 ± 0.87 | 1.3 ± 0.61 |
Ankom-96 h | 18.62 ± 0.76 | 33.75 ± 0.55 | 42.03 ± 3.04 | 2.92 ± 0.92 | 0.12 ± 0.17 | 0 ± 0 | 2.56 ± 0.65 |
Ankom-120 h | 25.56 ± 36.1 | 34.69 ± 5.61 | 34.69 ± 27.05 | 2.42 ± 2.3 | 0.09 ± 0.16 | 0 ± 0 | 2.55 ± 2.21 |
Ankom-Total 2 | 49.87 ± 31.59 | 17.7 ± 13.99 | 26.65 ± 17.42 | 3.82 ± 3.17 | 0.34 ± 0.92 | 0.11 ± 0.4 | 1.52 ± 1.27 |
RUSITEC PP-24 h | 81.2 ± 22.71 | 15.77 ± 21.41 | 2.12 ± 0.46 | 0.66 ± 0.73 | 0.18 ± 0.28 | 0 ± 0 | 0.07 ± 0.04 |
RUSITEC PP-48 h | 84.93 ± 2.24 | 11.03 ± 3.05 | 3.09 ± 0.98 | 0.67 ± 0.2 | 0.01 ± 0.02 | 0 ± 0 | 0.28 ± 0.21 |
RUSITEC PP-72 h | 76.16 ± 15.56 | 18.73 ± 13.27 | 3.89 ± 2.43 | 1.06 ± 0.62 | 0 ± 0 | 0 ± 0 | 0.17 ± 0.03 |
RUSITEC PP-96 h | 79.74 ± 4.39 | 14.45 ± 2.39 | 4.66 ± 1.83 | 0.96 ± 0.38 | 0.04 ± 0.07 | 0 ± 0 | 0.15 ± 0.04 |
RUSITEC PP-120 h | 69.8 ± 21.73 | 21.52 ± 17.63 | 6.5 ± 1.86 | 1.31 ± 1.21 | 0.27 ± 0.38 | 0 ± 0 | 0.6 ± 0.65 |
RUSITEC PP-Total 2 | 78.98 ± 13.45 | 15.93 ± 11.69 | 3.88 ± 1.98 | 0.9 ± 0.58 | 0.09 ± 0.19 | 0 ± 0 | 0.23 ± 0.26 |
RUSITEC Prime-24 h | 84.98 ± 10.96 | 10.97 ± 11.84 | 2.82 ± 0.97 | 0.98 ± 0.4 | 0.06 ± 0.03 | 0 ± 0 | 0.19 ± 0.1 |
RUSITEC Prime-48 h | 75.58 ± 19.68 | 20.38 ± 20.12 | 2.27 ± 1.75 | 1.41 ± 1.33 | 0 ± 0 | 0 ± 0 | 0.35 ± 0.22 |
RUSITEC Prime-72 h | 69.38 ± 17.51 | 23.48 ± 17.89 | 4.93 ± 3.36 | 1.26 ± 1.49 | 0 ± 0 | 0 ± 0 | 0.95 ± 1.4 |
RUSITEC Prime-96 h | 43.21 ± 23.97 | 37.08 ± 5.96 | 16.51 ± 17.07 | 1.57 ± 0.89 | 0.67 ± 1.16 | 0 ± 0 | 0.95 ± 0.91 |
RUSITEC Prime-120 h | 61.71 ± 38.46 | 27.37 ± 25.34 | 10.02 ± 13.82 | 0.73 ± 0.9 | 0 ± 0 | 0 ± 0 | 0.17 ± 0.16 |
RUSITEC Prime-Total 2 | 66.97 ± 24.96 | 23.86 ± 17.28 | 7.31 ± 10.09 | 1.19 ± 0.96 | 0.15 ± 0.52 | 0 ± 0 | 0.52 ± 0.74 |
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Shaw, C.A.; Park, Y.; Gonzalez, M.; Duong, R.A.; Pandey, P.K.; Brooke, C.G.; Hess, M. A Comparison of Three Artificial Rumen Systems for Rumen Microbiome Modeling. Fermentation 2023, 9, 953. https://doi.org/10.3390/fermentation9110953
Shaw CA, Park Y, Gonzalez M, Duong RA, Pandey PK, Brooke CG, Hess M. A Comparison of Three Artificial Rumen Systems for Rumen Microbiome Modeling. Fermentation. 2023; 9(11):953. https://doi.org/10.3390/fermentation9110953
Chicago/Turabian StyleShaw, Claire A., Yuna Park, Maria Gonzalez, Rich A. Duong, Pramod K. Pandey, Charles G. Brooke, and Matthias Hess. 2023. "A Comparison of Three Artificial Rumen Systems for Rumen Microbiome Modeling" Fermentation 9, no. 11: 953. https://doi.org/10.3390/fermentation9110953