The aim of this study was to evaluate the capability of different in vitro incubation systems to maintain a rumen microbiota similar to that inoculated to the system from the rumen. Batch cultures (DaisyII ANKOM incubator, DAI and Wheaton flasks, WB) and single-flow continuous-culture fermenters (CC) were tested with pooled rumen liquor from goats and using the same substrate across systems. Samples of filtered rumen content (0h) were kept and used to compare the microbial ecosystem with that obtained after incubation in the different systems for 48h in DAI; 24 and 72h in WB and 4 days in CC. In CC, samples from the effluents obtained on the 5th day of incubation were also taken to compare the microbiota with that developed in the fermenter contents the previous day. Microbial abundances and bacterial diversity were evaluated by real time-PCR (qPCR) and terminal-restriction fragment length polymorphism (T-RFLP), respectively. Total bacteria population declined (P≤0.015) in both batch cultures after 48h (DAI system) or 72h (WB) of incubation, but did not change (P≤0.134) after 24h of incubation in WB or 4 days of incubation in CC. Fibrobacter succinogenes, Ruminococcus flavefaciens and fungi decreased (P<0.001) in DAI and WB after 48 and 72h, respectively; however, there was an increase (P<0.001) of these microorganisms after 24h of incubation in WB and 4 days in CC. Protozoa density declined (P≤0.028) after 48h of incubation in DAI, after 72h of incubation in WB and after 4 days in CC. Methanogens increased (P=0.001) after 48h of incubation in DAI and WB and declined (P≤0.002) in numbers after 72h in WB or 4 days of incubation in CC. When the microbial abundances in fermenter effluents were compared to those in the contents, only declines (P≤0.05) in fibrolytic microorganisms and protozoa were observed. With regard to the overall bacterial diversity, the Shannon index and Pielou evenness were lower (P≤0.049) in the DAI and in WB, after 48 and 24h of incubation, respectively, compared to those in the original rumen liquor. On the other hand, no changes (P=0.469) in the Shannon index were observed among inoculum, fermenter contents and effluents in the CC system. The structure of the bacterial community that developed in the DAI and WB systems after 48 and 24h of incubation, respectively, was rather different compared to the population originally present in the inoculum. On the contrary, the bacterial community profile that developed in the CC was very similar to the original inoculum after 4 days of incubation. Effluents and fermenter contents had very similar profiles.The results of this study show that batch cultures displayed important changes in microbial abundances and bacterial species profile while continuous-culture fermenters maintain a more stable microbiota and bacterial community structure, throughout the incubation of the same type of rumen inoculum.
Bibliographical noteFunding Information:
This research was supported by Junta de Andalucía (Excellence Programme , Project P05-AGR-00408) and the Spanish National Research Council (“Proyecto Intramural”- CSIC-200740I021 ). E. C. Soto gratefully acknowledges support from CSIC for I3P grant. Thanks to J. Fernández, T. García and I. Jiménez for technical assistance.
- Bacterial community
- In vitro systems
- Microbial populations