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Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

ABSTRACT: The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate accumulation of protozoa and bacteria individually. When mixtures were dosed with a moderate concentration of glucose (4.62 or 5 mM) (n = 2 each), protozoa accumulated large amounts of reserve carbohydrate; 58.7% (standard error of the mean [SEM], 2.2%) glucose carbon was recovered from protozoal reserve carbohydrate at time of peak reserve carbohydrate concentrations. Only 1.7% (SEM, 2.2%) was recovered in bacterial reserve carbohydrate, which was less than that for protozoa (P < 0.001). When provided a high concentration of glucose (20 mM) (n = 4 each), 24.1% (SEM, 2.2%) of glucose carbon was recovered from protozoal reserve carbohydrate, which was still higher (P = 0.001) than the 5.0% (SEM, 2.2%) glucose carbon recovered from bacterial reserve carbohydrate. Our novel competition experiments directly demonstrate that mixed protozoa can sequester sugar away from bacteria by accumulating reserve carbohydrate, giving protozoa a competitive advantage and stabilizing fermentation in the rumen. Similar experiments could be used to investigate the importance of starch sequestration.

SUBMITTER: Denton BL

PROVIDER: S-EPMC4325151 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

Denton Bethany L BL Diese Leanne E LE Firkins Jeffrey L JL Hackmann Timothy J TJ

Applied and environmental microbiology 20141229 5

The aim of this study was to determine if rumen protozoa could form large amounts of reserve carbohydrate compared to the amounts formed by bacteria when competing for glucose in batch cultures. We separated large protozoa and small bacteria from rumen fluid by filtration and centrifugation, recombined equal protein masses of each group into one mixture, and subsequently harvested (reseparated) these groups at intervals after glucose dosing. This method allowed us to monitor reserve carbohydrate ...[more]

PMID: 25548053

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Project description:The rumen protozoa, alongside fungi, comprise the eukaryotic portion of the rumen microbiome. Rumen protozoa may account for up to 50% of biomass, yet their role in this ecosystem remains unclear. Early experiments inferred a role in carbohydrate and protein metabolism, but due to their close association with bacteria, definitively attributing these functions to the protozoa was challenging. The advent of 'omic technologies has created opportunities to broaden our understanding of the rumen protozoa. This study aimed to utilize these methods to further our understanding of the role that protozoa play in the rumen in terms of their metabolic capacities, and in doing so, contribute valuable sequence data to reduce the chance of mis or under-representation of the rumen protozoa in meta'omic datasets. Rumen protozoa were isolated and purified using glucose-based sedimentation and differential centrifugation, extracted RNA was Poly(A) fraction enriched and DNase treated before use in a phage-based, cDNA metatranscriptomic library. Biochemical activity testing of the phage library showed 6 putatively positive plaques in response to carboxymethyl cellulose agar (indicative of cellulose activity), and no positive results for tributyrin (indicative of esterase/lipase activity) or egg yolk agar (indicative of proteolysis). Direct sequencing of the cDNA was also conducted using the Illumina HiSeq 2500. The metatranscriptome identified a wealth of carbohydrate-active enzymes which accounted for 8% of total reads. The most highly expressed carbohydrate-active enzymes were glycosyl hydrolases 5 and 11, polysaccharide lyases and deacetylases, xylanases and enzymes active against pectin, mannan and chitin; the latter likely used to digest rumen fungi which contain a chitin-rich cell membrane. Codon usage analysis of expressed genes also showed evidence of horizontal gene transfer, suggesting that many of these enzymes were acquired from the rumen bacteria in an evolutionary response to the carbohydrate-rich environment of the rumen. This study provides evidence of the significant contribution that the protozoa make to carbohydrate breakdown in the rumen, potentially using horizontally acquired genes, and highlights their predatory capacity.

Dataset Information

Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

Publications

Accumulation of reserve carbohydrate by rumen protozoa and bacteria in competition for glucose.

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