Quantifying the Responses of Mixed Rumen Microbes to Excess Carbohydrate

Abstract

The aim of this study was to determine if a mixed microbial community from the bovine rumen would respond to excess carbohydrate by accumulating reserve carbohydrate, energy spilling (dissipating excess ATP energy as heat), or both. Mixed microbes from the rumen were washed with N-free buffer and dosed with glucose. Total heat production was measured by calorimetry. Energy spilling was calculated as heat production not accounted by (i) endogenous metabolism (heat production before dosing glucose) and (ii) synthesis of reserve carbohydrate (heat from synthesis itself and reactions yielding ATP for it). For cells dosed with 5 mM glucose, synthesis of reserve carbohydrate and endogenous metabolism accounted for nearly all heat production (93.7%); no spilling was detected (P ‫؍‬ 0.226). For cells dosed with 20 mM glucose, energy spilling was not detected immediately after dosing, but it became significant (P < 0.05) by approximately 30 min after dosing with glucose. Energy spilling accounted for as much as 38.7% of heat production in one incubation. Nearly all energy (97.9%) and carbon (99.9%) in glucose were recovered in reserve carbohydrate, fermentation acids, CO 2 , CH 4 , and heat. This full recovery indicates that products were measured completely and that spilling was not a methodological artifact. These results should aid future research aiming to mechanistically account for variation in energetic efficiency of mixed microbial communities.

Accumulation of Reserve Carbohydrate by Rumen Protozoa and Bacteria in Competition for Glucose

“…For the moderate glucose concentration (4.62 or 5 mM), ϳ60% of glucose carbon was recovered in the reserve carbohydrate of bacteria and protozoa combined (Table 1). This value compares with the 60% of carbon recovered for mixed rumen microbes given 5 mM glucose (19). For the high glucose concentration (20 mM), ϳ30% was recovered in the reserve carbohydrate of bacteria and protozoa combined (Table 1), which is lower than the 53% observed for mixed rumen microbes given 20 mM glucose (19).…”

“…When we washed mixed rumen microbes with N-free buffer and dosed them with 5 or 20 mM glucose, we recovered Ͼ50% of the glucose in reserve carbohydrate at the time of peak accumulation (19). Characterization of the reserve carbohydrate identified it as glycogen.…”

“…Local regression (LOCFIT package of R [20]) was used to fit time series data to smooth curves, as described previously (18,19). Original data are presented alongside the smooth curves in the figures, and smooth curves were used for calculations (e.g., carbon recovery and changes in reserve carbohydrate) and statistical analysis.…”

“…Chemical analyses, direct counts of protozoa and bacteria, and calculation of carbon recovery were done as previously described (18,19). Briefly, pellets were analyzed for reserve carbohydrate by using the anthrone method and for protein by using the Pierce BCA assay kit (product number 23227; Thermo Scientific, Rockford, IL).…”

“…Carbon recovery was calculated from concentrations of glucose, reserve carbohydrate, short-chain fatty acids, CO 2 , and CH 4 both before and after glucose dosing. CO 2 and CH 4 concentrations were determined by reaction stoichiometry (18,19).…”