Evaluation of methods to detect changes in reserve carbohydrate for mixed rumen microbes

• We compared methods to detect reserve carbohydrate in mixed rumen microbes.
• Methods were based on the anthrone reaction and amyloglucosidase hydrolysis.
• The anthrone method detected larger changes in reserve carbohydrate concentration.
• The anthrone method gave complete recovery of energy, carbon, and cell components.

Thin-layer chromatography and other analyses revealed that rumen microbes accumulated large amounts of glycogen. The aim of this study was to identify a method that would most accurately quantify accumulation and utilization of this reserve carbohydrate. For whole cells, the anthrone reaction detected more (P < 0.001) carbohydrate than did hydrolysis with amyloglucosidase, even after exhaustive extraction by bead beating (45 min) or KOH digestion (3 h). Less carbohydrate was detected after isolating reserve polysaccharide by ethanol precipitation. Compared to the amyloglucosidase hydrolysis method, the anthrone method detected a larger (P = 0.017) increase in cell carbohydrate when glucose (20 mM) was dosed in cultures. Additionally, it detected a larger (P = 0.049) decrease in cell carbohydrate after glucose was exhausted. This result indicated that the anthrone method detected more carbohydrate that functions as a reserve material, which accumulates during energy excess and is utilized for energy during starvation. For the anthrone method, recoveries for energy (97.5%), carbon (100.2%), and cell components (99.8%) were high, indicating carbohydrate was completely detected. For the amyloglucosidase hydrolysis method, recoveries of energy (88.9%), carbon (91.6%), and cell components (92.8%) were lower. Some authors have inferred from iodine staining that amyloglucosidase hydrolyzes all glycogen in cells. However, iodine did not stain glycogen remaining after cells were incompletely extracted intentionally. The anthrone method appeared to accurately quantify changes in reserve carbohydrate and shows merit for quantitative studies, whereas the amyloglucosidase hydrolysis method detected smaller changes and was less consistent with expected carbon and energy recovery.