Optimization of the bicinchoninic acid assay for quantifying carbohydrates of soil extracellular polymeric substances

Abstract

Background and aimsThe bicinchoninic acid (BCA) method was not yet applied on soil extracts of extracellular polymeric substances (EPS) to quantify polysaccharides, although this might be possible by introducing a cleavage step to produce monosaccharides. A pre-extraction with CaCl2 to remove interfering substances is usually performed before extracting EPS. The main objective of this study was to optimize the BCA assay for total carbohydrates quantification by applying a hydrolysis step to the EPS extracts, while also testing carbohydrate contents of CaCl2 pre-extracts.MethodsTotal carbohydrates were quantified with BCA in EPS extracts of three soils, after hydrolysis with H2SO4, using two acid concentrations (0.75 and 1.0 M), three hydrolysis temperatures (100, 120 and 130 °C), and five hydrolysis times (10, 30, 50, 70, and 90 min). EPS were extracted with the cation exchange resin (CER) method adapted to soils. Two versions of pre-extraction with CaCl2 were tested twice consecutively.ResultsMore carbohydrates were measured after hydrolysis with 0.75 M H2SO4 at below 100 °C and after 10 min for all soils. Decreasing values were seen after longer reaction times and higher temperatures. CaCl2 extracted no or negligible amounts of carbohydrates from the soil.ConclusionThe pre-extraction step can be done without in most cases. The BCA assay is free of toxicity and easily performed, while also tolerant to interferences from most compounds in EPS extracts. These characteristics highlight the potential of this method for a rapid quantification of carbohydrates in studies of extractable polymers in several areas of soil biogeochemistry.