Comparison of glomalin and humic acid in eight native US soils

Two important extractable fractions of soil organic matter (SOM) are humic acid (HA) and glomalin-related soil protein (glomalin). Optimizing the purity of each fraction is necessary to correlate fraction quantity and molecular characteristics with soil quality. Manipulation of extraction sequence and controlled precipitation of HA were used to evaluate co-extraction of HA and glomalin. Eight bulk soil samples (0 to 10 cm depth) were collected from four U.S. states (Colorado, Nebraska, Maryland, and Georgia). In Experiment 1, glomalin extraction (50 MM citrate, pH 8.0, at 121 degrees C) was followed by HA extraction (0.1 N NaOH at room temperature), and Experiment 2 used the reciprocal sequence. Experiment 2 HA was precipitated stepwise at pH levels 2.5, 2.0, and 1.0. Gravimetric weight, Bradford-reactive soil protein (BRSP), and immunoreactive soil protein (IRSP), along with percentages of C, N, H, and Fe, were used to compare glomalin and HA. The HA fraction from Experiment 2 contained 2-fold greater amounts of BRSP than HA from Experiment I and showed that pre-extraction of glomalin improved the purity of HA. The glomalin fraction from Experiment 1 contained 1.5 times the BRSP of glornalin from Experiment 2 and was twice the gravimetric weight. BRSP and gravimetric weight were concentrated in HA that precipitated at pH 2.5 or 2.0 and percentage IRSP was significantly higher in the pH 2.5 precipitate. The results indicate that glomalin should be extracted first and examined as a biomolecule separate from the humic acid mixture. Percentages C, H, N, and Fe in glomalin varied across soils and experiments. In seven soils, the changes in Fe percentage in glomalin from Experiment 1 to 2 were significantly correlated with the changes in glornalin weight and %C. Iron in glornalin from Experiment 1 was related to soil pH and clay content, whereas soil organic C was positively and significantly correlated with Experiment 1 glornalin BRSP and IRSP. In Experiment 1, a recalcitrant pool of glornalin was released by treating soil with NaOH, suggesting that a fraction of glomalin is difficult to remove from soil and glornalin extraction efficiency could be improved. Refinements to extraction and purification protocols such as pretreatment of soils with HCI and sequential extraction can facilitate studies on organic matter structure and function.