Soil Science Society of America Journal 65:1734-1735 (2001)
© 2001 Soil Science Society of America
DIVISION S-3 - SOIL BIOLOGY & BIOCHEMISTRY
A pressure cooker method to extract glomalin from soils
S. F. Wright* and
L. Jawson
USDA-ARS-Sustainable Agricultural Systems Lab., Bldg. 001, Rm. 140, BARC-W, 10300 Baltimore Ave., Beltsville, MD 20705
* Corresponding author (wrights{at}ba.ars.usda.gov)
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ABSTRACT
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Glomalin, a glycoprotein produced by arbuscular mycorrhizal fungi, is an important constituent of soil organic matter. Glomalin is insoluble in water or salt solutions commonly used in soil extractions. Solubilization of glomalin does occur in the presence of citrate at the temperature achieved by an autoclave (121°C, 103 kPa). Most soil-testing laboratories have access to all of the instruments required for glomalin analysis except an autoclave. Small (4-L) and large (14-L) pressure cookers were compared with a bench-top autoclave to test glomalin extraction from three soils using 50 mM citrate, pH 8.0 as the extracting agent. Glomalin concentrations, as measured by the Bradford protein assay, were identical for extracts from the autoclave and the 14-L pressure cooker when 103 kPa (121°C) was achieved in both vessels. The 4-L pressure cooker extracted less glomalin than the other vessels. A pressure cooker that will achieve 103 kPa is a low-cost substitute for an autoclave to extract glomalin from soils.
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INTRODUCTION
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THE RELATIONSHIP BETWEEN soil aggregation and total glomalin from arbuscular mycorrhizal fungi (AMF) has been demonstrated (Wright and Upadhyaya, 1998; Wright et al., 1999; Wright and Anderson, 2000). Native glomalin is insoluble. To extract and solubilize glomalin, a method was developed that uses citrate at the temperature attained by an autoclave (121°C) (Wright et al., 1996; Wright and Upadhyaya, 1996). Quantification of glomalin in extracts is by the modified Bradford protein assay or an immunoassay (Wright and Upadhyaya, 1996). Soil testing laboratories often are not equipped with an autoclave, and small autoclaves that cost several thousand dollars are not spacious enough to accommodate more than a few extraction tubes. The objective of this study was to determine whether a pressure cooker could be used as a low-cost alternative to an autoclave to achieve the temperature required to solubilize glomalin.
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Materials and Methods
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Soils used for comparison of extraction methods were collected from the following soil series and locations: Baltimore (fine-loamy, mixed, mesic Mollic Hapludalf), MD; Elliott (fine, illitic, mesic Aquic Argiudoll), IL; and Georgeville (kaolinitic, thermic Typic Kanhapludult), VA. Soil pH (in 0.01M CaCl2) was 5.02, 5.08 and 3.48 for Baltimore, Elliott, and Georgeville, respectively. Percent carbon was 3.1, 2.3 and 4.9 for Baltimore, Elliott, and Georgeville, respectively.
One-g samples from each soil (replicated for each vessel) were placed in 50 mL centrifuge tubes. Eight mL of 50 mM trisodium salt of citric acid, pH 8.0 (adjusted with HCl) was added to each tube, tubes were vortexed, and caps with small vent holes were placed on the tubes.
Soils were extracted for 1-h intervals in an autoclave (Market Forge Sterilmatic Model Stm-E), a 4-L pressure cooker (Presto Model #124), and a 14-L pressure cooker (Presto Model 01/CA16) equipped with a pressure gauge. The pressure cookers were placed on hot plates and timing was started when the vessels were fully pressurized. The 4-L cooker has a regulator cap that vibrates when the vessel is pressurized. The autoclave and 14-L cooker were allowed to reach 103 kPa. Vessels were depressurized according to operating instructions before samples were removed.
Samples were centrifuged at 6,850 x g for 10 min and the supernatant was poured off and retained. Samples were sequentially extracted by adding an additional 8 mL of citrate to the soil pellet, vortexing, and heating in the appropriate vessel for 1-h intervals. A 1-mL aliquot from the first extraction of autoclaved samples was removed and saved for analysis. Extractions were continued until the supernatant was a pale straw color (a total of 3 h for these soils), and the extracts were pooled. A 1-mL aliquot was removed from each pooled extract for analysis. The autoclaved 1-h aliquots and the pooled extract aliquots were centrifuged at 10,000 x g for 3 min in a microtube centrifuge then analyzed for glomalin using the Bradford protein assay as modified by Wright and Upadhyaya (1996). Protein concentrations in the pooled samples were subjected to analysis of variance (Statistix, Tallahassee, FL) to determine treatment differences within a soil at P < 0.05. Treatment means were separated by Scheffe's multiple range test.
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Results and Discussion
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Glomalin concentration was not different between the autoclave and 14-L pressure cooker in any of the soils, but was lower using the 4-L pressure cooker compared with other methods (Fig. 1) . An operator cannot quantify the pressure or temperature attained in the 4-L pressure cooker, and we do not recommend modifying the factory model to change the pressure or add a gauge due to safety concerns about using pressurized vessels. Since the results obtained with the 4-L pressure cooker were not comparable with the 14-L pressure cooker, we recommend purchasing the larger model equipped with a pressure gauge to be assured that the pressure reaches 103 kPa.
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Fig. 1. Total glomalin obtained by extracting soils in an autoclave (A), a 4-L pressure cooker (4-L PC) and a 14-L pressure cooker (14-L PC) using 50mM citrate, pH 8.0. Three sequential 1-h extractions were run, and extracts were pooled. Common letters indicate replicate treatment means within a soil are not significantly different at the 0.05 level by Scheffe's multiple range test. Aliquots of extracts from the first hour in the autoclave also were collected and analyzed for glomalin.
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Three sequential 1-h extractions were sufficient to extract total glomalin from the soils in this experiment with an autoclave or the larger cooker. The number of autoclave cycles required to extract glomalin from different soils varies from 2 to 7 h for the temperate soils we have tested in other experiments. Extracting for longer than 1 h per cycle is not recommended in the types of vessels described in this report because it is not possible to monitor the water level or add water while a vessel is pressurized.
We routinely analyze the combined supernatant from all extractions for total glomalin rather than analyzing each sequential extraction. Glomalin has a red-brown color, and concentration is related to the intensity of the color. Color comparisons among extracts give a qualitative assessment of glomalin concentrations and may be used to evaluate whether to run the more quantitative Bradford assay. The first autoclave extraction removed 56, 35, and 59% of the total glomalin from the Baltimore, Elliott, and Georgeville soils, respectively (Fig. 1), indicating that the first 1-h extract should not be used to make comparisons among different soils.
A 14-L pressure cooker with a pressure gauge costs approximately $80. A hot plate averages $300 but is common in most laboratories. The 4-L pressure cooker costs approximately $30 but was not adequate for glomalin extraction. The 14-L pressure cooker or similar size models can accommodate up to thirty-six 50 mL centrifuge tubes. An autoclave large enough to accommodate 72 to108 samples can average $2000–5000. Thus, as a low-cost alternative to an autoclave, a large pressure cooker that can achieve 103 kPa as determined by a pressure gauge can substitute for a small autoclave to extract glomalin from soils.
Centrifugation is required to separate the supernatant from soil. Most laboratories have a centrifuge as standard equipment. We use an intermediate speed centrifuge (at 6,850 x g) to pellet the soil between sequential extractions and a small high-speed micro centrifuge (10,000 x g) prior to performing the Bradford assay. Clay must be removed from the solution to prevent spurious Bradford protein assay results.
The Bradford protein dye reagent can be purchased from biochemical supply sources or made from purchased dye and other chemicals (Suelter, 1985). Cost of the reagent for a Bradford assay is nominal, but the reagent is considered hazardous due to the presence of phosphoric acid and methanol. The standard procedure for the Bradford assay requires a spectrophotometer, which is integral to most soil testing laboratories. We use an assay modified for 96-well microtiter plates. This requires a microplate reader equipped with a 590 or 595 nm filter, which is an additional expense. However, small assay volumes minimize hazardous waste disposal costs. Extracts can be stored at 4°C for 2 to 4 wk until laboratory equipment is available.
We have found little, if any, extraneous protein in glomalin extracts obtained by using citrate at autoclave temperature of 121°C. Evidence for this is that the same amount of glomalin from many crude extracts of a variety of soils give consistent banding on gel electrophoresis (Wright and Upadhyaya, 1996), and the bands correspond with an equivalent amount of protein from extracts of AMF hyphae (data not shown).
In conclusion, we recommend the use of a pressure-regulated pressure cooker that attains 103 kPa to extract glomalin from soil when an autoclave is not available. The Bradford assay is a widely available protein assay that can be used to quantify glomalin (Wright and Upadhyaya, 1996).
Received for publication October 28, 2000.
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REFERENCES
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- Suelter, C.H. 1985. A practical guide to enzymology. John Wiley & Sons, New York.
- Wright, S.F., M. Franke-Snyder, J.B. Morton, and A. Upadhyaya. 1996. Time-course study and partial characterization of a protein on arbuscular mycorrhizal hyphae during active colonization of roots. Plant Soil 181:193–203.
- Wright, S.F., and A. Upadhyaya. 1996. Extraction of an abundant and unusual protein from soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Sci. 161:575–586.
- Wright, S.F., and A. Upadhyaya. 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil 198:97–107.
- Wright, S.F., J.L. Starr, and I.C. Paltineanu. 1999. Changes in aggregate stability and concentration of glomalin during tillage management transition. Soil. Sci. Soc. Am. J. 63:1825–1829.[Abstract/Free Full Text]
- Wright, S.F., and R.L. Anderson. 2000. Aggregate stability and glomalin in alternative crop rotations for the central Great Plains. Biol. Fert. Soils 31:249–253.
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ABSTRACT
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