Soil Science Society of America Journal 65:347-351 (2001)
© 2001 Soil Science Society of America
DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY
Procedure for Determining the Biodegradation of Radiolabeled Substrates in a Calcareous Soil
Lena Ström,
Douglas L. Godbold and
David L. Jones
School of Agricultural and Forest Sci., Univ. Wales-Bangor, Gwynedd, LL57 2UW, UK
Corresponding author (Lena.Strom{at}planteco.lu.se)
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ABSTRACT
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Calcareous soils are frequently typified by a low availability of plant nutrients due to poor solubility of these elements at high pH. Calcicole plants have recently been shown to release organic acids in response to the nutrient deficient conditions prevailing in these soils. It has been speculated, however, that the efficiency of this nutrient mobilization mechanism may be significantly reduced by microbial degradation of the organic acids. In conventional methods, root exudate degradation is typically determined by the addition of 14C-radiolabeled substrates to soil and subsequent tracking of their fate with time by trapping evolved 14CO2 in a strong alkali trap. However, in calcareous soils, 14CO2 and H214CO3 produced by microbial decomposition may become trapped as Ca(H14CO3)2. The aim of this study was to develop and validate an experimental procedure for the accurate quantification of 14C-labeled substrate degradation rates in calcareous soils. Conventional methods for determining 14C-labeled substrate decomposition rates in calcareous soils are inaccurate due to incomplete recovery of 14CO2. Up to 49% of the 14CO2 produced during microbial degradation of 14C-labeled organic acids (malate, oxalate, citrate) was trapped as carbonate in this calcareous soil (pH 7.58). For an acid soil (pH 4.32) no detectable amount of 14CO2 was trapped. We describe a simple, accurate, and reliable method, which includes a postincubation HCl addition, for the accurate determination of 14CO2-evolution and substrate degradation in calcareous soils.
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INTRODUCTION
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CALCAREOUS SOILS cover more than 30% of the earth's land surface and are characterized by a high base status and pH between 7.5 and 8.5 depending on the quantity of CaCO3 present (Chen and Barak, 1982). These soils are typically low in availability of plant nutrients (e.g., Fe, Mn, Cu, Zn, and P) due to poor solubility of these elements at high pH and the formation of relatively insoluble Ca2+ complexes. This nutrient limitation can provide a serious constraint to plant growth, resulting in reduced crop yields, unless fertilizers are applied to alleviate nutrient deficiency (Marschner, 1995; Zaiter et al., 1993).
In natural and seminatural ecosystems where fertilization is unlikely, successful plant establishment and colonization of calcareous soils requires a mechanism for enhancing nutrient solubilization. Recent studies have shown that calcicole plants (i.e., those that can establish on calcareous soils) generally have enhanced concentrations of organic acids (oxalate, citrate) in their rhizosphere compared with calcifuge plants (those that do not establish on calcareous soils) (Ström et al., 1994; Tyler and Ström, 1995; Ström, 1997). However, the behavior of organic acids in the rhizosphere is complex (Jones, 1998). To fully evaluate the importance of this proposed nutrient mobilization mechanism requires a detailed understanding of the sorption, leaching, complexation, and biological reactions of organic acids in soil.
Once exuded, low molecular weight substances such as organic acids can lead to significant proliferation of microorganisms in the soil (Vancura and Hovadik, 1965; Vancura and Hanzlikova, 1972; Matsumoto et al., 1979). Furthermore, the degradation rates of organic acids have been reported to be rapid, at least in acidic Typic Fragiochrept soils (Jones et al., 1996). If calcicole plants are releasing organic acids in response to, for example, the nutrient deficient conditions prevailing in calcareous soils, it can be speculated that the impact of the nutrient mobilizing mechanism may be significantly reduced by microbial degradation of the mobilizing substance, as has been shown to occur for phytosiderophores (von Wirén et al., 1993).
In conventional methods the rate of root exudate degradation in soil is typically determined by the addition of 14C-radiolabeled substrates to soil and the subsequent tracking of their fate with time (Wolf et al., 1994; Jones and Darrah, 1994; Jones et al., 1996). In these studies, the rate of substrate degradation via microbial mineralization is measured by trapping any evolved 14CO2 in a strong alkali trap (e.g., 1 M NaOH) causing the formation of stable NaH14CO3, which can be determined by liquid scintillation counting (Jones et al., 1996). However, in calcareous soils, where the pH often exceeds 7 and carbonate equilibria dominate the chemistry of the soil solution, this method may not function satisfactorily. The pKa values of carbonic acid (H2CO3, pKa1 = 6.38 and pKa2 = 10.32) indicate that any 14CO2 or H214CO3 produced in an acid soil (pH 
6) will primarily be released from the soil as 14CO2, whereupon it can be successfully trapped by strong alkali. However, in an alkaline soil (pH 7) it is probable that significant quantities of 14CO2 and H214CO3 produced by the microorganisms will become trapped in the soil through the formation of Ca(H14CO3)2. It has been proposed that this potential underestimation of 14CO2 production through soil Ca(H14CO3)2 trapping can be prevented through a controlled acidification of the soil prior to experimentation (i.e., decalcification; Guerin, 1999). This method recommends that the soil pH be acidified to 
6 with HCl before addition of the substrate and estimation of microbial degradation by 14CO2 trapping in alkali. However, this acidification is likely to cause significant changes to the physiology of the microbial population as a result of direct acid addition (pH stress), increased salt stress (from liberated Ca2+ and added Cl-), changes in soil solution chemistry, and a disruption of membrane transport systems.
The aim of this study was therefore to develop and validate an experimental procedure for the accurate quantification of 14C-labeled substrate degradation rates in calcareous soils.
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MATERIALS AND METHODS
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Soils
Two soils of contrasting pH were used to develop and test the method: Soil 1 represents a calcareous Typic Rendoll, derived from Ordovician limestone and is located on the alvar of Öland in Sweden (56° 40'N, 16° 30'E). The site has a total annual rainfall of 388 mm, mean annual temperature of 7.1°C, slope of 2.4°, elevation of 30 m and is dominated by calcicole vegetation-for example, sagebrush (Artemisia campestris L.), melic (Melica ciliata L.), and sedum (Sedum album L.). Soil 2 represents an acidic Typic Fragiochrept derived from Ordovician mudstones and shale and is located in Abergwygregyn, North Wales, UK. The site has a total annual rainfall of 1250 mm, mean annual temperature of 10.6°C, slope of 38°, elevation of 500 m and is dominated by calcifuge vegetation—for example, bedstraw (Galium saxatile L.), sheep fescue (Festuca ovina L.), and bentgrass (Agrostis capillaris L.).
Soil samples were collected from the Ah horizon (0–10 cm) of each soil using a spade, sieved to pass 6 mm, and kept field moist at 10°C until required. Chemical and physical properties of the soils are provided in Table 1. All analyses were performed in triplicate.
Measurement of Carbon-14 Organic Acid Degradation
Step 1
A 14C-radiolabeled organic acid solution (0.50 mL, specific activity 0.34 kBq mL-1) was added to 5.0 g of field moist soil (100% of water holding capacity at 0.033 MPa) contained in 60-mL polypropylene tubes. Three individual organic acids, 14C-citric acid (1,5-14C; ICN Pharmaceuticals Inc., Irvine, CA; 3.7 GBq mmol-1), 14C-malic acid (1,2,3,4-14C; NEN Life Science Products, Boston, MA; 7.0 GBq mmol-1), and 14C-oxalic acid (1,2-14C; Sigma Chemical Co., St Louis, MO; 0.2 GBq mmol-1), were added to soil (0.1 mL g-1) at two concentrations representing low (0.005 µmol g-1 soil) and high (5 µmol g-1 soil) rhizosphere soil concentration levels (Jones, 1998). Following addition, the soils were briefly shaken to ensure soil mixing and incubated at 25°C for 1, 3, 6, and 24 h in hermetically sealed 60-mL polypropylene tubes. The 14CO2 produced by biodegradation of the organic acids was collected by placing a plastic scintillation vial containing 1.0 mL of 1 M NaOH inside the polypropylene tube, on top of the soil but separated from direct contact with the soil by a spacer. The apparatus is shown schematically in Fig. 1
. The 14CO2 trapped as NaH14CO3 in the NaOH was determined by liquid scintillation (Wallac 1404 scintillation counter, Wallac EG&G, Milton Keynes, UK) using alkali compatible scintillation fluid (Wallac Optiphase 3; Wallac EG&G).
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Fig. 1. Schematic representation of the 14CO2 recovery system. Step 1 involves the addition of radiolabeled compounds to soil, incubation, and trapping any 14CO2 evolved with a static NaOH trap. Step 2 involves the release of any additional 14CO2 held in the soil by application of HCl and trapping evolved 14CO2 with a series of forced-air NaOH traps. The figure is drawn to scale
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Step 2
After removal of the NaOH trap, 10 mL of 1 M HCl was added to the soil to release 14CO2 potentially trapped in the soil. The HCl addition leads, theoretically, to the release of 14CO2 from solid phase Ca(H14CO3)2. Any evolved 14CO2 was then captured as NaH14CO3 by passing air over the sample and subsequent flow through five successive 15-mL polypropylene tubes containing 5.0 mL of 1 M NaOH (Fig. 1, Step 2). Air was passed over the sample until CO2 emission from the soil following HCl addition had ceased (i.e., 60 min).
Measurement of Alkali Trap Efficiency
The efficiency of the alkali traps to capture evolved 14CO2 was determined by the addition of 10 mL of 1 M HCl to a NaH14CO3 solution (1 mM; 0.34 kBq mL-1; 1-14C; Sigma Chemical Co.; 0.2 GBq mmol-1) followed by the capture of 14CO2 in five successive alkali traps.
The efficiency of the soil acidification procedure (Step 2) was evaluated by adding 0.5 mL of NaH14CO3 (0.34 kBq mL-1) to 5 g of soil at rates of 0.001, 0.01, or 0.1 µmol g-1 soil according to the experimental setup shown in Fig. 1. The evolution of 14CO2 was determined using alkali traps during a 6-h period (Step 1), after which 10 mL of 1 M HCl was added to the soil and the procedure described above followed (Step 2).
Whether the soil acidification procedure in Step 2 resulted in any oxidation of added 14C-labeled organic acids directly to 14CO2 and, thus, the risk of capturing 14CO2 not released as a result of microbial biodegradation was determined. Hydrogen chloride (10 mL of 1 M) was added to 5 g of soil directly (i.e., 2 min) after organic acid addition and to pure solutions of organic acids (without soil) and evolved 14CO2 collected as described above.
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RESULTS AND DISCUSSION
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As predicted from the solution chemical equilibrium constants of carbonic acid, significant quantities of 14CO2 produced during the microbial degradation of 14C-labeled organic acids became trapped in the calcareous soil (Fig. 2a and 2b)
. This is presumably due to the pH of the soil (pH 7.6) being greater than the pKa1 of carbonic acid (6.38). In contrast, no significant 14CO2 trapping was observed in the acid soil as the soil pH (4.32) was well below the pKa1 value for carbonic acid. In the majority of substrate incubations performed in this study, the portion of 14CO2 trapped in the calcareous soil was greater than the amount recovered in the NaOH trap (Fig. 2a and 2b). In both soil types, organic acid degradation was rapid in agreement with previous studies on the mineralization of low molecular weight root exudate components (Coody et al., 1986). No consistent differences were apparent in the rate of organic acid degradation in either the acid or calcareous soil (sum of 14CO2 recovered in Steps 1 and 2), although in two cases the biodegradation was higher in the acid soil (0.05 mM malate and oxalate, P > 0.05, paired t-test, Fig. 2a).
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Fig. 2. Microbial degradation of organic acids as measured by microbially respired 14CO2 in a high pH calcareous Typic Rendoll and low pH Typic Fragiochrept soil. (A) Low (0.005 µmol g-1) and (B) high (5 µmol g-1) organic acid addition to the soil. Clear bars represent the amount of 14CO2 recovered in a static NaOH-trap (Step 1), while striped bars represent the amount of 14CO2 recovered after the addition of HCl to the soil (Step 2; see Materials and Methods for details). Values represent means ± standard error of the mean, n = 3 (n.d. indicates not detectable)
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The aim of this study was to develop an experimental procedure for the accurate quantification of 14C-labeled substrate degradation rates in calcareous soils. Relative to calcifuge plants calcicole plants have recently been shown to have higher concentrations of organic acids in their rhizosphere soil solution, presumably in response to the nutrient deficient conditions prevailing in calcareous soils (Ström, 1997). The release of organic acids by roots into the rhizosphere can be expected to cause an increase in the growth of preexisting rhizosphere microorganisms and act as chemoattractants stimulating the movement of motile microorganisms towards the root (Barbour et al., 1991; Zheng and Sinclair, 1996; Jones, 1998). Therefore, it may be hypothesized that the potential for microbial degradation of organic acids is higher in the rhizosphere relative to the bulk soil. Studying rhizosphere soil naturally results in small soil samples, and we therefore chose a relatively small sample size (5 g) for the development of this method. However, small sample sizes can result in difficulties in controlling water potential, so that for incubation periods longer than 24 h it might be necessary to pass moist air over the sample.
The method of applying HCl after incubation proved satisfactory in releasing CO2 trapped in the soil. The recovery of added NaH14CO3 (0.001, 0.01, and 0.1 µmol g-1 soil) in a static alkali trap from the acid soil ranged between 92 and 100% at the end of 6 h incubation. Upon soil acidification with HCl to release any trapped 14CO2 only a very small additional amount of 0.06 to 1.3% was retrieved (Fig. 3)
. In contrast, the recovery of 14CO2 in the calcareous soil by a static alkali trap only ranged from 41 to 46%. However, upon HCl acidification a further 41 to 49% was liberated, indicating significant trapping of CO2 in the soil probably as Ca(H14CO3)2. In all cases the recovery was not 100%, possibly due to the uptake of some NaH14CO3 into microbial cells and subsequent conversion to other compounds incapable of liberation by HCl (Thomsen and Kristensen, 1997). The addition of 10 mL of 1 M HCl to 5 g of soil was clearly sufficient to achieve significant acidification resulting in a drop of pH to between 2 and 2.5 in the calcareous soil and <1 in the acid soil. During the acidification procedure (Fig. 1, Step 2) the presence of five successive NaOH collection traps proved sufficient to recover 100% of the 14CO2 released from the calcareous soil upon HCl addition (Table 2). This was also confirmed by collecting the 14CO2 liberated from the acidification of a NaH14CO3 solution by HCl (data not presented). In addition to a static trap (Step 1), a flow-through system was chosen, since air needs to be passed over the sample to collect emitted 14CO2 following the HCl addition in Step 2. This can be achieved without a substantial 14CO2 loss by interconnecting several traps. As is shown in Table 2, the first trap collected only 55% of emitted 14CO2, whereas five interconnected traps collected 100%. The addition of HCl did not result in any oxidation of organic acids directly to 14CO2 as the recovery of 14CO2 was below the detection limit following both HCl addition to soil 2 min after organic acid addition, and directly to the organic acid solutions.
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Fig. 3. Recovery (percentage of added NaH14CO3) of 14CO2 following the addition and incubation of a 14C-labeled bicarbonate (NaH14CO3) solution (0.001, 0.01, and 0.1 µmol g-1 soil) in a high pH calcareous Typic Rendoll (T. Rend.) or low pH Typic Fragiochrept (T. Frag.) soil. Hydrogen chloride (1 M) was added to the soil after 6 h (denoted by arrow). Values represent means ± standard error of the mean, n = 3
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Table 2. Amount of 14CO2 recovered in five successive alkali traps (1 M NaOH) following the acidification of NaH14CO3 by HCl (10 mL 1 M) after addition to the calcareous Typic Rendoll soil. Values represent means ± SEM, n = 3
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Our results clearly show that measurement of the microbial consumption of 14C-radiolabeled substrates in this calcareous soil was greatly underestimated using standard procedures (Wolf et al., 1994; Domsch et al., 1973; Behera and Wagner, 1974; Anderson, 1982; Alef, 1995). In all soils containing significant quantities of CaCO3, and where the soil pH is above pKa1 of carbonic acid, concentrated acid should be added to the soil in order to fully recover any 14CO2 trapped as carbonates and bicarbonates.
Furthermore, our results confirm that the traditional method of capturing 14CO2 using a strong alkali trap inside the incubation vial (Fig. 1, Step 1; Jones et al., 1996) functions satisfactory for the acid soil tested (Fig. 2). Again this relationship probably holds true as long as the pH of the soil studied is sufficiently below the pKa1 of carbonic acid. However, the exact pH point where HCl treatment starts to be of importance remains to be determined but is probably not determined solely by pH and/or CaCO3 content. In addition to these factors, others might also be of importance, for example, dominant cations, texture, and CaCO3 particle size.
The protocol described here gave an efficient recovery of trapped 14CO2 for the calcareous soil upon applying 10 mL of 1 M HCl to 5 g of soil. However, if calcareous soils are evaluated with a higher carbonate content than that employed here (204 g kg-1) the efficiency of the acidification step should be validated either by measuring the final pH of the soil or, if possible, by determining 14CO2 recovery from added NaH14CO3 as described above. In addition, due to the volume of CO2 released from more calcareous soils than that used here, the volume and number of NaOH traps may have to be modified to ensure 100% 14CO2 recovery.
In conclusion, conventional methods for determining 14C-labeled substrate decomposition rates in calcareous soils appear to be highly inaccurate due to the incomplete recovery of 14CO2. The modified method described here, however, provides a simple and reliable method for accurately determining 14CO2 evolution and substrate degradation in calcareous soils.
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ACKNOWLEDGMENTS
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This work was supported by the Leverhulme Trust, Overseas Development Program, Grant no. F/174/N. We would also like to thank Andrew Owen and Germund Tyler for their experimental support.
Received for publication March 30, 2000.
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REFERENCES
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ABSTRACT
INTRODUCTION
