V. SOIL ORGANIC MATTER ANALYSES Section Editors: E.G. Gregorich and M.H. Beare ß 2006 by Taylor & Francis Group, LLC. ß 2006 by Taylor & Francis Group, LLC. Chapter 45 Carbon Mineralization D.W. Hopkins Scottish Crop Research Institute Dundee, Scotland, United Kingdom 45.1 INTRODUCTION Organic matter in soils is the complex mixture of organic compounds derived from the dead and decaying remains of plants, animals, and microorganisms, and their corpses and meta- bolic wastes at different stages of decomposition. Mineralization of organic carbon (C) is the conversion from the organic form to inorganic compounds as a result of decomposition reactions carried out by decomposer organisms, the vast majority of which are microorgan- isms (bacteria and fungi) (Gregorich et al. 2001). In the process of utilizing soil organic matter, heterotrophic soil organisms release CO 2 during respiration. The release of CO 2 as a metabolic by-product of organic matter decomposition is referred to as C mineralization. Because soil organic matter is a complex mixture of organic compounds of different biological origins and at different stages of decay, C mineralization is the result of a complex set of biochemical processes conducted by a wide range of organisms. Despite the fact that it is a simplification of the actual process, C mineralization measurements are commonly used in investigations of soils and the data have a wide range of applications in agriculture, forestry, ecology, and the environmental sciences. One reason for this is the relative ease with which CO 2 can be measured in the laboratory. There are a wide range of methods for measuring CO 2 production in the field and at the landscape scale, but this chapter is concerned with measuring C mineralization under controlled laboratory conditions and only limited reference is made to field methods to illustrate some principles. Data on mineralization of soil C may be used in two ways. The rate of C mineralization measured over periods from a few days to a few weeks is commonly used as an indicator of general biological activity because it is an integrated measure of the combined respiration rate of all the organisms active in the soil under specific conditions. However, with time and without inputs of fresh organic matter, the rate of C mineralization declines as the most readily available soil organic matter is depleted. The total CO 2 -C released when the rate of production subsides is an index of the readily mineralizable fraction of organic C in soil. Given enough time, however, all, or virtually all, soil organic matter will be mineralized and therefore the total mineralizable C fraction is equivalent, or close, to the total organic C content of the soil. It is important to distinguish between the total amount of C that will be mineralized eventually and the fraction readily mineralized during the initial period of rapid ß 2006 by Taylor & Francis Group, LLC. decomposition when the most easily utilized and accessible components are decomposed. This chapter focuses on the readily mineralizable fraction of the soil organic matter, which is believed to be a biologically meaningful, albeit operationally defined, fraction of the soil organic matter. However, defining biologically meaningful fractions is fraught with diffi- culties (Hopkins and Gregorich 2005). Because the readily mineralizable C is one such operationally defined fraction, the conditions under which it is measured need to be carefully specified. It should also be recognized that there is no inherent linkage between the size of the readily mineralizable C fraction and the rate of C mineralization measured over the short- term. Two soils may contain the same amount of readily mineralizable C, but because of more favorable conditions for decomposition, one may have a much faster initial rate of C mineralization than the other. 45.2 SOIL PREPARATION AND INCUBATION CONDITI ONS Before the start of the mineralization assay, some degree of sample preparation is inevitable, but in general, this should be kept to a minimum consistent with being able to prepare a representative and suitable sample. Soil is usually sieved (<2 mm) in the field moist state to enable representative sampling and to remove stones and large pieces of plant material. Drying and grinding the soil should be avoided because these lead to substantial increases in mineralization, commonly referred to as a ‘‘flush’’ of respiration. The flush is caused by the mineralization of nonbiomass released from physical protection and the C from organi- sms killed by drying and rapid rehydration (Powlson 1980; Wu and Brookes 2005). Even sample collection and preparation without harsh treatments such as drying and grinding lead to a short-lived (3–4 days) flush of respiration. It is recommended that soil be preincubated under the same temperature and moisture conditions to be used in the C mineralization assay for a period of 7–10 days to allow equilibration before the start of the assay. Incubation temperatures in the range 208C–258C are frequently used (e.g., Hopkins et al. 1988; S ˇ imek et al. 2004), but the actual temperature used can be set to match the objectives of the particular study. If the aim of the investigation is specifically to determine the effect of temperature on mineralization, the incubation temperature is of paramount importance. Recent papers have drawn attention to the possibility that mineralization of different fractions of the soil organic matter may (or may not) respond differently to incubation temperature (Bol et al. 2003; Fang et al. 2005; Fierer et al. 2005), with obvious implications for predicting the effects of climate change on soil organic C reservoir. If a stable tempera- ture is required throughout an incubation, as is often the case, then it is necessary to use a temperature-controlled room or incubator. Similar to temperature, the moisture content of the soil during incubation needs careful consideration. Moisture contents between 50% and 60% water holding capacity (e.g., Rey et al. 2005; Wu and Brookes 2005) are commonly used because the optimum moisture content for mineralization usually falls in this range. However, alternative moisture contents are used when the aim of the investigation is to determine the effect of moisture or wet–dry cycles on mineralization (e.g., Rey et al. 2005; Chow et al. 2006; Hopkins et al. 2006). 45.3 INCUBATION AND DETECTION METHODS Three incubation approaches to measure C mineralization in soils in the laboratory are described. In two of them, the soil is enclosed in a sealed vessel and the CO 2 produced is either allowed to accumulate in the headspace and then determined, or the CO 2 is trapped ß 2006 by Taylor & Francis Group, LLC. as it is produced (usually in alkali solution) and then determined. In the third, the soil is incubated in a flow-through system in which the headspace is replaced by a stream of CO 2 - free air and the CO 2 released from the soil is trapped or measured continuously as the air flows out of the chamber. The particular choice of approach will depend on the equipment and other resources (e.g., financial) available to the investigator and a consideration of the advantages and disadvantages of the different methods (Table 45.1). The method of CO 2 analysis is determined by a combination of the incubation approach adopted and the instrumentation available. Four methods commonly used to determine CO 2 produced from soil are outlined below. 45.3.1 ACID–BASE TITRATIONS Carbon dioxide can be trapped in alkali (typically KOH or NaOH) and then determined by backtitration of the excess alkali with a dilute acid (Hopkins et al. 1988; Schinner et al. 1996). In its simplest form, this can be done by a manual titration using a burette with a pH indicator. Automatic titrators that measure pH with an electrode and deliver acid from a mechanized burette can increase the precision, although rarely the sample throughput. TABLE 45.1 Some Advantages and Disadvantages of Different Approaches to Determining C Mineralization in Soils under Laboratory Conditions Approach Advantages Disadvantages Closed chamber incubation with CO 2 accumulation in the headspace Inexpensive Common equipment requirements Easily replicated IRGA of CO 2 can be very rapid Composition of the atmosphere changes because of O 2 depletion and CO 2 enrichment, therefore unsuitable for long-term incubations (i.e., >5–10 days) unless the headspace is flushed Not suitable for soils with pH above neutrality because some CO 2 is absorbed in the soil solution Usually only suitable for short-term incubations Closed chamber incubation with CO 2 trapping Can be inexpensive Can have simple equipment requirements Usually easily replicated Usually suitable for both short- and long-term incubations Composition of the atmosphere changes because of O 2 and CO 2 depletion, therefore may unsuitable for long-term incubations if there is a large O 2 demand Automated, multichannel respirometers are expensive Manual titration of alkali traps can be time consuming and produce toxic waste products that require disposal Open chamber incubation with continuous flushing and CO 2 trapping Suitable for both long- and short-term incubations More expensive More complex equipment Less easily replicated ß 2006 by Taylor & Francis Group, LLC. 45.3.2 INFRARED G AS A NALYSIS Carbon dioxide absorbs radiation in the infrared region and detection of this absorbance is at the heart of infrared gas analyzers (IRGAs) used to determine CO 2 in both closed and open chamber incubation systems (e.g., Bekku et al. 1995; Schinner et al. 1996; Rochette et al. 1997; King and Harrison 2002). There are a range of IRGAs commercially available, and many of those used for measuring CO 2 from soil are modifications of systems used for photosynthesis measurements. 45.3.3 CONDUCTIOMETRY Carbondioxidetrappedinalkalicanbedeterminedconductiometricallyontheprinciplethatthe impedance of the alkali solution declines as CO 2 is absorbed. Although stand-alone conductio- metric systems can be assembled (Chapman 1971; Anderson and Ineson 1982), this method of CO 2 detection is usually an integral part of multichannel respirometers (Nordgren 1988) which are expensive, but permit a high degree of replication and near-continuous measurements. 45.3.4 G AS C HROMATOGRAPHY Gas chromatography (GC) provides very precise analysis, but is suitable only for incubation approaches in which CO 2 accumulates. There is wide variety of GCs available for CO 2 determination and a review of the different types is beyond the scope of this chapter. However, the commonest GC methods involve separation on packed columns and detection using either a thermal conductivity (i.e., hot-wire) detector (e.g., Hopkins and Shiel 1996; Schinner et al. 1996). One advantage of GC is that the instruments are very versatile and can be modified for use in many types of analyses other than CO 2 determination by reconfiguring the injector, column, and detector. 45.4 CLOSED CHAMBER INCUBATION WITH ALKALI CO 2 TRAPS 45.4.1 M ATERIALS AND REAGENTS 1 Incub ation jars with gast ight lids (Ma son or Kil ner types ; Figur e 45.1 ) 2 Glass vials (20–50 mL) for the alkali solution and water 3 M NaOH solution 4 0.5 M HCl solution 5 Phenolphthalein solution 6 1 M BaCl 2 7 Pipettes 8 Burette or automatic titrator 9 Magnetic stirrer (optional) 10 Incubator or co ntrolled environment room (optional) ß 2006 by Taylor & Francis Group, LLC. 45.4.2 PROCEDURE Weigh 100–150 g (dry weight equivalent) into jars and record the weight of each jar plus soil without its lid. Place one vial containing 10 mL of 1 M NaOH and one vial containing water into each jar and seal them with the lids (Figure 45.1). Incubate the jars in the dark and at the desired temperature. The CO 2 can be assayed at intervals of 3–10 days typically. For each mole of CO 2 trapped in the NaOH, 2 moles of NaOH will be converted to Na 2 CO 3 (Equation 45.1). Therefore, the total CO 2 produced is twice the depletion of NaOH in the trap. Remove the vials of water and NaOH and then backtitrate the excess NaOH with HCl (Equation 45.2) using phenolphthalein as an indicator after having removed dissolved CO 2 and carbonates by precipitation with the addition of 2 mL of BaCl 2 . 2NaOH þ CO 2 ! Na 2 CO 3 þ H 2 O (45:1) NaOH þ HCl ! NaCl þ H 2 O (45:2) For example, if 5 mL of 0.5 M HCl was required to backtitrate the excess NaOH in an alkali trap that originally contained 10 mL of 1.0 M NaOH after precipitating the carbonates with BaCl 2 , then the CO 2 content of the traps would be calculated as CO 2 in trap ¼ 0:5  (((V NaOH  C NaOH )=1000) À ((V HCl  C HCl )=1000)) (45:3) where V NaOH is the initial volume of NaOH (mL), C NaOH is the initial molar concentration of NaOH, V HCl is the volume of HCl used in the titration (mL), and C HCl is the molar concentration of HCl used in the titration. So, CO 2 in the trap ¼ 0:5  [((10  1:0)=1000) À ((5  0:5)=1000)] ¼ 0:00375 mol C H 2 O Alkali Soil FIGURE 45.1. Closed incubation vessel with NaOH traps for CO 2 . ß 2006 by Taylor & Francis Group, LLC. Where the incubation involved 100 g of dry weight equivalent soil and an incubation time of 48 h, the C mineralization rate would be calculated as C mineralization rate ¼ CO 2 in the trap =(soil mass in g  incubation time in h) ¼ 0:00375 =(100  48) ¼ 0:00000078 mol C g À1 soil h À1 or 0:78 mmol C g À1 soil h À1 (45:4) If the incubation is to be continued, wipe any condensation from the inside of the jar and the lid, weigh the jars, and correct for any weight loss by addition of water. Then put fresh NaOH and water vials in the jars, reseal them, and continue the incubation. 45.4.3 COMMENTS The method given here is very general and may be adapted to address a wide range of specific research questions. Among other factors, the amount of soil, the temperature and moisture conditions, the concentration and amount of NaOH, and the incubation time can all be adjusted to suit particular applications. It is, however, important to be sure that the headspace in the jars is large enough to avoid the risk of anaerobiosis during long-term incubations. Typically, 100– 150 g soil in a 1000 mL vessel is suitable for 3–4 days incubation intervals. It is also important to ensure that the amount of NaOH is adequate to trap all the CO 2 produced. If the amount of CO 2 produced is small, reducing the NaOH concentration will increase the sensitivity of the assay. Carbonic anhydrase can be added to the analyte to catalyze the dissolution of CO 2 in water and allow titration between two pH endpoints, 8.3 to 3.7 (Underwood 1961). An automatic titrator and a magnetic stirrer can be used to help improve the precision of the titration. However, these are not essential as the assays can be carried out satisfactorily using manual equipment provided the operator is careful and skilful. Commonly used protocols that employ closed chamber incubations to measure soil bio- logical activity and to quantify the amount of readily mineralizable C in soil are given below. Closed chamber techniques involving alkali traps for measuring CO 2 production in the field have also been described by Anderson (1982) and Zibilske (1994). 45.5 CLOSED CHAMBER INCUBATION WITH CO 2 ACCUMULATION 45.5.1 M ATERIALS AND REAGENTS 1 Mini aturized incub ation vessels (Figure 45.2a an d Figure 45.2b) 2 1% CO 2 gas standard mixtur e 3 Gas chromatograph 4 Incubator (optional) 45.5.2 PROCEDURE This procedure is based on that of Heilmann and Beese (1992) as modified by Hopkins and Shiel (1996). Weigh 10–15 g (dry weight equivalent) soil into glass vials, put them into the ß 2006 by Taylor & Francis Group, LLC. incubation chambers, and set the volume of the incubation chamber by adjusting the plunger before closing the three-way tap (Figure 45.2a). After 2–3 days, remove a sample of the headspace gas using the smaller sampling syringe, flushing it several times to ensure mixing. Analyze the gas sample by GC. Many GC configurations can be used. In the method of Hopkins and Shiel (1996), a GC fitted with a 1.32 m long  3 mm internal diameter stainless steel column packed with 80 =100 mesh Poropak Q and a thermal conductivity detector was used. After sampling the gas from the headspace, the air in the incubation chambers should be replenished before they are resealed and the incubation continued. The incubation chamber shown in Figure 45.2b is an adaptation of the chamber used in Figure 45.1, which can be used for CO 2 accumulation. 45.5.3 C OMMENTS Soils may contain CO 2 sinks, such as alkaline soil solution in which bicarbonate may accumulate (Martens 1987) and chemoautotrophic bacteria which reduce CO 2 (Zibilske 1994). The importance of these sinks is often overlooked, but in alkaline soils, where the capacity for CO 2 dissolution is large or where the respiratory CO 2 flux is small they may lead to underestimates of C mineralization, methods in which CO 2 is trapped may be preferable. The incubation chambers can be assembled from easily available materials; however, because some grades of plastic are permeable to CO 2 and the joints between components may leak, it is advisable either to check plastic materials before starting or to use glass equipment. If plastic syringes are used, care should be taken to ensure that the insides of the syringe barrels and the plungers do not get scored by soil particles as this will cause them to leak. Because the headspace volume is relatively small, prolonged incubation without replenishing the headspace is not advisable as this will increase the chance of anaerobiosis and will also increase the risk of leakage. Glass vial Soil 60 mL syringe Three-way valve 1 mL syringe Hypodermic needle Septum H 2 O Soil (a) (b) FIGURE 45.2. Two different (a and b) closed incubation vessels in which CO 2 can accumulate. ß 2006 by Taylor & Francis Group, LLC. 45.6 OPEN CHA MBER INCUB ATION The system outlined in Figure 45.3 is suitable for collecting CO 2 in an open chamber incubation in which the airflow is maintained either by a suction pump or vacuum line to draw air through the apparatus, or by air pumps (such as a diaphragm aquarium pump) or compressed gas cylinders to force air through the apparatus. Depending on the source of the air, it is necessary to consider the purity of the gas and if necessary use supplementary concentrated H 2 SO 4 scrubbers to remove organic contaminants from the compressed gas cylinder or carried over from the pumps. The CO 2 bubble traps on the upstream side of the soil can be replaced with soda lime traps. After the incubation, the contents of the NaOH traps are quantitatively transferred to a beaker and the CO 2 produced is determined by titration as described in Section 45.4.2. The main advantages of this approach are that there is no risk of anaerobiosis or leakage of accumulated CO 2 , and soil drying is reduced by the air flowing through the water bottle immediately upstream of the incubation chamber. The equipment can be assembled from easily available laboratory glassware. However, for replicated measurements multiple systems will be required and this will increase the amount of laboratory space required. 45.7 COND UCTIOMETRIC RESPIROMETERS There is a range of dedicated multichannel respirometers, which can be used to measure CO 2 production (and in some cases other gases) in soils, sediments, composts, animals, and cell cultures (including microorganisms). A systematic account of the operation of these instru- ments is beyond the scope of this chapter. The instrument which appears to be most widely used in soil research is the Respicond instrument (Nordgren 1988). This instrument com- prises up to 96 chambers (Figure 45.4) in which CO 2 is trapped in KOH. Absorbed CO 2 leads to a fall in the conductance of the trap as the KOH concentration falls. This change in Soil NaOH H 2 O NaOH NaOH Enlarged view Airflow Airflow To pump Multiple NaOH traps FIGURE 45.3. Open incubation vessel in which CO 2 released into a CO 2 -free air stream is trapped in NaOH traps. (Adapted from Zibilske, L.M., in R.W. Weaver, S. Angle, P. Bottomly, D. Bezdieck, S. Smith, A. Tabatabai, and A. Wollum (Eds), Methods of Soil Analysis, Part 2—Microbiological and Biochemical Processes, Soil Science Society of America, Madison, Wisconsin, 1994.) ß 2006 by Taylor & Francis Group, LLC. [...]... microbial biomass and organic matter caused by air-drying and rewetting of a grassland soil Soil Biol Biochem 37: 50 7 51 5 Zibilske, L.M 1994 Carbon mineralization In R.W Weaver, S Angle, P Bottomly, D Bezdieck, S Smith, A Tabatabai, and A Wollum, Eds Methods of Soil Analysis, Part 2—Microbiological and Biochemical Processes, SSSA Book Series No 5 Soil Science Society of America, Madison, WI, pp 8 35 863 Chapter... quite sensitive to sample pretreatment, particularly in the early phase of incubation (see Section 46.2 .5) 2 Mix 15 50 g of soil with sand at a soil: sand ratio of 1:1 for medium-textured soils and 1:2 for fine-textured soils It may be helpful to apply a light mist of water to prevent particle=aggregate size segregation during transfer to the leaching tubes 3 Sand soil mixture is supported in the leaching... for the continuous, long-term monitoring of soil respiration rate in large numbers of samples Soil Biol Biochem 20: 955 – 957 ß 2006 by Taylor & Francis Group, LLC Powlson, D.S 1980 The effects of grinding on microbial and non-microbial organic matter in soil J Soil Sci 31: 77– 85 Rey, A., Petsikos, C., Jarvis, P.G., and Grace, J 20 05 Effect of temperature and moisture on rates of carbon mineralization... review of more than 65 published papers, Gregorich et al (2006) showed that for agricultural mineral soils, the amount of soil C and N accounted for in POM is usually much greater than that in the LF On average, POM (50 –2000 mm diameter) accounted for 22% of soil organic C and 18% of total soil N In contrast, LF organic matter (specific gravity . 46.2 .5) . 2 Mix 15 50 g of soil with sand at a soil: sand ratio of 1:1 for medium-textured soils and 1:2 for fine-textured soils. It may be helpful to apply a light mist of water to prevent particle=aggregate. 955 – 956 . Wu, J. and Brookes, P.C. 20 05. The proportional mineralization of microbial biomass and organic matter caused by air-drying and rewetting of a grassland soil. Soil Biol. Biochem. 37: 50 7 51 5. Zibilske,. . 20: 955 – 957 . Powlson, D.S. 1980. The effects of grinding on microbial and non-microbial organic matter in soil. J. Soil Sci. 31: 77– 85. Rey, A., Petsikos, C., Jarvis, P.G., and Grace, J. 20 05.