Measurement of Microbial Biomass by Fumigation-Extraction in Soil ...

Published November, 1994

Measurement of Microbial Biomass by Fumigation-Extraction in Soil Stored Frozen Julien P. Winter,* Zhiyuan Zhang, Mario Tenuta, and R. Paul Voroney ABSTRACT This research examined the effect of freezing on soil microbial C and N measured by CHClj fumigation-extraction (CFE). A soil was stored frozen ( — 15°C) and sampled after 1, 7, and 170 d. Compared with a nonfrozen soil, freezing had no effect on microbial C or N, except after 7 d when C increased by 24% and N increased by 34%. The soil was also subjected to freeze-thaw cycles (18/6 h). Microbial C and N were unaffected by one cycle, increased with two, and decreased with three cycles. In a second experiment, soil was amended with 14C-glucose and incubated for 1, 7, or 35 d (25°C), then stored frozen for either 1, 7, 21, or 35 d. Soil incubated for 1 d showed a decline in microbial "C (measured by CFE) after freezing for 7 to 35 d. Soil incubated for 7 and 35 d contained a 14C microbial population that was unaffected by freezing. At first glance, these observations indicated that the CFE assay was not greatly affected by freezing soils; however, closer examination indicated that microbial mortality had been masked by a freezing-induced improvement in CFE efficiency. Increased efficiency may have resulted from improved aggregate dispersal following freezing. Homogenization of soil samples improved extraction of microbial C by 40%. In conclusion, we don't recommend that soils be frozen prior to CFE. Also, the physical disruption of soil during CFE must be severe to remove any interference of aggregate stability on the results of this assay.

M

EASUREMENT OF MICROBIAL BIOMASS by fumiga-

tion-extraction (Brookes et al., 1985; Vance et al., 1987) is a relatively simple laboratory technique used in examining nutrient transformations in soils (Bottner, 1985). Despite the simplicity of the assay, soil samples can become too numerous to analyze in their fresh state, and might sometimes be stored frozen. Microbial biomass has been measured on soils stored frozen using a fumigation-incubation technique (Jenkinson and Powlson, 1976; Ross et al., 1980; Ladd et al., 1981) similar to fumigationextraction. Freezing is also an important consideration for Department of Land Resource Science, Univ. of Guelph, Guelph, ON, Canada NIG 2W1. Received 3 June 1992. ""Corresponding author.

Published in Soil Sci. Soc. Am. J. 58:1645-1651 (1994).

winter sampling of soils from cool climates that undergo freeze-thaw cycles, which affect microbial biomass and its measurement. Freezing soils may cause mortality in the soil microbial population (Morley et al., 1983), thus affecting measurements of the microbial biomass. The degree of microbial mortality may be affected by the storage temperature, rate of freezing and thawing, microbial nutritional status, microbial population age structure, time stored frozen, and soil pretreatment such as drying-wetting history (MacLeod and Calcott, 1976). During frozen storage, although ice forms in the soil solution, some microbes become supercooled but are not frozen, even at — 16°C (MacLeod and Calcott, 1976). In supercooling, cell membranes may be excessively permeable, and lethal quantities of ions and organic solutes exude from the cytoplasm. Freezing damage can be caused by ice crystal formation, or by low osmotic potentials resulting from the exclusion of cytoplasmic solutes from ice (MacLeod and Calcott, 1976). Many reports of freezing injury to soil microbes come from plate counts. Commonly, numbers of microbes have declined by 30 to 50% (Mack, 1963; Campbell et al., 1970; Morley et al., 1983; Skogland et al., 1988), but increases in bacteria and actinomycetes have been observed (Ivarson and Mack, 1962; Mack, 1963; Morley et al., 1983) and were attributed to unproved extraction caused by soil aggregate dispersal during freezing. Although many studies have linked freezing to disaggregation of soil (Lehrsch et al., 1991), it can also cause aggregation (Bisal and Nielsen, 1964; Lehrsch et al., 1991). Disaggregation may be more likely to occur in recently dried soils (Lehrsch et al., 1991), or when soils are subjected to freeze-drying (Bisal and Nielsen, 1964). The fumigation-extraction procedure may similarly be affected by freezing. Besides the release of solutes from damaged cells, the efficiency of fumigation-extraction could be affected by aggregate dispersal. Ross (1988) reported that better dispersal of aggregates by more

1646

SOIL SCI. SOC. AM. J., VOL. 58, NOVEMBER-DECEMBER 1994

violent shaking in the K2SO4 solution caused an increase in the quantity of organic C extracted from fumigated soils, but noticed no improvement for unfumigated soils. After thawing soils, extractable carbohydrates (Ivarson and Gupta 1967; Ross 1972), and amino acids (Ivarson and Sowden 1966, 1970) increased in some studies. However, other studies measured no increase in waterextractable C (Rolston and Liss 1989) or NO§~ and NHtf (Gasser, 1958; Campbell et al., 1970; Ross and Bridger, 1978) following thawing of soils. Solutes extracted from thawed soils can be reduced by precipitation or microbial metabolism. Ivarson and Sowden (1970) observed a substantial decline in water-soluble amino acids and hexoses during 12 wk of storage at -14°C. They theorized that in the concentrated unfrozen portion of the soil solution, microbial solutes became chemically bound or adsorbed. Also, if extraction of soils is not done immediately, microbial solutes may be metabolized during the burst of microbial activity occuring during the first 2 to 3 d after thawing (Jenkinson and Powlson, 1976; Skogland et al., 1988). The purpose of this study was to evaluate the effect of frozen storage on the fumigation-extraction technique used hi measuring microbial biomass. Three experiments were conducted. One experiment investigated the effect of frozen storage and freeze-thaw cycles. A second experiment examined the effect of frozen storage on 14C-labeled microbial populations of increasing age. Since freezing soil may disperse soil aggregates, a third experiment examined the potential to improve the efficiency of the fumigation-extraction technique by increasing physical disruption of soil samples. THEORY Measurement of microbial C and N extracted after fumigation may be affected by the mortality of microbial biomass resulting from freezing, because microbial solutes released by freezing may come from part of the same pool as those released by fumigation (Jenkinson and Powlson, 1976). Thus freezing should increase C and N in unfumigated soils, and reduce estimates of microbial C and N released by fumigation. However, physical and chemical changes that may occur in the soil during freezing could affect the efficiency of the fumigationextraction procedure, and obscure freezing damage to the microbial population. In order to understand the implications of any of the above factors on the fumigation-extraction method, four hypothetical cases are presented. Using C fractions as an example, the following terms can be defined: x = freezing treatment, such as duration of storage or number of freeze-thaw cycles. For nonfrozen fresh soils, x = 0. C f j = K2SO4-extractable organic C in fumigated soil, subjected to freezing treatment x. CNF,* = K2SO4-extractable organic C in unfumigated soil, subjected to freezing treatment x. CMB,* = actual total microbial biomass C in soil subjected to freezing treatment x. CZj = C in the microbial population of F or NF soils damaged by freezing treatment x and releasing organic solutes to the soil solution. CN-M^ = nonmicrobial C: K2SO4-extractable organic C not from microbial cytoplasm.

CE., =

extracted organic C released by fumigation of a soil subjected to freezing treatment x. ECfl = extraction efficiency: CE released after fumigation of a nonfrozen soil as a fraction of total microbial C. The original equation from Vance et al. (1987) for determining microbial C from the CE released by fumigation of fresh soil (x = 0) was CMB.O = (C-F,o — CNF,O)/£C,O [1] Vance et al. (1987) found an average extraction efficiency, Ec,o = 0.35, for a range of soil types. If freezing treatment (x) affects the extraction efficiency (Ec), then Eq. [1] will not give an accurate estimate of total microbial C. Case A Consider a hypothetical situation where freezing caused a microbial mortality of 40%, but had no effect on the extractability of soil C fractions (Fig. 1, Case A). If the organic solutes released by freezing damage came from the same pool as those released by fumigation (Jenkinson and Powlson, 1976), CF,* would be composed of microbial C released by fumigation and by freezing damage, plus the background of nonmicrobial extractable C (Cp^ = CF.O). Thus, for Case A, CFjc was unaffected by freezing. In nonfumigated soils, extracted C would be composed of background nonmicrobial C, plus a contribution from freezing-damaged cells. In Case A, microbial solutes released by freezing damage caused a decrease in the CE attributed to fumigation. In Fig. 1, Case A, increasing the severity of the freezing treatment reduced the microbial biomass by 40% of its prefreezing level and if extraction efficiency was unaffected, there was a similar 40% reduction in CE,*. Case B Freezing may change the efficiency of CE^ extraction. To illustrate this, consider another hypothetical case (Fig. 1, Case B) where freezing improved C extraction (Ec,*), but did not cause microbial mortality (Cz,* = 0 and CMB,* = CMB.O). Case C In a third hypothetical case (Fig. 1, Case C), freezing caused microbial damage (Cz>1) and changed the extractability of microbial C. In a thawed soil, extractable C after fumigation would include microbial C released by freezing and fumigation, nonmicrobial C, and increases in extractable C caused by freezing. If the decline of CMB.O to CMB,* caused by freezing damage was compensated by an increase in £c,*, it is possible that no net change in CE would be observed (Fig. 1, Case C). CaseD Finally, consider a fourth hypothetical case (Fig. 1, Case D) where all the attributes of Case C applied, yet there was also freezing-induced change in the extraction of nonmicrobial C (CN-M,*). In this case, CN-M = CN-M.O + CN-M,*. Nonmicrobial C contributed to both CF,* and CNF,*, thus it cancels out when calculating extracted microbial C (CE,*). The result is the same as Case C. The above discussion has used C as an example, but the same principles may also apply to N, and to isotopes of C and N. Isotopes of C and N may respond differently from total C or N if the labeling is unevenly distributed between young and old microbial cells, and if freezing differentially affects the extractability of different microbial subpopulations.

1647

WINTER ET AL.: MICROBIAL BIOMASS MEASUREMENT IN SOILS STORED FROZEN

MATERIALS AND METHODS Experiment 1. Long-Term Storage and Freeze-Thaw Cycles Samples of a Conestoga silt loam (fine-loamy, mixed, mesic Typic Hapludalf; 30% sand, 50% silt, 20% clay; 25 g organic C kg"1) were taken from the surface 10 cm of a summer-fallow field, previously in long-term continuous corn (Zea mays L.). Field-moist soil (0.25 kg water kg"' oven-dry soil) was sieved through a 4-mm mesh and thoroughly mixed. Subsamples of 25 g wet weight were weighed into 75-mL glass vials and capped with an air-tight plastic lid. These vials became the experimental units subjected to freezing treatments. The first half of this experiment examined the effect of storage on microbial biomass. Capped glass vials containing soil were taken from room temperature (22 °C) and placed in a freezer at -15°C for 18 h (referred to as the 1-d treatment) or 7 or 170 d. At the end of storage, the glass vials were removed from the freezer and thawed for 6 h on a laboratory bench. Microbial C and N were determined (see below) and compared with measurements on untreated, fresh soil. The second half of this experiment examined the ability of the microbial biomass to withstand repeated freezing stress. Capped glass vials containing soil were subjected to one, two, or three freeze-thaw cycles. A single cycle consisted of 18 h in the freezer at - 15°C, followed by 6 h at room temperature on a laboratory bench. Following freeze-thaw treatment, microbial C and N were measured. All treatments were replicated five times. Experiment 2. Frozen Storage of 14C-labeled Microbial Biomass

•100 u

(C)

•3 75 I

50 25

100

(d) 75 50

Soil for the second experiment was from the plow layer of

a sandy loam (Typic Hapludalf; 15 g organic C kg"1), growing corn. Soil was air dried, sieved (2-mm mesh), moistened with a N-P-S nutrient solution to 0.1 kg water kg"1 oven-dry soil, and preincubated at 25°C for 10 d. After preincubation, 14 C-glucose solution (1.6 g C kg"1 soil with a specific activity of 500 Bq mg"1 glucose C) was sprayed with a fine mister and mixed into the soil. Total C, N, P, and S amendments were at a ratio of 10:1:0.4:0.4. Amended soil was adjusted to a moisture content of 0.16 kg water kg"' oven-dry soil (-45 kPa) and incubated at 25°C. After 1, 7, or 35 d of incubation, 15 subsamples (20 g each) were taken; three of these were analyzed for 14C immediately, the others after freezing at -15°C for 1, 7, 21, or 35 d. The 14C content was determined by fumigation-extraction (see below). Microbial Biomass C and N Microbial biomass C (after Vance et al., 1987) and N (after Brookes et al., 1985) were determined using the fumigationextraction method. One of a pair of soil samples was fumigated with CHC13 (stabilized with a nonvolatile hydrocarbon, Caladon Laboratories, Georgetown, ON) to lyse microbial cells and extracted (50 mL of 0.5 M K2SO4, on a rotary shaker at 240 revolutions min"1 for 1 h, then filtered through a Whatman no. 5 filter paper) and organic C and total N measured. A second matching soil sample was not fumigated before extraction. Organic C and total N in extracts were measured using the Technicon Autoanalyzer II system (Alfa-Laval, Stockholm, Sweden) following Technicon Industrial Methods no. 45576W/A and 759-841, respectively. In Exp. 2, 14C in 2 mL of the K2SO4 filtrates was determined by liquid scintillation counting (Beckman LS250 counter, Beckinan Instruments, Irvine, CA) in 15 mL of Picofluor-40 (Packard Instruments, Mississauga, ON). The 14C counting efficiency was 85%. Ex-

25 0

Severity of freezing (cycles or duration) Fig. 1. Hypothetical effects of soil freezing treatment (x) on total microbial biomass C (CMBj), and measurement of extractable organic C in fumigated (Cr,*) and nonfumigated (CNF,*) soil, the extractable microbial C due to fumigation (CEj), and nonmicrobial soil organic matter (CvBMj): (a) Case A, freezing killed some microbes; (b) Case B, freezing had no effect on microbes but improved CE,X extraction; (c) Case C, freezing killed some microbes and improved extraction of CE,« (d) Case D, same as Case C, plus improved extraction of CVBM,.-

tracted microbial C (CE), 14C (14CE), and N (NE) were determined using the formula (using C as an example): CE = CF - CNF. Experiment 3. Effect of Soil Homogenization on Extraction of C Aggregates (9-19 mm, 0.11 kg water kg"1 oven-dry soil) from a Conestoga silt loam, cultivated to bromegrass (Bromus inermus L.) for 6 yr, were analyzed by the fumigation-extraction procedure used in Exp. 1 and 2, except for the following modifications. After adding 50 mL of 0.5 M K2SO4, each soil sample was subjected to a homogenization treatment. Soil samples were homogenized in glass vials for 5 s using a Brinkman PT 10-35 tissue homogenizer with a saw-tooth PT 20ST probe generator (Kinematica GmbH, Switzerland). Homogenization treatments were none, or 3.8, 4.3, 4.9, or 5.5 on the machine power level dial. Fumigated samples were replicated five times, and nonfumigated samples 10 times.

1648


After homogenization treatment, soil was shaken and filtered, and soil organic C was measured as in Exp. 1, above. Statistical Analysis All experiments were treated as completely randomized designs. Statistics were performed using PC-SAS software (SAS Institute, 1988). RESULTS AND DISCUSSION Experiment 1. Long-term Storage

and Freeze-Thaw Cycles Frozen Storage In the soil stored frozen for 1 to 170 d, the extracted microbial C and N ranged from 92 to 124% and 100 to 134% of the control unfrozen fresh soils, respectively (Fig. 2). However, only on Day 7 of freezing were values significantly (P < 0.05) increased from fresh soils, since freezing increased the C and N extracted from fumigated soil. Duration of frozen storage had a significant effect on the amount of C extracted from nonfumigated soils (Fig. 2a), declining to 66% of the value of fresh soil after 7 d of storage. However, this decline in CNF was of little importance relative to the size of increase in C extracted from fumigated soils. Thus, changes in CNF with freezing had only a small effect on calculating microbial biomass C. Duration of frozen storage produced a significant (P < 400

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Duration of freezing (d) Fig. 2. The effect of duration of storage at - 15°C on the extraction of (a) organic C and (b) total N in fumigated (F) and nonfumigated (NF) soil, and the extractable microbial C or N due to fumigation (E). Least significant difference (LSD, P < 0.05). 'Values significantly different (P < 0.05) from those of fresh soil.

0.05) linear increase in NNF and a quadratic increase in NF (P < 0.001) (Fig. 2b). A simple explanation for the increase in NF and NNF is an improvement in the extraction of nonmicrobial N. However, it seems likely that N extraction increased as a result of microbial mortality and improved efficiency of fumigation-extraction (Case C, Fig. 1). During the 1- to 7-d period, an improvement in En,x resulted in a larger increase in NF than the increase in NNF caused by microbial mortality and resulted in a significantly larger NE,?. Beyond Day 7, increasing microbial mortality may have been narrowing the difference between NF and NNF so that after 170 d of storage, the flush of microbial N was sunilar to that of fresh soil (NE,i7o = NE,o). Using Case C, the 21% increase in Np.no above NF,O could have been caused by improved extraction of microbial N (£N,i7o). Following the assumptions of Case C, we can express microbial mortality as a percentage of NMB.O: Nz.. 100 _ NMB.O

~ NNF.Q) 100 - NNF,O

[2]

The numerator on the right side of the equation calculates microbial N released by freezing damage to solution in the nonfumigated soil. The denominator on the right side calculates microbial biomass N while accounting for improved extraction efficiency by freezing. If there had been no effect on the efficiency of microbial N extraction (Case A), then the microbial mortality percentage could easily be calculated from the change in NE from fresh soil: 100 _ (NE>I - NE!O) 100 N,MB.O N:E,0

[3]

From the long-term storage experiment (Fig. 2b), we compared Cases A and C scenarios for estimating microbial mortality. After 1 d of frozen storage, Cases A and C predicted no change in microbial N; after 7 d of storage, Case A was +34% and Case C was —11%; for 170 d of storage, Case A was +3% and Case C was -41%. These calculations predict that for Case A, freezing increased microbial biomass, which is unlikely. On the other hand, Case C's prediction of a 41 % decline in microbial biomass after 170 d of storage is more in line with findings from previous researchers (Mack 1963; Campbell et al., 1970; Morley et al., 1983; Skogland et al., 1988), thus is the more likely scenario. Like microbial C, microbial N extracted after fumigation would be derived from microbial cytoplasm, and one would thus expect freezing treatments to produce sunilar trends in CF and NF, and CNF and NNF. The only similarity between the C and N data was the high extractability of microbial C and N that occurred after 7 d of freezing (Fig. 2). The upward trend in NF and NNF was not observed for CF and CNF, and the values for CF.HO, CNF.HO, and CE,™ were not significantly different (P < 0.05) from those of nonfrozen soil. The lack of difference between these values observed at Day 17, and fresh soil implies no freezing mortality, and no change in extraction efficiencies. These conclusions could

WINTER ET AL.: MICROBIAL BIOMASS MEASUREMENT IN SOILS STORED FROZEN

be false since they are quite contrary to the 41 % mortality calculated from N data using the Case C scenario. Microbial respiration could account for the absence of an upward trend in CF and NFc. After freezing, soils were placed on the laboratory bench where they quickly rose to room temperature (22 °C). During the 6 h on the laboratory bench, post-thaw respiration would have occurred (Skogland et al., 1988). When soils thaw and return to room temperature, there is characteristically a transient burst of respiration (Ivarson and Mack, 1962; Ross, 1972; Jenkinson and Powlson, 1976) that can be most intense after thawing to 25°C (Skogland et al., 1988). During the next 2 to 3 d, CO2 production declines to normal, prefreezing rates. This increase in respiration has been associated with the repair of cellular damage. In soils high in organic matter, Ivarson and Sowden (1970) and Ross (1972) reported average increases in COa evolution ranging from 1.25 to two times the basal respiration of fresh soil. Skogland et al. (1988) suggested that greater quantities of C could be lost during the first hours of thawing, but this has not yet been measured in mineral soils. In this research, respiration in intact cells may have decreased CF. Lysis and exudation of solutes (CzJ) could increase CNF,*, but this contribution to CNF,* could have been diminished by reimmobilization and respiration of CC>2 while the soil thawed. Because CNF,* declined with duration of frozen storage, Eq. [2] was not used to estimate microbial mortality.

1649

thaw cycles did not greatly reduce the extracted microbial C and N until the third cycle (Fig. 3). If microbial cells were lysed by the first two freeze-thaw cycles, then one could have expected a decrease in CEjJ and NE,^ due to an increase in CNF,* and NNF,* values. While this may indicate that microbial cells were resistant to at least two cycles of freezing and thawing, the absence of any significant diiference in CE,* and NE)J from fresh soil might be explained by a combination of improved efficiency of the fumigation-extraction procedure and respiration of microbial exudates in thawed soil. With the third cycle, CF and NF declined to 70 and 82% of the fresh soil values, respectively, but without concomitant increases hi CNF and NNF. The failure of CNF and NNF to increase may be attributable to precipitation of organic solutes, respiration (for CNF), or denitrification (for NF), which may be stimulated by freezing and thawing (McGarity, 1962).

Experiment 2. Frozen Storage of 14C-labeled Microbial Biomass When the Brookston soil was incubated with 14Cglucose, 14C was incorporated into a growing microbial population during the first day. As the new cells aged and died, the remaining 14C would have been reimmobilized into a steady-state microbial population (Ladd and Paul, 1973). To a large extent, this process appeared to have occurred by 7 d of incubation at 25°C (Fig. 4) 600

Freeze-Thaw Cycles Past research has reported that the greatest decline in microbial numbers occurs during the first freeze-thaw cycle, with only a small decline with subsequent cycles (Biederbeck and Campbell 1971; Shields et al., 1974; Skogland et al., 1988). In this research, repeated freeze-

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Freeze-thaw cycles Fig. 3. The effect of freeze-thaw cycles on the extraction of (a) organic C and (b) total N in fumigated (F) and nonfumigated (NT) soil, and the extractable microbial C or N due to fumigation (E). Within fumigation treatment, least significant difference (LSD, P < 0.05). •Values significantly different (P < 0.05) from those of fresh soil.

10

Duration of freezing (d) Fig. 4. The effect of duration of storage at — 15° C on the extraction of organic 14C from a soil preincubated with wC-glucose for (a) 1, (b) 7, and (c) 35 d. Least significant difference (LSD, P < 0.05). *Values significantly different (P < 0.05) from those of fresh soil.

1650


since soil frozen after 7 and 35 d of incubation behaved similarly, demonstrating no significant change (P < 0.05) in 14CE with duration of frozen storage (Fig. 4b and 4c). Freezing damage of 14C microbes appeared to be most severe in soil incubated only 1 d with 14C-glucose (Fig. 4a). The 14CF,i was greater than 14CF,o, suggesting a freezing-induced improvement in extraction efficiency. As the duration of freezing increased through 7 to 35 d, 14CF^ declined from the 14CF,i maximum. In this soil, aggregation may have been improved with prolonged freezing, or precipitation of microbial solutes may have negated the initial improvement in extraction efficiency. Since the general effect of freezing was to improve extraction efficiency, Eq. [2] was adapted to calculate microbial mortality (Table 1, Eq. [4]) and compared with changes in 14CE^ (Table 1). The estimate of microbial mortality from freezing was greater if improved extraction efficiency was considered (Case C; Table 1, Eq. [4]) than if only the change in 14CE^ was used (Case A; Table 1, Eq. [5]). The discrepancy between the two methods of estimating mortality was greatest after only 1 d of freezing. As the duration of storage increased, the improvement hi extraction efficiency may have decreased (Fig. 4a; c.f. decline in 14Cp,* from Days 1 to 35) causing the 14CE^ to decrease also. Equation [4] may be the more accurate estimate of microbial mortality if freezing affected microbial-C extraction, such as through aggregate dispersal. If prolonged freezing caused precipitation of microbial solutes from the soil solution, then both equations may underestimate microbial mortality. Experiment 3. Soil Homogenization Effect on C and N Extraction In both the Brookston and Conestoga soils, freezing caused an increase in microbial biomass measured by extraction of C or N released after soil fumigation, possibly because of inadequate aggregate dispersal when fresh soils were shaken in K2SO4 solution. To test the hypothesis that more severe physical disruption of soil could improve the efficiency of C extraction, a Conestoga silt loam was placed in K2SO4 solution and subjected to an increase in homogenization intensity before the standard shaking treatment (Fig. 5). Table 1. Estimates of microbial mortality in a Brookston soil incubated at 25 °C with l4C-glucose for 1, 7, and 35 d, then frozen at - 15°C for periods up to 35 d.

The standard shaking treatment was insufficient to optimize organic-C extraction (Fig. 5). As homogenization power increased, CF increased asymptotically to a maximum at the higher power levels. The value of CNF was also increased by homogenizing intensity, reaching a maximum at power setting 4.8, and then declining (P < 0.05) at 5.5. The CNF increase was relatively small, only 5% of the CF of unhomogenized soil. Net CE increased 36 to 43% (P < 0.05) above unhomogenized soil. Thus, there was the potential for freezing to improve the extraction efficiency of microbial solutes by improving aggregate dispersion. CONCLUSIONS After 170 d of frozen soil storage (-15°C), the extracted microbial C or N was no different from that of fresh soil, implying that freezing had caused no damage to the microbial biomass. However, freezing may have improved the extractability of biomass C and N and compensated for microbial mortality. When improved extractability was considered, a possible 41% loss of microbial biomass was calculated. Microbial biomass undergoing growth on recently added glucose was more susceptible to freezing damage than a steady-state microbial population. We do not recommended that soils be frozen pending analysis for microbial biomass by the fumigation-extraction method. This is especially true if isotopic tracers are being used because subpopulations of the microbial biomass may vary in labeling, age, and susceptibility to freezing. Some soils may store better at 4°C; Ross (1991) observed no change in microbial C in a high smectite soil refrigerated for 14 mo. Any soil property (e.g., clay content or organic matter) or environmental event (e.g., wetting-drying or freezing-thawing) that affects soil aggregation can affect the efficiency of microbial biomass measurement. In our soil, homogenization unproved the efficiency of extraction of microbial biomass C by 40%. Further research is necessary to determine the best homogenization procedure for minimizing potential interference from aggregate stability in microbial biomass determinations. A 400

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