DIVISION S-8—NOTES

COLD STORAGE OF A TROPICAL SOILDECREASES NITRIFICATION

POTENTIAL

Louis V. VERCHOT*

AbstractRecommendations for soil handling when determining microbial

attributes often include cold storage in the interim between collectingand executing an assay, even if the storage period is likely to be short.Cold storage may not be appropriate for isothermic tropical soilswhere microbial communities are not adapted to low temperatures.The objective of this experiment was to determine the effects of ashort period of cold storage on nitrifying bacterial activity in an Oxisolfrom eastern Amazonia. Soil samples were collected from primaryforest, active pasture, and secondary forest in eastern Amazonia;half were stored at 4°C for 5 d and the other half were incubatedimmediately. Nitrification potential was determined using a slurryincubation assay. Cold storage reduced the absolute value obtainedfrom the nitrification potential incubations (P = 0.05). However,relative ranking of the ecosystems was the same for stored and freshsamples. Therefore, storage for short periods at room temperaturemay be the best treatment for tropical soils. However, when theobjective is to obtain a relative index of nitrification, cold storagemay simply reduce the sensitivity of the method.

ESTIMATIONS of microbial activity and microbial bio-mass in soils are becoming routine in a wide variety

of areas of scientific research. Soil microbial processesare important in regulating the availability of nutrientsfor plant growth, weathering of minerals in soils, andcycling of elements between terrestrial and aquatic eco-systems or between terrestrial ecosystems and the atmo-sphere. For studies of microbial activity in soils, mea-surements on fresh samples are generally preferred, butthis may not always be practical. When immediate analy-sis is not feasible, recommendations for soil handlingoften include refrigeration or freezing in the interimbetween collecting and executing the assay, even if thestorage period is likely to be short (Wollum, 1994; Hartet al., 1994). This recommendation was developed fromexperience with temperate soils where microbial popu-lations are adapted to large temperature shifts over thecourse of a year that often include periods of freezingtemperatures. The logic behind refrigeration of samplesis that cooling slows microbial growth and decreasesdisturbance effects associated with sampling.

Some authors have shown that enzyme activity, mi-crobial biomass, microbial population numbers andother parameters used to characterize microbial corn-

institute of Ecosystem Studies, P.O. Box AB, Millbrook, NY 12545.Received 23 Nov. 1998. * Corresponding author (verchotl@ecostudies.org).

Published in Soil Sci. Soc. Am. J. 63:1942-1944 (1999).

munities remain relatively constant during short storageperiods (3-7 wk) at 4°C (Wollum, 1994; Petersen andKlug, 1994). In some instances, freezing has been shownto be preferable to cold storage. For example, Stenberget al. (1998) compared several microbiological processesin soil samples stored for different periods at 2°C andat -20°C. They showed that long-term cold storageresulted in decreases in microbial biomass, basal respira-tion, potential ammonium oxidation, and potential deni-trification, but, with the exception of basal respiration,these properties were unchanged in frozen soils. In thesame study, N mineralization rates decreased after 1 mostorage in both frozen and cold stored samples, butreturned to pre-storage values after 3 mo of storage andremained there for 13 mo in cold-stored soils. In frozensamples stored for 3 mo or more, N mineralization rateswere elevated above values obtained from freshsamples.

The observation of increased N mineralization ratesfollowing freezing is consistent with observations byother researchers (Ross, 1991) and with field studiesthat show high mineralization and nitrification rates fol-lowing soil thawing (for a review see" Edwards and Cres-ser, 1992). For this reason, cold storage is recommendedfor soils before laboratory measurements of N mineral-ization or nitrification are taken. However, cold storagemay not be appropriate for isothermic tropical soilswhere microbial communities are not adapted to lowtemperatures. In fact, cooling may perturb soil microbialcommunities in these soils and decrease microbial func-tions of interest. The objective of this experiment wasto determine the effects of a short period of cold storageon nitrifying bacteria activity in an Oxisol from east-ern Amazonia.

Materials and MethodsSoils

For this experiment, soils were collected from two areas ina primary forest, a secondary forest, and an active pasture atFazenda Vitoria (Victory Ranch), near the town of Paragomi-nas in Para state, Brazil (2°59' S lat; 47°31' W long). Themean annual rainfall is 1850 mm yr"1 and the mean annualtemperature is 25 °C. Soils in the region developed on Pleisto-cene terraces cut into the Belterra clay and Tertiary Barreirasformations (Sombroek, 1966; Clapperton, 1993). These sedi-ments consist primarily of kaolinite, quartz, and hematite andare widespread at elevations below 200 m in the Amazon basin(Clapperton, 1993). Soils are classified as Kaolinitic YellowLatosols in the Brazilian classification system or Typic Haplus-tox (clayey, kaolinitic, isothermic) according to U.S. Depart-ment of Agriculture taxonomy (Sombroek, 1966). Oxisolscover 40% of the Amazon basin (Richter and Babbar, 1991)and are concentrated in eastern and southern Amazonia,where rainfall is seasonal.

Most primary forest stands in the region, including the onesampled here, are remnant stands that have been influencedby surrounding human activities, such as hunting and harvest-

1942

NOTES 1943

Table 1. Selected physical and chemical characteristics of the surface 10 cm of soil in the ecosystems sampled at Fazenda Vitoria.

Ecosystem

Primary forestSecondary forestActive pasture

Moisturecontentkg kg-'

0.350.340.31

Bulkdensityg cm-3

0.990.961.25

pH-H2Of

4.45.55.7

Total NtMg N ha '

2.22.63.6

NOr-N§kg N ha'1

6.201.411.99

Total C$Mg C ha-'

24.529.031.7

C:N

11.111.28.8

11:2.5 soil:H,O.t Total C and total N data from D. Markewitz and E.A. Davidson, unpublished data (1998); analyses were done on a Perkin-Elmer (Norwalk, CT)

CHN analyzer.§ Determination made from soil extracts (15 g of soil in 100 mL 2 M KC1, following Bundy and Meisinger, 1994).

ing of single trees. While human influences are pervasive, thisforest stand has the complex floristic structure of a primaryforest that has probably not undergone major disturbance (i.e.,clearing or fire) during the last few centuries. An inventory ofa 5-ha plot of primary forest at Fazenda Vitoria by Nepstad(1989) identified 171 species with diameters at breast height>20 cm; and aboveground biomass in the forest was 264Mg ha"1.

The secondary forest was regenerating naturally from apasture that had been abandoned in 1976. The area had beenused with moderate intensity during the 1970s as a pasture(1-3 head of cattle ha~') and had been burned periodically.At the time of measurement, stand height was patchy, withsome areas as high as 13 to 16 m, and the aboveground biomasswas 50 Mg ha"1. This forest is floristically much simpler thanthe primary forest with 75 tree species found on twelve 10-by 10-m plots (W. Stanley, personal communication, 1998).

The active pasture was first cleared in 1969 and had beenplanted to Panicum maximum Jacq. and later to Brachiariahumidicola (Rendle) Schweick. The pasture was heavilygrazed until the early 1980s (2-3 head of cattle ha"1). Thepasture was then reformed, meaning that it was cleared,burned, disked, fertilized with P, and planted to the foragegrass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. At thetime of sampling, the active pasture had few woody invaders.

In February 1997, at two different areas within each ecosys-tem, an area of 100 m2 was sampled. At each site, eight soilsamples were collected to a depth of 10 cm with a bucketauger (8-cm diam.). Details of the characteristics of the soilsfor each ecosystem can be found in Table 1. After returningto the lab, each soil sample was hand-sorted to remove rocksand roots. Samples from each site were randomly divided intotwo groups so that there were four samples from each site ineach group. Nitrification potential measurement was begunimmediately (18 h after collection) on the first group, while thesecond group was stored for 5 d at 4°C before measurement ofnitrification potential.

Incubation MethodNitrification potentials were determined using the shaken

slurry method of Hart et al. (1994). This method assesses themaximum rate (Fmax) of nitrification for a soil sample. A 15-gsubsample was taken from each soil sample and mixed with100 mL of a solution containing 1.5 mM NH^ and 1 mMPC>4~ in a 250-mL Erlenmeyer flask to make a slurry. Wedeviated from the described method in that we did not adjustthe pH of the buffer solution prior to mixing with the soil.Experience with these soils has shown that the buffering capac-ity of the soil far exceeds the buffering capacity of the solution.

Slurries were shaken on an orbital shaker at 180 rpm tomaintain aerobic conditions. After 2 h and 24 h, a 15-mLsample was taken from each slurry for analysis for NO3-N.Owing to the dispersion of clay particles, it was impossible tofilter the solutions, and we did not have access to a centrifuge

with enough power to rapidly settle the solids. Therefore, wemixed the slurry samples with 15 mL of 4 M KC1 to stop thereaction and to flocculate the colloids, and then let the solutionsettle overnight at 4°C. This resulted in a solution with a finalconcentration of 2 M KC1. After pipetting the supernatantinto sample vials, the samples were frozen for later analysis.Analysis was done via a flow-injection autoanalyzer (Alpkem,Wilsonville, OR) using a modified Griess-Illosvay procedurefor determining NO3-N + NO2-N, which was reported asNO3-N (Bundy and Meisinger, 1994). Nitrification potentialwas calculated from the rate of increase in NO3-N concentra-tion in the slurry and results were expressed as NO3-N produc-tion on a daily basis.

Statistical AnalysisThe experimental design was a split-plot, where the ecosys-

tem constituted the main plot and had two replications. Thetest of the hypothesis for differences between ecosystems wasdone using the whole-plot error term (Error A) that wasestimated by the mean square of the interaction between repli-cation and ecosystem. The refrigeration treatment constitutedthe split-plot and was replicated four times within each sub-plot. The test of the hypothesis for differences between re-frigeration treatments was done using the split-plot error term(Error B) that was estimated by the mean square of the three-way interaction between replication, ecosystem, and refrigera-tion treatment. This analysis was done using the GLM proce-dure in SAS (SAS, 1992).

Results and DiscussionResults of the nitrification potential measurements

are presented in Table 2. The ANOVA (Table 3)showed that the differences among ecosystems weresignificant at the P = 0.01 level. Mean nitrification po-tential values followed the order: primary forest > sec-ondary forest = active pasture. Means of the nitrifica-tion potentials in the stored samples were consistentlylower than the samples that were not stored. The resultsof the ANOVA confirm that this reduction was signifi-cant at the P = 0.05 level (Table 3). In most cases,

Table 2. Results of nitrification potential incubations for threetropical ecosystems. ____________ _________

Fresh Cool storage Reduction

Primary forestSecondary forest

Active pasture

SitelSite 2SitelSite 2SitelSite 2

8.32 (1.90)t5.12 (0.80)3.06 (0.72)3.51 (0.87)3.85 (1.18)4.76 (2.67)

ioir1 d-' ——1.62 (1.15)4.68 (3.27)0.50 (0.42)0.52 (0.89)0.19 (0.18)0.20 (0.14)

80.58.6

83.785.195.195.8

t Mean values (standard error).

1944 SOIL SCI. SOC. AM. J., VOL. 63, NOVEMBER-DECEMBER 1999

Table 3. Split-plot analysis of variance with nitrification potentialas the dependent variable. The test of the hypothesis thatnitrification potential does not differ among ecosystems wasmade using the interaction term between replication and eco-system to estimate the whole-plot error (Error A). The testof the hypothesis that nitrification potential does not differbetween refrigerated samples and nonrefrigerated samples wasmade using the three-way interaction term between replication,ecosystem, and refrigeration treatment (Error li).

SourceReplicationEcosystem (whole-plot)

Error ARefrigeration (split-plot)Ecosystem X refrigeration

Error B

At

122123

Mean square0.504

44.0520.288

145.9521.796

13.343

F value

153.0810.940.13

P> F

0.00650.04550.8791

cold storage resulted in decreases in mean nitrificationpotentials within a site by more than 80%. At the onesite that we did not observe this marked decrease; oneof the stored samples had a very high rate of NO3-Nproduction during the incubation, which offset the lowerrates measured in the other three samples.

We conclude that cold storage of soil for 5 d reducesthe absolute value obtained from the nitrification poten-tial incubations. Considering the fact that the growthrate of nitrifying bacteria is relatively slow (Schmidt,1982), storage for short periods at room temperaturemay be the best treatment for tropical soils.

Storage of soils did not affect the relative results ofthe incubation at these sites. For both incubations, nitri-fication potentials for the primary forest were greaterthan for the other two ecosystems, and there was nodiscernable difference between the other ecosystems.There was some variation in the degree to which coldstorage decreased nitrification potential, notably at oneof the forest sites where nitrification potential decreasedby <10%. As we noted earlier, the high mean for thestored samples from this site was due to one elevatedobservation. At all other sites, the decrease in nitrifica-tion potential was fairly uniform. Therefore, when theobjective is to obtain a relative index of nitrification,cold storage may simply reduce the sensitivity of themethod to distinguish differences, but with an adequatenumber of samples the method will provide a reliablerelative index. If the objective is to determine the maxi-

mum rate of nitrification, Vmax determined on cold storedsamples will underestimate a true Vmax.

AcknowledgmentsFunding for Louis V. Verchot through an appointment to

the Global Change Distinguished Postdoctoral Fellowships,sponsored by the U.S. Department of Energy, Office of Healthand Environmental Research, and administered by the OakRidge Institute for Science and Education is gratefully ac-knowledged. Funding for the field work was provided throughgrants from NASA (NAGW-3772 and NAG5-6277). Wewould like to thank Dr. Eduardo Makluff and the staff of thesoils laboratory at Empresa Brasileira de Pesquisa Agropecu-aria/Centro de Pesquisa Agropecuaria do Tropico Umido,and the Instituto de Pesquisa Ambiental da Amazonia forlogistical support.