Grundmann Et Al.: Spatial Modeling of Nitrifier Microhabitats in Soil

plexity of soil affects the probability of bacteria encountering appropriate substrates or other bacteria. The Soil bacteria function in the three-dimensional space in heterogequantitative assessment of bacteria in soil is mostly conneous soil complex and their activities depend in part on encountering substrates at the microbial scale. The bacterial density per gram of fined to population density or biomass measurements soil, which is generally measured, does not indicate if bacteria are (Atlas and Bartha, 1981; Schmidt, 1982; Powlson, 1994), all in the same location or spread throughout the soil complex. We but rarely is the spatial organization of the cells taken characterized spatial distribution for how dispersed or aggregated into account (Hattori, 1973). For example, do bacteria nitrifiers (NH 4 and NO 2 oxidizers) were at a submillimeter scale. in a gram of soil coalesce in a few spots or are they The spatial approach was based on the relationship, obtained experidistributed evenly across the soil complex? Dispersion mentally, between the percentage of microsamples (50–500 m diam.) is not available for microorganisms but is routinely meaharboring nitrifiers and the volume of the microsamples. The smallest sured for macroscopic organisms because it determines sample size (50m diam.) was considered as an approximation of the frequency of encountering food and other organmicrohabitat. The simulated spatial pattern of NO 2 oxidizer microisms. Characterizing the spatial distribution of microhabitats in soil were compared with experimental data. The simulated pattern of NO 2 oxidizer distribution suggested that microhabitats habitats is important if there is to be progress in microaveraged seven NO 2 oxidizers and occurred in preferentially colobial ecology in soils. Also, a better understanding of the nized patches that had about a 250m diam. These were ranspatial arrangement of bacterial habitats should lead to domly distributed and occupied 5.5% of the soil volume. They were the development of more appropriate bioremediation functionally connected through microporosity and hence diffusion techniques (increasing probability of bacteria encounprocesses probably controlled the spatial distribution of nirifiers. The tering substrates) and the optimization of soil functions nitrifier spatial pattern enabled efficient nitrification because NH 4 (Holden and Firestone, 1997). and NO 2 oxidizers were near one another. The results showed the Bacterial activities have been reported to be unevenly potential of our method to study spatial distribution of bacteria at distributed in soil, leading to the concept of hot spots the microhabitat scale. that are linked to local, transient available C for microbial growth and activity (Parkin, 1987; Robertson et al., 1988; Beare et al., 1995). Most microbiological research B are responsible for major biogeochemical is carried out on macro scales grams of soil, but bacteria transformations of organic and mineral constitcells exist and interact at the micro scale. Information uents in soils (Atlas and Bartha, 1981; Paul and Clark, of the spatial distribution of bacteria in soil is very lim1989). Soil bacteria live in a complex three-dimensional ited, with microhabitats being poorly defined (Harris, habitat of a porous heterogeneous medium (Stotzky and 1994). Hattori (1973) reported results of several early Burns, 1982; Tisdall and Oades, 1982; Crawford and studies on spatial patterns of bacteria in soil and Harris Young, 1998; Young and Ritz, 1998). The geometric com(1994) mentioned that they were mostly based on microscopic observations. The lack of quantitative data on G.L. Grundmann and A. Dechesne, Laboratoire d’Ecologie microthe spatial patterns of bacteria at the microhabitat scale bienne. U.M.R. C.N.R.S. 5557. UCB Lyon I. 43 Bd du 11 Novembre 1918. 69622 Villeurbanne, Cedex. France. F. Bartoli, Centre de Pédo(Hattori, 1973, for total microflora) is because of limitalogie Biologique. UPR 6831 CNRS-Université Henri-Poincaré, tions for sampling and sample processing methods. Nancy I. 17 rue Notre Dame des Pauvres BP5, 54 501 VandoeuvreThe two main locations for active bacteria are beLes-Nancy. J.P. Flandrois, J.L. Chassé, and R. Kizungu, Laboratoire lieved to be soil pores (Hattori and Hattori, 1976; Hatde Biométrie et Biologie Evolutive. U.M.R. C.N.R.S. 5558. UCB Lyon I. 43 Bd du 11 Novembre 1918. 69622 Villeurbanne, Cedex. tori, 1988; Pievetz and Steenhuis, 1995), (within the surFrance. Received 18 March 2001. *Corresponding author (grundman@ rounding water film), in regions of preferential flow biomserv.univ-lyon1.fr). Abbreviations: VU, volumetric unit; MDT, mean detection time. Published in Soil Sci. Soc. Am. J. 65:1709–1716 (2001). 1710 SOIL SCI. SOC. AM. J., VOL. 65, NOVEMBER–DECEMBER 2001 the laboratory, a 3-cm fragment was carefully taken from (Harris, 1994; Kinsall et al., 1995), or alternatively eninside the fragment with a sterile scalpel and kept in a humid trapped within the soil matrix (Foster, 1988; Paul and container to prevent desiccation. The soil water content at Clark, 1989). These points have not yet been clearly sampling was 16 g kg . Over 2 d, the small soil fragment was established, nor are the mechanisms of microhabitat gradually subdivided with a sterile scalpel under a binocular colonization understood. microscope into eight aggregates of about 0.5 cm, to represenThe objective of this work was to characterize the tatively sample the fragment. Each aggregate was then carespatial distribution of bacteria at the microhabitat scale. fully dissected on a calibrated grid under the binocular microIt was not to find the exact three-dimensional coordiscope with a sterile scalpel. The calibrated grid was used to nates but rather to determine how dispersed or clustered select volume units (VU) fitting into 50-, 100-, 250-, or 500m squares. They were considered to be cubes of 1.25 10 , bacteria were at the microscale. We used nitrifiers as a 1 10 , 1.6 10 , or 0.125 mm volumes, which are referred bacterial model on an undisturbed soil sample. Other to as the sizes 50, 100, 250, and 500, respectively. A series of research disciplines have shown that the probability of 96 replicates of each VU size (4 VU sizes 96 replicates) a process or organism being detected at several scales were sampled randomly from the aggregates. Although the VU is linked to spatial patterns (Madden and Hughes, 1999). dried rapidly once dissected, they were immediately placed We used a microsampling procedure to measure the in a culture medium to allow for bacteria survival. It was relationship between the percentage of the studied bacassumed that the bacteria were trapped in the dissected VU terial type present and the sample volume. A simulation and allowed for unbiased tests of the presence or absence of procedure, projecting the soil to a three-dimensional grid the nitrifiers. was then applied to estimate the type of distribution. Nitrifiers oxidize NH 4 and NO 2 stepwise to produce Testing for Ammonium and Nitrite Oxidizers NO 3 in soil. Ammonium oxidizers are obligate lithoin Volumetric Units trophs, while NO 2 oxidizers are mostly lithotrophic with Each VU was taken up with a sterile plugged glass capillary some having heterotrophic capabilities (Schmidt, 1982; that had been dipped in sterile, biologically inert silicon oil Prosser, 1989) and there is little redundancy in soil (Mar(SV40), and transferred to a 2-mL defined culture medium illey and Aragno, 1999). The tendency of nitrifier cells by swirling the tip of the capillary in the medium. Ninety-six to cluster in soil is based on speculation (Schmidt, 1982; replicates of each VU size were cultured in NH 4 oxidizer Berg and Rosswall, 1986; Keen and Prosser, 1988). They culture medium (Schmidt and Belser, 1994) (1 mM NH 4 final are attractive to study because their substrates or prodconcentration) and 96 other replicates of each VU size in ucts are easily measured; NO 2 by Griess-Ilosvay reagent NO 2 oxidizer culture medium (Schmidt and Belser, 1994) (Keeney and Nelson, 1982) and H by measuring pH (1 mM NO 2 final concentration), for 12 wk at 28 C in the changes. dark, in the wells of micro-culture plates containing 24 wells (Schmidt and Belser, 1994). Nitrite (5 mM final concentration) was added to each culture that tested positive for NH 4 oxiMATERIALS AND METHODS dizers and the culture was further tested for NO 2 oxidizers, Preparation of Soil Volumetric Units to assess for the presence of both NH 4 and NO 2 oxidizers in each VU. This first series of 8 96 VU replicates (4 sizes The agricultural soil studied was under maize (Zea Mays L.) for NO 2 and NH 4 oxidizers) is referred to as Exp. A. A cultivation for 10 yr. It is a sandy loam, developed from a second set of replicates (24, 48, or 72 VU depending on VU recent glacial drift (Riss) (Typic Hapludalf) with the following size) was taken from the same soil clod (kept at 5 C) 2 wk characteristics: bulk density, 1.3 Mg m 3 (Angulo et al., 1997); later (Exp. B) (Table 1). The presence of NO 2 oxidizers was clay, 17.0%; silt, 39.2%; sand, 40.4%; organic C, 1.4%; pH scored positive if there was no NO 2 left in the culture medium. (H2O), 6.4 (Grundmann et al., 1995); and weakly structured This was determined each week by the Griess–Ilosvay spot (Ranjard et al., 1997). A 30-cm intact soil ped was taken at the 5-cm depth in June from an area without vegetation. In test (Keeney and Nelson, 1982) on one drop of medium. The Table 1. Percent of NO 2 and NH 4 oxidizers in different of volume unit sizes. NO 2 and NH 4 oxidizer simultaneous distribution NO 2 ( ) NO 2 ( ) NO 2 ( ) NO 2 ( ) Number of and and and and VU size Experiment† VU tested NO 2 ( )‡ NH 4 ( ) NO 2 ( )§ NH 4 ( ) NH 4 ( ) NH 4 ( ) NH 4 ( ) 2