Microbial Ecology Spatial Stratification of Soil Bacterial Populations in Aggregates of Diverse Soils Daniel Mummey 1, William Holben1, Johan Six 2 and Peter Stahl3 (1) Division of Biological Sciences, University of Montana, Missoula, MO, USA (2) Department of Plant Sciences, University of California-Davis, Davis, CA, USA (3) Department of Renewable Resources, University of Wyoming, Laramie, WY, USA Received: 23 December 2004 / Accepted: 1 January 2005 / Online publication: 6 April 2006 Abstract influenced by, the environment. Such an understanding can only be attained by analysis at scales relevant to those Most soil microbial community studies to date have at which processes influencing microbial diversity actu- focused on homogenized bulk soil samples. However, it ally operate [29]. However, because of the biotic and is likely that many important microbial processes occur abiotic complexity exhibited by most soils at nearly all in spatially segregated microenvironments in the soil scales, determining soil microbial diversity patterns leading to a microscale biogeography. This study at- remains a formidable challenge for soil microbiologists tempts to localize specific microbial populations to dif- [54]. ferent fractions or compartments within the soil matrix. The vast majority of soil microbial analyses are Microbial populations associated with macroaggregates conducted on bulk soil samples, and often composited and inner- versus total-microaggregates of three diverse bulk soil samples, which averages localized heterogeneity soils were characterized using culture-independent, mo- and provides little insight into the spatial origins of lecular methods. Despite their relative paucity in most detected strains. Moreover, as several studies indicate surveys of soil diversity, representatives of Gemmatimo- that molecular approaches tend to be biased toward the nadetes and Actinobacteria subdivision Rubrobacteridae more abundant organisms at the time of sampling [5, 20, were found to be highly abundant in inner-microag- 41], stratified sampling strategies, which restrict sample gregates of most soils analyzed. By contrast, clones af- area to definable habitats, may increase numbers of filiated with Acidobacteria were found to be relatively detectable organisms in a given habitat [14], and increase enriched in libraries derived from macroaggregate frac- the power of detecting differences by decreasing variabil- tions of nearly all soils, but poorly represented in inner- ity within a given sample [25]. Such approaches should microaggregate fractions. Based upon analysis of 16S also provide insights into the controlling factors and rRNA, active community members within microag- functions in different segments of the soil microbial gregates of a Georgian Ultisol were comprised largely of community. Increased understanding of relationships Gemmatimonadetes and Rubrobacteridae, while within between microbial community composition and soil microaggregates of a Nebraska Mollisol, Rubrobacteridae microhabitats may facilitate the integration of biotic data and Alphaproteobacteria were the predominant active with the copious soil physical and chemical information bacterial lineages. This work suggests that microaggre- available in the literature, potentially providing for better gates represent a unique microenvironment that selects understanding of soil functioning. for specific microbial lineages across disparate soils. Soil consists of a complex physical framework that largely determines solute, substrate, and energy flows. Introduction Therefore, soil provides a heterogeneous habitat for microorganisms characterized by different substrate, Understanding soil microbial diversity and the environ- nutrient, water, and oxygen concentrations, as well as mental processes it controls requires an in-depth under- pH and the size of pores available for microbial habitation standing of how microbial diversity influences, and is [13, 17, 18, 42]. These structural aggregates are classified hierarchically in terms of size and relative stability [40, Correspondence to: Daniel Mummey; E-mail: [email protected] 55]. Macroaggregates (diameter 9 250 mm) are formed 404 DOI: 10.1007/s00248-006-9020-5 & Volume 51, 404–411 (2006) & * Springer Science+Business Media, Inc. 2006 D. MUMMEY ET AL.: STRATIFICATION OF SOIL BACTERIAL POPULATIONS IN AGGREGATES OF DIVERSE SOILS 405 by temporary associations of microaggregates, minerals, compare whole and inner microaggregate communities and particulate organic matter, predominantly through across the five soils; and (3) determine relationships be- enmeshment by fungal hyphae and plant roots [39]. tween organismal presence and activity for inner-micro- Water-stable microaggregates (diameter G 250 mm), aggregate communities based on phylogenetic analysis of generally the most stable soil secondary structures, both 16S rRNA (rRNA) and 16S rRNA genes (rDNA). typically form within macroaggregates through micro- bially mediated processes [39, 51] and are largely Materials and Methods dependent upon persistent organic binding agents for structural stability [55]. Because soil structure is a Sites and Soils dominant factor controlling microbial diversity and processes [59], analysis at the aggregate and subaggregate Georgia. Soil samples were collected from no-tillage levels will be required to elucidate the principle biotic agricultural plots at the Horseshoe Bend Long Term and abiotic controls on soil microbial diversity. Environmental Research site (HSB) near Athens, GA A number of studies have reported contrasting (33-540N, 83-240W). The site has been under continuous distributions of microbial biomass, specific species or summer grain crops (Sorghum bicolor or Glycine max) populations, and functional attributes in different aggre- and winter crops (Secale cerale or Trifolium incarnatum) gate size classes [4, 6, 15, 28, 56]. A number of authors since 1978. Soil at the site is a fine loamy, siliceous, have suggested partitioning soil microorganisms into thermic Rhodic Kanhapludult. Annual precipitation (125 inner- and outer-aggregate fractions based on soil micro- cm) is relatively evenly distributed throughout the year. site physicochemical differences and presumed microbial Management history and site characteristics are described functional differences [3, 16, 17], and a number of in detail by Hendrix [19]. microscopy, molecular, and cultivation-based studies have explored microbial community structure in inner Nebraska. This site is located at on the High Plains and outer macroaggregates [7, 17, 18, 37, 44, 45, 57]. Agricultural Laboratory (HPAL), 8.3 km north of Sidney, However, despite the fundamental importance of micro- NE (41-140N, 103-000W). Soil samples were collected aggregates to soil structure and function, including the from long-term, no-tillage plots under a wheat–fallow sequestration of organic matter [22, 52], microbial com- cropping system. Soil at the site is classified as a fine silty, munity structure within this size class has received very mixed mesic Pachic Haplustoll. Average annual little study, particularly with regard to the outer- and precipitation (38 cm) predominantly (75%) occurs inner-aggregate distribution of microbial populations. between April and August. Management history and site Although habitats existing within microaggregates characteristics are described by Lyon et al. [30]. offer relatively stable water availability and protection against predation, they are typically described as oligo- Montana. Soil samples were collected from a gently trophic environments, containing limited amounts of sloping undisturbed grassland site (MGS) 10 km north of biologically recalcitrant organic matter having much Missoula, MT (46-4500000N, 114-0703000W). The site has a slower turnover rates than are observed for soil as a semiarid climate, receiving on average 34–42 cm whole [53]. Inner-microaggregate organisms, principally precipitation per year. Soil at the site is classified as a bacteria and archaea, would be expected, due to pore size cobbly loam Argixeroll. constraints, to largely escape predation by soil fauna [57, Soil samples from all sites were collected to a depth of 58] and perhaps exposure to fungal antibiotics. Due to 10 cm and placed on ice in sterile plastic bags in the field. pore size and diffusion constraints, inner-microaggregate All samples were frozen within 6 h of collection and stored organisms might also be expected to be of smaller size at _20-C prior to fractionation and nucleic acid isolation. and have slower turnover rates than their outer-aggregate or macropore counterparts [24]. Soil Fractionation. Field-moist soils were immersed in A previous study [36] provided evidence for spatial deionized H2O on top of three-tiered, nested sieves of stratification of bacterial division- and subdivision-level progressively finer mesh (2000, 250, and 53 mm). After an lineages within soil microaggregates of two different soils, initial hydration period (5 min), large macroaggregates an Aridisol and a mine spoil (Entisol). The present study were gently pressed through the 2000-mm sieve while expands on that work to examine whether similar spatial gently raising and lowering the sieves. The 2000-mm sieve stratification is observable across a broad range of was then removed, and the two remaining nested sieves geographically separated soil types, differing greatly in slowly raised and lowered (3 cm) to separate stable climate and soil forming factor influences. To accom- microaggregates from