Microbial population structures in soil particle size fractions of a long-term fertilizer field experiment

Abstract

Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil particle size fractions have been well studied, little information is available on the structure of microbial populations in microhabitats. In this study, topsoil samples of different fertilizer treatments of a long-term field experiment were analyzed. Size fractions of 200 to 63 microm (fine sand fraction), 63 to 2 microm (silt fraction), and 2 to 0.1 microm (clay fraction) were obtained by a combination of low-energy sonication, wet sieving, and repeated centrifugation. Terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes were used to compare bacterial community structures in different particle size fractions. The microbial community structure was significantly affected by particle size, yielding higher diversity of microbes in small size fractions than in coarse size fractions. The higher biomass previously found in silt and clay fractions could be attributed to higher diversity rather than to better colonization of particular species. Low nutrient availability, protozoan grazing, and competition with fungal organisms may have been responsible for reduced diversities in larger size fractions. Furthermore, larger particle sizes were dominated by alpha-Proteobacteria, whereas high abundance and diversity of bacteria belonging to the Holophaga/Acidobacterium division were found in smaller size fractions. Although very contrasting organic amendments (green manure, animal manure, sewage sludge, and peat) were examined, our results demonstrated that the bacterial community structure was affected to a greater extent by the particle size fraction than by the kind of fertilizer applied. Therefore, our results demonstrate specific microbe-particle associations that are affected to only a small extent by external factors.

FIG. 1

16S rDNA-based HaeIII-HhaI T-RFLP fingerprint patterns obtained with three different particle size fractions of soils amended with sewage sludge.

FIG. 2

UPGMA dendrogram generated from all representative T-RFLP sample profiles. The scale indicates the distance level.

FIG. 3

Neighbor-joining phylogenetic tree based on 382 nucleotides of the 16S rRNA gene of clones showing highest similarity with bacteria belonging to the Holophaga/Acidobacterium division. Sequences obtained in this study are printed in boldface letters. The percentages of 100 bootstrap replicates are shown at the left nodes when at least 50%. The tree demonstrates the tremendous diversity of clones belonging to this group rather than definitive phylogenetic relationships. The accession numbers of the 16S rDNA sequences used are AF337850 (D99), AF013527 (C028), Z95735 (mb2430), Y12597 (DA008), AJ232842 (LRS25), Z95728 (iii1–15), Z95725 (ii3–15), AF078262 (saf2 414), Z95721 (RB40), AB015560 (BD4–10), AJ009461 (SJA-36), AB013269 (NKB17), D26171 (Acidobacterium capsulatum), X77215 (Holophaga foetida), and U41563 (Geothrix fermentans). AF155147 (Alcaligenes faecalis) was used as an outgroup.