Impact of protozoan grazing on bacterial community structure in soil microcosms

Abstract

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.

FIG. 1.

Numbers of protozoa (a) and bacteria (b) and amount of inorganic nitrogen (c) in microcosms amended either with sterilized wheat roots or with a nutrient solution (sol.) and with (+P) and without (−P) a mixed protozoan community. The number of bacteria was estimated both by direct epifluorescence microscopy (AODC) and by plate counts (CFU). The error bars represent 1 standard error. Values marked with different letters are significantly different (P < 0.05; Tukey). For the bacterial counts, the letters a and b refer only to AODC values and the letters x, y, and z refer only to CFU values. For inorganic nitrogen (panel c), the error bars and letters (a, b, and c) refer to values for total inorganic nitrogen (NH₄⁺ + NO₃⁻).

FIG. 2.

Numbers of protozoa (a) and bacteria (b) and amount of inorganic nitrogen (c) in microcosms without protozoa (control), with a mixed community of eight isolates of soil protozoa (mixed), or with only one protozoan isolate (Acanth., Acanthamoeba; Cerc. 1 and 2, Cercomonas sp. morphotypes 1 and 2; Thaum., Thaumatomonas sp.; Spum., Spumella sp.). The data refer to the sampling after 52 days. For further details, see the legend to Fig. 1.

FIG. 3.

Frequency distribution of bacterial size classes in experiment 1 after 25 (a) and 52 days (b). The mean bacterial cell length (± standard error) for each treatment is given in parentheses. Values followed by different letters are significantly different (P < 0.05; Tukey). +P and −P, with and without a mixed protozoan community.

FIG. 4.

DGGE analysis of 16S rRNA gene fragments amplified from DNA extracted from three replicate microcosms of the four treatments of experiment 1 using eubacterial primers. Sterile soil microcosms were amended with either sterile wheat roots (Roots) or repeated additions of a liquid nutrient solution (Nut. sol.) containing various carbohydrates, organic acids, and amino acids (see the text) and inoculated with a mixture of soil bacteria (−P) or with bacteria and a mixture of eight soil protozoan isolates (+P). Bands marked with letters to the left of the lane correspond to bands that were excised from an ethidium bromide-stained gel with the same amplified DNA.

FIG. 5.

DGGE analysis of 16S rRNA gene sequences amplified from DNA extracted from three replicate microcosms amended with nutrient solution and inoculated with bacteria only (control), bacteria and a mixture of eight soil protozoan isolates (mixed), or bacteria and one of five individual protozoan isolates (Acanth., Acanthamoeba sp.; Cerc. 1 and 2, Cercomonas sp. morphotypes 1 and 2; Thaum., Thaumatomonas sp.; Spum., Spumella sp.). The bands marked with letters are presumed to be similar to the excised bands marked in Fig. 4. The bands marked with arrows are bands that respond differently in the different treatments.

FIG. 6.

Correspondence analysis plot of the DGGE banding patterns obtained from soil microcosms inoculated with bacteria alone (−P), with mixed protozoa (+P), or with one of the five individual protozoan isolates (see the legend to Fig. 5). The error bars represent the least significant difference (Tukey); hence, the coordinates are significantly different (P < 0.05) when the error bars do not overlap the mean from another treatment.

FIG. 7.

Principal-component (PC) plot of the community level physiological profile of soil microcosms amended with sterile roots and with (+P) or without (−P) the presence of a mixed protozoan community and soil microcosms amended with a nutrient solution (nut. sol.) without protozoa, with mixed protozoa, or with either Acanthamoeba (+Acanth. sp.) or Cercomonas sp. morphotype 1 (+Cerc. sp.1). The error bars represent the least significant difference (see the legend to Fig. 6).