Rapid method of determining factors limiting bacterial growth in soil

Abstract

A technique to determine which nutrients limit bacterial growth in soil was developed. The method was based on measuring the thymidine incorporation rate of bacteria after the addition of C, N, and P in different combinations to soil samples. First, the thymidine incorporation method was tested in two different soils: an agricultural soil and a forest humus soil. Carbon (as glucose) was found to be the limiting substance for bacterial growth in both of these soils. The effect of adding different amounts of nutrients was studied, and tests were performed to determine whether the additions affected the soil pH and subsequent bacterial activity. The incubation time required to detect bacterial growth after adding substrate to the soil was also evaluated. Second, the method was used in experiments in which three different size fractions of straw (1 to 2, 0.25 to 1, and <0.25 mm) were mixed into the agricultural soil in order to induce N limitation for bacterial growth. When the straw fraction was small enough (<0.25 mm), N became the limiting nutrient for bacterial growth after about 3 weeks. After the addition of the larger straw fractions (1 to 2 and 0.25 to 1 mm), the soil bacteria were C limited throughout the incubation period (10 weeks), although an increase in the thymidine incorporation rate after the addition of C and N together compared with adding them separately was seen in the sample containing the size fraction from 0.25 to 1 mm. Third, soils from high-pH, limestone-rich areas were examined. P limitation was observed in one of these soils, while tendencies toward P limitation were seen in some of the other soils.

FIG. 1

Flow diagram of the thymidine and leucine incorporation technique for detecting limiting substances for bacterial growth in soil.

FIG. 2

Relative bacterial activities (thymidine incorporation) at different times after the addition of C and N to the agricultural soil (A) and the forest humus (B). The activity of the nonamended control sample was set to 1. Bars indicate standard errors (SEs) (n = 2) obtained from ANOVA for each separate time. C, N, or CxN over the graph indicates significant effects of carbon, nitrogen, or the interaction of carbon and nitrogen. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

FIG. 3

Relative bacterial activities (thymidine incorporation) 48 h after the addition of C, N, and P separately and in different combinations. The activity of the nonamended control sample was set to 1. Bars indicate SEs for three different experiments. (A) Agricultural soil. (B) Forest humus.

FIG. 4

Comparison between the relative effects of C, N, and P addition in different combinations on the thymidine (open bars) and leucine (stipled bars) incorporation rate of soil bacteria. The activity of the nonamended control sample was set to 1. (A) Agricultural soil. (B) Forest humus.

FIG. 5

Relative bacterial activity (thymidine incorporation) 48 h after the addition of different concentrations of N-containing substances. The activity of the nonamended control sample was set to 1. (A) Agricultural soil. (B) Forest humus.

FIG. 6

The relation between relative bacterial activity (thymidine incorporation) and soil pH 48 h after the addition of different concentrations of N-containing substances to the forest humus. The activity of the nonamended control sample was set to 1.

FIG. 7

Relative bacterial activity (thymidine incorporation) 48 h after C and N addition to the agricultural soil. The activity of the nonamended control sample was set to 1. Straw was added at the beginning of the experiments. (A) Straw size fraction 1 to 2 mm. (B) Straw size fraction 0.25 to 1 mm. (C) Straw size fraction <0.25 mm. The bars indicate SEs (n = 2) obtained from ANOVA for each separate sampling date. C, N, or CxN over the graph indicates significant effects of carbon, nitrogen, or the interaction of carbon and nitrogen. ∗, P < 0.05; ∗∗, P < 0.01.

FIG. 8

Relative bacterial activity (thymidine incorporation) 64 h after the addition of C and P to different calcarious soils (A to G). The activity of the nonamended control sample was set to 1. 10xP indicates a separate experiment in which the effect of a 10-times-higher addition than normal was compared with the control. The bars indicate SEs obtained from ANOVA.