Population dynamics of phenol-degrading bacteria in activated sludge determined by gyrB-targeted quantitative PCR

Abstract

A method for quantifying bacterial populations introduced into an activated-sludge microbial community is described. The method involves extraction of DNA from activated sludge, appropriate dilution of the extracted DNA with DNA extracted from nonintroduced activated sludge, PCR amplification of a gyrB gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. The adequacy of the method was examined by analyzing the population dynamics of two phenol-degrading bacteria, Pseudomonas putida BH and Comamonas sp. strain E6, that had been introduced into phenol-digesting activated sludge. The density of each of the two populations determined by the PCR method immediately after the introduction was consistent with the density estimated from a plate count of the inoculum. This quantitative PCR method revealed different population dynamics for the two strains in the activated sludge under different phenol-loading conditions. The behavior of both of these strains in the activated sludge reflected the growth kinetics of the strains determined in laboratory axenic cultures.

FIG. 1

PCR amplification of the gyrB fragments of strains BH and E6. For the upper part of each gel 10-fold serial dilutions of each pure culture were mixed with the activated sludge, and DNA extraction from the mixed samples and PCR amplification of the extracted DNA were carried out. (a) Strain BH. Lane 1, 50-2500 DNA size marker (FMC Corporation); lane 2, 2.5 × 10⁷ cells per ml; lane 3, 2.5 × 10⁶ cells per ml; lane 4, 2.5 × 10⁵ cells per ml; lane 5, 2.5 × 10⁴ cells per ml; lane 6, 2.5 × 10³ cells per ml; lane 7, 2.5 × 10² cells per ml; lane 8, 2.5 × 10¹ cells per ml; lane 9, 2.5 × 10⁰ cells per ml; lane 10, uninoculated activated sludge; lane 11, pure BH culture. (b) Strain E6. Lane 1, 50-2500 DNA marker; lane 2, 1.9 × 10⁷ cells per ml; lane 3, 1.9 × 10⁶ cells per ml; lane 4, 1.9 × 10⁵ cells per ml; lane 5, 1.9 × 10⁴ cells per ml; lane 6, 1.9 × 10³ cells per ml; lane 7, 1.9 × 10² cells per ml; lane 8, 1.9 × 10¹ cells per ml; lane 9, 1.9 × 10⁰ cells per ml; lane 10, uninoculated activated sludge; lane 11, pure E6 culture. For the lower part of each gel DNA was extracted from activated sludge containing strains BH and E6 at densities of 2.5 × 10⁷ and 1.9 × 10⁷ cells per ml, respectively, before 10-fold serial dilution of the extracted DNA with DNA extracted from the uninoculated activated sludge. PCR amplification was carried out by using the diluted samples. (a) Strain BH. (b) Strain E6. Lanes 1, 50-2500 DNA size marker; lanes 2, undiluted DNA; lanes 3, 10-fold dilution; lanes 4, 10²-fold dilution; lanes 5, 10³-fold dilution; lanes 6, 10⁴-fold dilution; lanes 7, 10⁵-fold dilution; lanes 8, 10⁶-fold dilution; lanes 9, 10⁷-fold dilution; lanes 10, uninoculated activated sludge; lanes 11, pure culture.

FIG. 2

Standard curves for determining the densities of BH and E6 populations in laboratory unit sludge. A quantitative PCR was carried out as described in the legend to Fig. 1, except that a DNA quantity standard was used to compare the results obtained in different gels. The ordinate indicates the intensity of the band of each PCR product relative to the intensity of the DNA quantity standard. The means of three determinations are shown, and the error bars indicate standard deviations.

FIG. 3

Specificity and sensitivity of the PCR method for detecting the BH population in Ohdaira activated sludge. Lane 1, 50-2500 DNA size marker; lane 2, 3.5 × 10⁶ cells per ml; lane 3, 3.5 × 10⁵ cells per ml; lane 4, 3.5 × 10⁴ cells per ml; lane 5, 3.5 × 10³ cells per ml; lane 6, 3.5 × 10² cells per ml; lane 7, 3.5 × 10¹ cells per ml; lane 8, 3.5 × 10⁰ cells per ml; lane 9, uninoculated activated sludge; lane 10, pure BH culture.

FIG. 4

Dynamics of the BH (a) and E6 (b) populations introduced into phenol-digesting activated sludge under stable operational conditions at a phenol-loading rate of 0.4 g per liter per day. Symbols: ○, population density in total activated sludge; •, population density in activated-sludge flocs (per milliliter of liquid in the aeration tank); dashed line, theoretical washout curve at a T_r of 10 days; dotted line, theoretical washout curve at a T_r of 0.5 day. The theoretical curves were drawn by using the following equation: S_t = S₀ · exp[−t/T_r], where S_t is the population density at time t and S₀ is the initial population density. The means of three determinations are shown, and the error bars indicate standard deviations.

FIG. 5

Dynamics of BH (a) and E6 (b) populations in phenol-digesting activated sludge that was subjected to phenol shock loading as described in the text. Dotted bars, population densities in total activated sludge; cross-hatched bars, population densities in activated-sludge flocs (per milliliter of liquid in the aeration tank). The means of three determinations are shown, and the error bars indicate standard deviations.