Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community

Abstract

We used a culture-independent approach, namely, thermal gradient gel electrophoresis (TGGE) analysis of ribosomal sequences amplified directly from community DNA, to determine changes in the structure of the microbial community following phenol shocks in the highly complex activated sludge ecosystem. Parallel experimental model sewage plants were given shock loads of chlorinated and methylated phenols and simultaneously were inoculated (i) with a genetically engineered microorganism (GEM) able to degrade the added substituted phenols or (ii) with the nonengineered parental strain. The sludge community DNA was extracted, and 16S rDNA was amplified and analyzed by TGGE. To allow quantitative analysis of TGGE banding patterns, they were normalized to an external standard. The samples were then compared with each other for similarity by using the coefficient of Dice. The Shannon index of diversity, H, was calculated for each sludge sample, which made it possible to determine changes in community diversity. We observed a breakdown in community structure following shock loads of phenols by a decrease in the Shannon index of diversity from 1.13 to 0.22 in the noninoculated system. Inoculation with the GEM (Pseudomonas sp. strain B13 SN45RE) effectively protected the microbial community, as indicated by the maintenance of a high diversity throughout the shock load experiment (H decreased from 1.03 to only 0.82). Inoculation with the nonengineered parental strain, Pseudomonas sp. strain B13, did not protect the microbial community from being severely disturbed; H decreased from 1.22 to 0.46 for a 3-chlorophenol-4-methylphenol shock and from 1.03 to 0.70 for a 4-chlorophenol-4-methylphenol shock. The catabolic trait present in the GEM allowed for bioprotection of the activated sludge community from breakdown caused by toxic shock loading. In-depth TGGE analysis with similarity and diversity algorithms proved to be a very sensitive tool to monitor changes in the structure of the activated sludge microbial community, ranging from subtle shifts during adaptation to laboratory conditions to complete collapse following pollutant shocks.

FIG. 1

Validation experiment showing TGGE banding patterns of 16S rDNA fragments amplified from activated sludge DNA of three different untreated model sewage plants after scanning of the original gel and normalization to the reference standard. The time course of the experiments is indicated above the lanes in days. (A) Validation experiment. (B and C) Untreated controls (plant 3) of shock load experiments conducted with the GEM (B) and its parental strain Pseudomonas sp. strain B13 (C). S, standard reference pattern.

FIG. 2

Validation experiment showing analysis of the TGGE banding patterns from Fig. 1. (A) Dendrogram calculated on the basis of the Dice coefficient of similarity with the clustering algorithm of Ward. (B) Shannon index of diversity: ⧫, validation experiment; ■, GEM experiment; •, B13 experiment.

FIG. 3

GEM experiment showing original TGGE gels of amplified 16S rDNA fragments from activated sludge microbial communities given shock loads of 3CP-4MP (1 mM each) and simultaneously inoculated with GEM (plant 1) or lacking the GEM (plant 2). Plant 3 was an untreated control. The sampling time is indicated in days after the start of the phenol shock load. Lanes: S, standard reference pattern; GEM, pure culture of the GEM; 1, shock-loaded plant inoculated with the GEM; 2, shock-loaded plant lacking the GEM; 3, untreated control plant. Panels A, B, and C show individual TGGE gels.

FIG. 4

GEM experiment showing normalization of the TGGE gels from Fig. 3 to the reference standard. Gel strips are sorted in ascending time order for each plant separately. The sampling time is indicated above the gel strips. S, Standard reference pattern. (A) Plant 1, shock-loaded plant 1 inoculated with the GEM; (B) plant 2, shock-loaded plant 2 lacking the GEM; (C) plant 3, untreated control.

FIG. 5

GEM experiment showing analysis of the TGGE banding patterns from Fig. 4. (A) Dendrogram calculated on the basis of the Dice coefficient of similarity with the clustering algorithm of Ward. The terms “bioprotected,” “undisturbed,” and “collapsed” were assigned to the clusters to describe the status of the microbial communities during the shock load experiment. (B) Shannon index of diversity, H (shaded symbols), and oxygen uptake rate, Q_O2 (open symbols), of the activated sludge microbial communities during the GEM experiment: •, ○, shock-loaded plant 1, inoculated with the GEM; ⧫, ◊, shock-loaded plant 2, lacking the GEM; ■, □, plant 3, untreated control. The duration of the phenol shock load is indicated by the dotted line.

FIG. 6

B13 experiment showing original TGGE gels of amplified 16S rDNA fragments from activated sludge microbial communities given shock loads of substituted phenols (1 mM each individual phenol) and simultaneously inoculated with Pseudomonas sp. strain B13. The sampling time is indicated in hours or days relative to the start of the phenol shock. Lanes: S, standard reference pattern; GEM, pure culture of the GEM; 1, plant 1, inoculated with B13 and given a shock load of 3CP-4MP; 2, plant 2, inoculated with B13 and given a shock load of 4CP-4MP; 3, plant 3, untreated control plant.

FIG. 7

B13 experiment showing analysis of TGGE banding patterns from Fig. 6. (A) Dendrogram calculated on the basis of the Dice coefficient of similarity with the clustering algorithm of Ward. The terms “intermediate,” “collapsed,” and “undisturbed” were assigned to the clusters to describe the status of the microbial communities during the shock load experiment. (B) Shannon index of diversity, H (shaded symbols), and oxygen uptake rate, Q_O2 (open symbols), of the activated sludge microbial community during the B13 experiment: •, ○, plant 1, inoculated with B13 and amended with the 3CP-4MP mixture; ⧫, ◊, plant 2, inoculated with B13 and amended with the 4CP-4MP mixture; ■, □, plant 3, untreated control. The duration of the phenol shock load is indicated by the dotted line.