Bacterial community structure and physiological state within an industrial phenol bioremediation system

Abstract

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the alpha, beta and gamma subclasses of the class Proteobacteria [alpha-, beta-, and gamma-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the gamma-Proteobacteria. The alpha subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the beta subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The gamma subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the gamma-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted.

FIG. 1

Schematic diagram of the wastewater-processing system under investigation. P1 A and B, waste-receiving reservoir section 1, subsections A and B; P2 A and B, waste-receiving reservoir section 2, subsections A and B; INTER, intermediate reservoir; B, BITMAC influent; INLET, holding and mixing reservoir for VR; VR, Vitox biological reactor; TIDAL, tidal storage tank prior to discharge. Connecting pipelines and distances are shown in bold whilst the volume of each processing section is indicated in italics. Approximate flow rate into the biological reactor (VR) is also indicated.

FIG. 2

DGGE profiles obtained for all the treatment sections (corresponding to Fig. 1) after gel digitization and profile extraction. DGGE profile peaks represent band positions and intensities within the gel; the top of the gel is represented by lower pixel positions whilst increasing pixel position values represent distance down the gel.

FIG. 3

Unweighted pair group method using mathematic averages dendrogram of DGGE profiles obtained in Fig. 2 for each compartment within the processing system, indicating the similarity between the fingerprints by pairwise comparisons of DGGE band presence and position.

FIG. 4

Total cell counts obtained by DAPI staining and community structure by fluorescent in situ hybridization counts (% of total DAPI count) for populations within processing system compartments. (A) Total DAPI count. (B) Community composition based upon the percentage of the total DAPI cells assigned to the respective subclasses within the Proteobacteria by specific probes α 1b, β 42a, and γ 42a. (C) Group-specific probes: CF319a for members of the Cytophaga-Flavobacterium cluster; Ps, percentage of pseudomonds in the total DAPI cells detected.

FIG. 5

The relationship between total DAPI count and phenolic concentration measured throughout the processing system. Treatment compartment values are indicated as follows: P1A, ●; P1B, ■; P2A, ○; P2B, □; BITMAC, ×; INTER, ◊; INLET, ▴; VR, ▵; TIDAL, ⧫.

FIG. 6

(A) EUB338 probe fluorescence (as an indicator of ribosome content) for probe-positive members members of the domain Bacteria within the process compartments and its relationship with total phenolic concentration over the nine compartments. (B) Relationship between probe γ 42a fluorescence and phenolic concentration. (C) Relationship between probe γ 42a fluorescence and thiocyanate concentration. Note that the γ 42a data is restricted to five compartments (see Methods and Materials). Only relationships significant at P < 0.05 are indicated. Treatment compartment position in all the plots are indicated as follows: P1A, ●; P1B, ■; P2A, ○; P2B, □; BITMAC, ×; INTER, ◊; INLET, ▴; VR, ▵; TIDAL, ⧫.