Bacterial community dynamics during start-up of a trickle-bed bioreactor degrading aromatic compounds

Abstract

This study was performed with a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds (Solvesso100). The starter culture for the bioreactor was prepared in a fermentor with a wastewater sample of a care painting facility as the inoculum and Solvesso100 as the sole carbon source. The bacterial community dynamics in the fermentor and the bioreactor were examined by a conventional isolation procedure and in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotides. Two significant shifts in the bacterial community structure could be demonstrated. The original inoculum from the wastewater of the car factory was rich in proteobacteria of the alpha and beta subclasses, while the final fermentor enrichment was dominated by bacteria closely related to Pseudomonas putida or Pseudomonas mendocina, which both belong to the gamma subclass of the class Proteobacteria. A second significant shift was observed when the fermentor culture was transferred as inoculum to the trickle-bed bioreactor. The community structure in the bioreactor gradually returned to a higher complexity, with the dominance of beta and alpha subclass proteobacteria, whereas the gamma subclass proteobacteria sharply declined. Obviously, the preceded pollutant adaptant did not lead to a significant enrichment of bacteria that finally dominated in the trickle-bed bioreactor. In the course of experiments, three new 16S as well as 23S rRNA-targeted probes for beta subclass proteobacteria were designed, probe SUBU1237 for the genera Burkholderia and Sutterella, probe ALBO34a for the genera Alcaligenes and Bordetella, and probe Bcv13b for Burkholderia cepacia and Burkholderia vietnamiensis. Bacteria hybridizing with the probe Bcv13b represented the main Solvesso100-degrading population in the reactor.

FIG. 1

Schematic diagram of the experimental set-up of the trickle-bed bioreactor. T, temperature sensor; TC, temperature control; FID, flame ionization detector.

FIG. 2

Measured specific elimination capacity (defined as the difference of gas inlet and outlet concentrations divided by the mean gas residence time) versus specific pollutant load (defined as the gas inlet concentration divided by the mean gas residence time). The days when the samples for the microbial examinations were taken (days 127 and 227) are indicated.

FIG. 3

Measured degree of conversion (defined as the difference of gas inlet and outlet concentrations divided by the gas inlet concentration) versus specific pollutant load. The days when the samples for the microbial examinations were taken (days 127 and 227) are indicated.

FIG. 4

Comparison of bacterial composition of the samples from the wastewater of a car factory (inoculum I), the fermentor, and the trickle-bed bioreactor (biofilm and liquid phase) as determined by in situ hybridization. Error bars, standard deviations.

FIG. 5

In situ hybridization with fluorescein- and tetramethylrhodamine-labeled probes. Phase-contrast (upper panels) and epifluorescence micrographs (middle and lower panels) are shown for identical microscopic fields. Bar, 5 μm (all photomicrographs). (A) A sample from the fermentor at day 48 (inoculum II) was hybridized with fluorescein-labeled probe GAM42a (middle panel) and tetramethylrhodamine-labeled probe Ppu56a (lower panel). (B) A sample of the trickle-bed bioreactor biofilm at day 127 was hybridized with tetramethylrhodamine-labeled probe BET42a (middle panel) and fluorescein-labeled probe Bcv13b (lower panel).

FIG. 6

16S rRNA sequence alignment showing target regions of probe SUBU1237 for a selection of reference strains. Nucleotides are only identified for mispairings; pairings are indicated by dots. Lowercase letters indicate weakly destabilizing mispairing. Uppercase letters indicate strongly destabilizing mispairings.