Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor

Abstract

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.

FIG. 1.

Schematic representation of the FF reactor.

FIG. 2.

Reactor monitoring. The PCP load (closed squares; micromolar per day) was determined at the influent port of the reactor. The hydraulic retention times were 28 to 35 h. Concentrations of acetate (lozenges) and 3-CP (open circles) were determined at the effluent port. Gas production (open squares) was monitored by water displacement. Arrows indicate the times when the PCP load in the influent was changed.

FIG. 3.

Bacteria and Archaea profiles by PCR-DGGE. DNA was extracted from reactor biofilm samples. 16S rRNA gene sequences were PCR amplified with primers specific for Bacteria or Archaea, and PCR products were transferred onto a 20 to 70% urea-formamide DGGE gel. (A) Bacteria. Lanes: 1 and 9, original granular sludge; 2 to 8, biofilm samples taken after 7, 14, 35, 56, 81, 116, and 180 days of operation, respectively. Lanes 7 to 9 are from another DGGE gel and were adjusted on the basis of the profiles of the original sludge. (B) Archaea. Lane 1, original granular sludge; lanes 2 to 5, biofilm samples taken after 7, 14, 56, and 81 days of operation, respectively.

FIG. 4.

Spatial arrangement of Bacteria, Archaea, and D. hafniense in the anaerobic FF reactor. Biofilm samples were fixed, permeabilized, hybridized with fluorescently labeled oligonucleotides, and then counterstained with YOYO-1 before being examined by CLSM. FISH was performed with EUB338-Cy3 (Bacteria, panel A), ARC915-Cy3 (Archaea, panel B), and PCP-1-4-Cy3 and PCP-1-8-Cy5 (D. hafniense, panel C). Panels A, B, and C are biofilms taken after 56, 116, and 225 days of operation, respectively. Bars correspond to 20 μm.