Microstructure of Anaerobic Granules Bioaugmented with Desulfitobacterium frappieri PCP-1

Abstract

Oligonucleotide probes were used to study the structure of anaerobic granular biofilm originating from a pentachlorophenol-fed upflow anaerobic sludge bed reactor augmented with Desulfitobacterium frappieri PCP-1. Fluorescence in situ hybridization demonstrated successful colonization of anaerobic granules by strain PCP-1. Scattered microcolonies of strain PCP-1 were detected on the biofilm surface after 3 weeks of reactor operation, and a dense outer layer of strain PCP-1 was observed after 9 weeks. Hybridization with probes specific for Eubacteria and Archaea probes showed that Eubacteria predominantly colonized the outer layer, while Archaea were observed in the granule interior. Mathematical simulations showed a distribution similar to that observed experimentally when using a specific growth rate of 2.2 day ؊1 and a low bacterial diffusion of 10 ؊7 dm 2 day ؊1 . Also, the simulations showed that strain PCP-1 proliferation in the outer biofilm layer provided excellent protection of the biofilm from pentachlorophenol toxicity.Bioaugmentation of a natural bacterial community is a potentially efficient approach for maximizing the degradation performance of a biological system. However, the fate of a newly introduced strain strongly depends on its interactions with the members of the indigenous bacterial consortium. As a result, the disappearance of introduced strains was observed as often as their retention (4, 5, 7). Bioaugmentation attempts are most successful when a newly introduced strain establishes commensal or mutualistic relationships within a natural consortium, e.g., by degrading substances otherwise toxic to the consortium.The anaerobic degradation of chlorinated compounds by indigenous microorganisms often results in incomplete dechlorination and thus can benefit from bioaugmentation. In particular, bioaugmentation of an upflow anaerobic sludge bed (UASB) reactor with Dehalospirillum multivorans led to a transformation of perchloroethylene (PCE) to dichloroethylene, an improvement compared to a noninoculated control, which only transformed PCE to trichloroethene (17). Ahring et al.(1) introduced a 3-chlorobenzoate dechlorination activity in a UASB reactor by bioaugmentation with Desulfomonile tiedjei. Also, Christiansen and Ahring (7) demonstrated inoculation of a UASB reactor containing sterilized anaerobic granules with a pentachlorophenol (PCP)-degrading strain of Desulfitobacterium hafniense.In our previous work, a UASB reactor degrading PCP was successfully inoculated with the PCP-degrading strain Desulfitobacterium frappieri PCP-1 (28). This strain is the only isolated anaerobic microorganism capable of PCP dehalogenation to 3-chlorophenol (3). Proliferation of strain PCP-1 allowed a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day with a PCP removal efficiency of 99% and a dechlorination efficiency of not less than 91%. Following inoculation, the density of strain PCP-1 was found to increase from 10 6 to 10 10 cells/g of volatile suspended solids (V...

Desulfitobacterium hafniense Is Present in a High Proportion within the Biofilms of a High-Performance Pentachlorophenol-Degrading, Methanogenic Fixed-Film Reactor

“…In this respect, D. hafniense could have used chemotaxis toward pyruvate producers to scatter throughout the biofilm. We previously demonstrated that D. hafniense PCP-1 forms microcolonies and is mostly present at the surface of PCP-fed UASB reactors (27). However, in this particular case, strain PCP-1 was added to already formed granules.…”

“…They were then washed again in PBS for 5 min and stored at 4°C. For hybridization, biofilm samples were first dehydrated in a graded series of 50, 80, and 95% ethanol solutions for 5 min each and then soaked in an acetylation solution as described previously (27). The slides were then mounted on microscope slides for hybridization experiments.…”

“…The slides were then mounted on microscope slides for hybridization experiments. (27). Autofluorescence controls were prepared with hybridization buffer containing no fluorescent probe.…”

“…However, once added to autoclaved granules of a PCP-fed reactor, Desulfitobacterium hafniense DCB-2 showed a uniform distribution in the granules (11). Finally, we have previously demonstrated in a PCP-fed UASB bioreactor augmented with D. hafniense (formerly D. frappieri) PCP-1 that strain PCP-1 cells colonized the outer section of granules (27). This spatial organization suggests that strain PCP-1 protects other members of the consortium from PCP toxicity, thus supporting our observation of enhanced efficiency when PCP-1 bacteria are present.…”

Microbial Ecology of Fermentative Microbes in Anaerobic Granules

“…Alternatively, exogenously grown microorganisms (pure culture or mixed culture) with known hydrolysis function may be introduced into UASB and test whether they can survive and incorporate into anaerobic granules Lanthier et al, 2002).…”