Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Abstract

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the ␦ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.

Desulfoluna spongiiphila sp. nov., a dehalogenating bacterium in the Desulfobacteraceae from the marine sponge Aplysina aerophoba

“…We have previously demonstrated that the sponge Aplysina (synonym Verongia) aerophoba, which produces a brominated tyrosine derivative, harbours anaerobic, reductively dehalogenating bacteria (Ahn et al, 2003). From Aplysina sponge material, we maintained stable anaerobic enrichment cultures by using lactate as an electron donor and bromophenols as electron acceptors.…”

“…These compounds may serve as a chemical defence against predators and may inhibit biofouling (Weiss et al, 1996). In addition, Aplysina sponges harbour large amounts of bacteria, which can amount to 40 % of the biomass of the animal, and it has been hypothesized that some of the organobromine compounds may in fact be synthesized by bacteria associated with the sponge (Hentschel et al, 2001(Hentschel et al, , 2003.As the marine environment is a particularly rich source of biogenic organohalides, it is not surprising that dehalogenating bacteria have been isolated from marine environments. Two dehalogenating bacteria in the genus Abbreviations: 2BP, 2-bromophenol; APS, adenosine 59-phosphosulfate; DGGE, denaturing gradient gel electrophoresis; TRF, terminal restriction fragment; T-RFLP, terminal restriction fragment length polymorphism.…”

“…Sun et al (2000) isolated a halobenzoate-dehalogenating Desulfomonile limimaris strain from marine sediments. Propionigenium maris, isolated from bromophenol-producing worms, is capable of debrominating 2,4,6-tribromophenol to 4-bromophenol (Steward et al, 1995;Watson et al, 2000).We have previously demonstrated that the sponge Aplysina (synonym Verongia) aerophoba, which produces a brominated tyrosine derivative, harbours anaerobic, reductively dehalogenating bacteria (Ahn et al, 2003). From Aplysina sponge material, we maintained stable anaerobic enrichment cultures by using lactate as an electron donor and bromophenols as electron acceptors.…”

“…Nucleosides were analysed by using a model 1100 series chromatograph (Agilent Technologies), with a reversed-phase C18 column (Partisil ODS-3, 4.66250 mm, particle size 5 mm; Whatman) and a solvent of phosphate/methanol (90 : 10), and absorbance was monitored at 254 nm. Phosphate buffer (pH 4.0) was prepared by dissolving KH 2 PO 4 (0.049 M) in distilled water and adjusting the pH with 85 % H 3 PO 4 .Phenol and halogenated aromatic compounds were analysed by HPLC (1100 series; Agilent) with a C18 column (Spherisorb, 4.66250 mm, particle size 5 mm; Phenomenex), at a flow rate of 1 ml min 21 , with an eluent of methanol/water/acetic acid (40 : 58 : 2) and a UV detector set to 280 nm (Ahn et al, 2003). Organic acids were analysed by HPLC (LC-10AS; Shimadzu Corp.) with an HPX-87H column (Bio-Rad) at 60 u C, at a flow rate of 0.6 ml min 21 , with an eluent of 0.004 mM sulfuric acid and a UV detector set to 210 nm.…”

“…The amplified product was digested with restriction enzyme MnlI and the 59-labelled terminal restriction fragment (TRF) was analysed with an ABI310 sequencer (Applied Biosystems) with GENESCAN software. DGGE was performed with a D gene system (Bio-Rad Laboratories) as described previously (Ahn et al, 2003).…”