Young-Beom Ahn

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 δ 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.

Detection and characterization of a dehalogenating microorganism by terminal restriction fragment length polymorphism fingerprinting of 16S rRNA in a sulfidogenic, 2-bromophenol-utilizing enrichment

ABSTRACT Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

Biostimulation and bioaugmentation to enhance dechlorination of polychlorinated dibenzo-p-dioxins in contaminated sediments

Dechlorination of spiked 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) was investigated in sediment microcosms from three polychlorinated dibenzo-p-dioxin and dibenzofuran (CDD/F)-contaminated sites: River Kymijoki, Finland; Gulf Island Pond, Maine; and Lake Roosevelt, Washington. Dechlorination was stimulated by addition of electron donor and halogenated priming compounds, and bioaugmentation by a mixed culture containing Dehalococcoides ethenogenes strain 195. Amendment with 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) promoted rapid dechlorination of 1,2,3,4-TeCDD to 2-monochlorodibenzo-p-dioxin (2MCDD) in Gulf Island Pond and River Kymijoki sediments, however, only slow dechlorination to 1,4-dichlorodibenzo-p-dioxin was observed in Lake Roosevelt sediments. The dechlorination pathway in 1,2,3,4-TeCB-amended microcosms proceeded mainly via 1,3-dichlorodibenzo-p-dioxin, with less production of 2,3-dichlorodibenzo-p-dioxin in comparison with other treatments. Microbial community analyses indicated that Dehalococcoides-like bacteria were enriched with 1,2,3,4-TeCB. Quantitative real-time PCR analysis of Dehalococcoides-specific 16S rRNA genes and the D. ethenogenes strain 195 dehalogenase gene, tceA, showed at least an order of magnitude higher gene copy numbers in the bioaugmented than in the nonbioaugmented microcosms. An active-dechlorinating population is present in the River Kymijoki and biostimulation may enhance both native Dehalococcoides spp. and the bioaugmented D. ethenogenes strain 195.

Anaerobic dehalogenation of organohalide contaminants in the marine environment.

Microbially mediated dehalogenation processes contribute to the global cycling of both biogenic and anthropogenic halogenated organic compounds. Detailed information on biodegradation mechanisms for a variety of organohalides and on the microorganisms mediating these processes has greatly increased our understanding of the cycling and fate of these unique and widespread compounds in our environment. The marine environment appears to be a particularly rich source of dehalogenating microorganisms. It is well established by laboratory and field studies that anaerobic dehalogenation of sediment contaminants, such as PCBs, pesticides, and dioxins, occurs intrinsically and can be enhanced via various methods. Specific dehalogenating bacterial populations can be enriched on various organohalides. Biodehalogenation processes are likely to be significantly affected by the prevailing terminal electron-accepting condition, and thus, biotransformation of organohalide contaminants in marine and estuarine environments will vary as a function of the redox conditions within the sediment profile. Fundamental knowledge of the activities and interactions of dehalogenating microorganisms is providing a strong basis for development of new bioremediation technologies for removal of harmful halogenated compounds from our environment.

Degradation of Phenol via Phenylphosphate and Carboxylation to 4-Hydroxybenzoate by a Newly Isolated Strain of the Sulfate-Reducing Bacterium Desulfobacterium anilini

ABSTRACT A sulfate-reducing phenol-degrading bacterium, strain AK1, was isolated from a 2-bromophenol-utilizing sulfidogenic estuarine sediment enrichment culture. On the basis of phylogenetic analysis of the 16S rRNA gene and DNA homology, strain AK1 is most closely related to Desulfobacterium anilini strain Ani1 (= DSM 4660T). In addition to phenol, this organism degrades a variety of other aromatic compounds, including benzoate, 2-hydroxybenzoate, 4-hydroxybenzoate, 4-hydroxyphenylacetate, 2-aminobenzoate, 2-fluorophenol, and 2-fluorobenzoate, but it does not degrade aniline, 3-hydroxybenzoate, 4-cyanophenol, 2,4-dihydroxybenzoate, monohalogenated phenols, or monohalogenated benzoates. Growth with sulfate as an electron acceptor occurred with acetate and pyruvate but not with citrate, propionate, butyrate, lactate, glucose, or succinate. Strain AK1 is able to use sulfate, sulfite, and thiosulfate as electron acceptors. A putative phenylphosphate synthase gene responsible for anaerobic phenol degradation was identified in strain AK1. In phenol-grown cultures inducible expression of the ppsA gene was verified by reverse transcriptase PCR, and 4-hydroxybenzoate was detected as an intermediate. These results suggest that the pathway for anaerobic degradation of phenol in D. anilini strain AK1 proceeds via phosphorylation of phenol to phenylphosphate, followed by carboxylation to 4-hydroxybenzoate. The details concerning such reaction pathways in sulfidogenic bacteria have not been characterized previously.

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

A reductively dehalogenating, strictly anaerobic, sulfate-reducing bacterium, designated strain AA1T, was isolated from the marine sponge Aplysina aerophoba collected in the Mediterranean Sea and was characterized phenotypically and phylogenetically. Cells of strain AA1T were Gram-negative, short, curved rods. Growth of strain AA1T was observed between 20 and 37 degrees C (optimally at 28 degrees C) at pH 7-8. NaCl was required for growth; optimum growth occurred in the presence of 25 g NaCl l(-1). Growth occurred with lactate, propionate, pyruvate, succinate, benzoate, glucose and sodium citrate as electron donors and carbon sources and either sulfate or 2-bromophenol as electron acceptors, but not with acetate or butyrate. Strain AA1T was able to dehalogenate several different bromophenols, and 2- and 3-iodophenol, but not monochlorinated or fluorinated phenols. Lactate, pyruvate, fumarate and malate were not utilized without an electron acceptor. The G+C content of the genomic DNA was 58.5 mol%. The predominant cellular fatty acids were C14:0, iso-C14:0, C14:0 3-OH, anteiso-C15:0, C16:0, C16:1omega7c and C18:1omega7c. Phylogenetic analysis based on 16S rRNA gene sequence comparisons placed the novel strain within the class Deltaproteobacteria. Strain AA1T was related most closely to the type strains of Desulfoluna butyratoxydans (96% 16S rRNA gene sequence similarity), Desulfofrigus oceanense (95%) and Desulfofrigus fragile (95%). Based on its phenotypic, physiological and phylogenetic characteristics, strain AA1T is considered to represent a novel species of the genus Desulfoluna, for which the name Desulfoluna spongiiphila sp. nov. is proposed. The type strain is AA1T (=DSM 17682T=ATCC BAA-1256T).

Kangiella spongicola sp. nov., a halophilic marine bacterium isolated from the sponge Chondrilla nucula

A novel halophilic bacterium of the genus Kangiella was isolated from a marine sponge collected from the Florida Keys, USA. Strain A79(T), an aerobic, Gram-negative, non-motile, rod-shaped bacterium, grew in 2-15 % (w/v) NaCl, at a temperature of 10-49 °C and at pH 4.5-10. Phylogenetic analysis placed strain A79(T) in the family Alcanivoraceae in the class Gammaproteobacteria. Strain A79(T) showed 98.5 % 16S rRNA gene sequence similarity to Kangiella japonica KMM 3899(T), 96.6 % similarity to Kangiella koreensis DSM 16069(T) and 95.6 % similarity to Kangiella aquimarina DSM 16071(T). The major cellular fatty acids were iso-C(11 : 0), iso-C(11 : 0) 3-OH, iso-C(15 : 0), iso-C(17 : 0) and iso-C(17 : 1)ω9c and the G+C content of the genomic DNA was 44.9 mol%. On the basis of physiological, chemotaxonomic and phylogenetic comparisons, strain A79(T) represents a novel species in the genus Kangiella, for which the name Kangiella spongicola sp. nov. is proposed. The type strain is A79(T) ( = ATCC BAA-2076(T) = DSM 23219(T)).

Pleiotropic and Epistatic Behavior of a Ring-Hydroxylating Oxygenase System in the Polycyclic Aromatic Hydrocarbon Metabolic Network from Mycobacterium vanbaalenii PYR-1

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.

Effect of sterilized human fecal extract on the sensitivity of Escherichia coli ATCC 25922 to enrofloxacin

The ingestion of antimicrobial residues in foods of animal origin has the potential risk of exposing colonic bacteria to small concentrations of antibiotics and inducing resistance in the colonic bacteria. To investigate whether human intestinal contents would influence resistance development in bacteria, Escherichia coli ATCC 25922 (MIC of enrofloxacin <0.03 μg ml−1) was exposed to 0.01 to 1 μg ml−1 of enrofloxacin in media supplemented with glucose, sucrose, sodium acetate or sterilized human fecal extract. In the first passage, only the medium containing sterilized fecal extract supported the growth of E. coli at an enrofloxacin concentration equal to the MIC. In the second and third passages following exposure to sub-inhibitory concentrations of the drug, the bacteria in media containing sterilized fecal extract grew at 0.1 μg ml−1 of enrofloxacin. The efflux pump inhibitors, reserpine and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), increased the sensitivity of bacteria to 0.1 μg ml−1 of enrofloxacin in the medium containing sucrose, but their effect was not observed in the medium supplemented with 2.5% sterilized fecal extract. The proportions of unsaturated and saturated fatty acids in E. coli grown in the medium with 2.5% sterilized fecal extract differed from those grown in the medium alone. Fecal extract may contain unknown factors that augment the ability of E. coli to grow in concentrations of enrofloxacin higher than MIC, both in the presence and absence of efflux pump inhibitors. This is the first study showing that fecal extract affects the level of sensitivity of E. coli to antimicrobial agents.

ANAEROBIC DEHALOGENATION OF HALOGENATED ORGANIC COMPOUNDS: NOVEL STRATEGIES FOR BIOREMEDIATION OF CONTAMINATED SEDIMENTSOF CONTAMINATED SEDIMENTSOF CONTAMINATED SEDIMENTSOF CONTAMINATED SEDIMENTS

greatest challenges for restoration of estuaries. Halogenated organic compounds constitute one of the largest groups of environmental pollutants and their use has resulted in widespread dissemination and environmental contamination, with freshwater, estuarine and marine sediments as significant sinks. Consequently, the management of sediments contaminated with toxic organic compounds, including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), pesticides and brominated flame retardants, is a major problem with far-reaching economic and ecological consequences. Anaerobic dehalogenating populations appear to be abundant in estuarine and marine sediments and many chlorinated and brominated aromatic compounds are readily dehalogenated, potentially leading to complete degradation and mineralization. For example, dechlorination of chlorinated dioxins and dibenzofurans is readily promoted in sediments from several sites. The biodegradability of organohalides is affected by available electron donors and acceptors, and the dehalogenating microbial populations active in different redox zones are distinct. Co-amendment with halogenated analogues enhanced dechlorination of spiked PCDD/Fs in estuarine sediments under a variety of conditions. Enhancement of microbial dehalogenation is an attractive remediation alternative that could potentially detoxify sediments and avoid the problematic redistribution of contaminants that is associated with dredging. Microbial reductive dechlorination is an important environmental process because it has the potential of decreasing the toxicity of PCDD/Fs if lateral chlorines are removed. These fundamental studies are providing an understanding of how dehalogenation processes are incorporated into a global 505 Remediation of sediments contaminated with toxic chemicals is one of the I. Twardowska et al. (eds.), and Water Pollution Monitoring, Protection and Remediation, 3–23.