Michael Bunge

Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) are among the most notorious environmental pollutants. Some congeners, particularly those with lateral chlorine substitutions at positions 2, 3, 7 and 8, are extremely toxic and carcinogenic to humans. One particularly promising mechanism for the detoxification of PCDDs and PCDFs is microbial reductive dechlorination. So far only a limited number of phylogenetically diverse anaerobic bacteria have been found that couple the reductive dehalogenation of chlorinated compounds—the substitution of a chlorine for a hydrogen atom—to energy conservation and growth in a process called dehalorespiration. Microbial dechlorination of PCDDs occurs in sediments and anaerobic mixed cultures from sediments, but the responsible organisms have not yet been identified or isolated. Here we show the presence of a Dehalococcoides species in four dioxin-dechlorinating enrichment cultures from a freshwater sediment highly contaminated with PCDDs and PCDFs. We also show that the previously described chlorobenzene-dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 (ref. 3) is able to reductively dechlorinate selected dioxin congeners. Reductive dechlorination of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) demonstrates that environmentally significant dioxins are attacked by this bacterium.

Evidence for methane production by saprotrophic fungi

Methane in the biosphere is mainly produced by prokaryotic methanogenic archaea, biomass burning, coal and oil extraction, and to a lesser extent by eukaryotic plants. Here we demonstrate that saprotrophic fungi produce methane without the involvement of methanogenic archaea. Fluorescence in situ hybridization, confocal laser-scanning microscopy and quantitative real-time PCR confirm no contribution from microbial contamination or endosymbionts. Our results suggest a common methane formation pathway in fungal cells under aerobic conditions and thus identify fungi as another source of methane in the environment. Stable carbon isotope labelling experiments reveal methionine as a precursor of methane in fungi. These findings of an aerobic fungus-derived methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase cultures.

Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

Anaerobic reductive dehalogenation of polychlorinated dioxins

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are among the most harmful environmental contaminants. Their widespread distribution due to unintentional or unknown release coincides with environmental persistence, acute and chronic toxicity to living organisms, and long-term effects due to the compounds’ tendency for bioaccumulation and biomagnification. While microbial aerobic degradation of PCDD/Fs is mainly reported for the turnover of low chlorinated congeners, this review focuses on anaerobic reductive dehalogenation, which may constitute a potential remediation strategy for polychlorinated compounds in soils and sediments. Microorganisms in sediments and in microcosms or enrichment cultures have been shown to be involved in the reductive dechlorination of dioxins. Bacteria related to the genus Dehalococcoides are capable of the reductive transformation of dioxins leading to lower chlorinated dioxins including di- and monochlorinated congeners. Thus, reductive dehalogenation might be one of the very few mechanisms able to mediate the turnover of polychlorinated dioxins by reducing their toxicity and paving the way for a subsequent breakdown of the carbon skeleton.

Properties of a trichlorodibenzo‐p‐dioxin‐dechlorinating mixed culture with a Dehalococcoides as putative dechlorinating species

An anaerobic mixed culture enriched over 16 transfers (1/10) from Saale river sediment reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) to di- and monochlorinated congeners in the presence of pyruvate (or lactate) and fumarate as cosubstrates. Besides TrCDD, tetrachloroethene and 1,2,3-trichlorobenzene were dechlorinated. Dioxin dehalogenation was sensitive to pasteurization, but was not remarkably influenced by inhibitors of methanogens, sulfate-reducing bacteria or Gram-positive bacteria. The rate of 1,3-dichlorodibenzo-p-dioxin formation increased with rising initial concentrations of 1,2,4-TrCDD (1-250 microM) from 0.05 to 5.4 micromol l(-1) day(-1). Two isolates, belonging to Sulfurospirillum and Trichococcus, did not show reductive dehalogenation. 16S rDNA-targeted methods further revealed the presence of Acetobacterium, Desulfitobacterium, Desulfuromonas and Dehalococcoides. Nested polymerase chain reaction (PCR) indicated the presence of Dehalococcoides in highest most probable number (MPN) dilutions that were positive for dioxin dechlorination.

Effects of silver nanoparticles on microbial growth dynamics

Engineered metal nanoparticles are increasingly used in consumer products, in part as additives that exhibit advantageous antimicrobial properties. Conventional nanoparticle susceptibility testing is based largely on determination of nontemporal growth profiles such as measurements of inhibition zones in common agar diffusion tests, counting of colony‐forming units, or endpoint or regular‐interval growth determination via optical density measurements. For better evaluation of the dynamic effects from exposure to nanoparticles, a cultivation‐based assay was established in a 96‐well format and adapted for time‐resolved testing of the effects of nanoparticles on micro‐organisms.

Regiospecific dechlorination of spiked tetra- and trichlorodibenzo-p-dioxins by anaerobic bacteria from PCDD/F-contaminated Spittelwasser sediments

Samples were taken from sediment of the River Spittelwasser (district Bitterfeld, Germany), which is highly polluted with PCDD/Fs and other chloroorganic compounds. The sediment cores were separated into 10-20 cm thick layers, spiked with 50 microM of 1,2,3,4-tetrachlorodibenzo-p-dioxin and incubated for 8 months under anaerobic conditions in the presence of cosubstrates. Reductive dechlorination of the tetrachlorinated congener and formation of tri- and dichlorinated products were observed in all biologically active incubations. Analysis of subcultures spiked with 1,2,3- and 1,2,4-trichlorodibenzo-p-dioxin, respectively, revealed two different dechlorination pathways within the sediment cores. Pathway M was characterized by the simultaneous dechlorination of peri- and lateralchlorine atoms, whereas sequence SP was restricted to the dechlorination at positions flanked by chlorine atoms on both sides.

The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles

Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

Antimicrobial Precious-Metal Nanoparticles and their Use in Novel Materials

Nanotechnology offers powerful new approaches to controlling unwanted microorganisms and other potential biohazards. Engineered nanoparticles with antifungal, antimicrobial, and antiviral properties are now being developed for a variety of applications, including manufacture and maintenance of sterile surfaces, prevention and control of biological contamination, food and water safety, and treatment of infectious diseases and cancer. The great potential of antimicrobial precious-metal nanoparticles is reflected by the high number of recent publications and patent applications, which is summarized, at least in part, in this paper. This review should provide an overview and offer guidance to the scientific community interested in nano(bio)technology, nanomedicine, and nanotoxicology, and may also be of interest to a broader scientific audience. Furthermore, this review covers specific topics in research and development addressing the effects of nanoparticles on microorganisms as well as novel nanotechnology-based approaches for controlling potentially pathogenic microorganisms.