Anders R. Johnsen

Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

ABSTRACT We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.

Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of 13C-Labeled Cell Lipids

ABSTRACT Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [14C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [13C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The 13C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The 13C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in 13C, suggesting that actinomycetes metabolized phenanthrene in this soil. The 13C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess 13C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [13C]phenanthrene.

Diffuse PAH contamination of surface soils: environmental occurrence, bioavailability, and microbial degradation.

The purpose of this review is to recognize the scientific and environmental importance of diffuse pollution with polycyclic aromatic hydrocarbons (PAHs). Diffuse PAH pollution of surface soil is characterized by large area extents, low PAH concentrations, and the lack of point sources. Urban and pristine topsoils receive a continuous input of pyrogenic PAHs, which induces a microbial potential for PAH degradation. The significance of this potential in relation to black carbon particles, PAH bioaccessibility, microbial PAH degradation, and the fate of diffuse PAHs in soil is discussed. Finally, the state-of-the-art methods for future investigations of the microbial degradation of diffuse PAH pollution are reviewed.

Evaluation of Fluorescently Labeled Lectins for Noninvasive Localization of Extracellular Polymeric Substances in Sphingomonas Biofilms

ABSTRACT Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms andSphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.

Strong Impact on the Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated Soil Dominated by Mycobacteria

ABSTRACT Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.

Fungal PAH-metabolites resist mineralization by soil microorganisms.

This study investigated the mineralization of water-soluble polycyclic aromatic hydrocarbon (PAH) metabolites produced by the soil fungus Cunninghamella elegans. Eleven soil fungi were screened for their ability to metabolize (14)C-phenanthrene, (14)C-fluoranthene, and (14)C-pyrene into water-soluble compounds. Eight fungi produced water-soluble metabolites from all or some of the PAHs. The composition of the water-soluble PAH-metabolites from the most effective solubilizer C. elegans was analyzed by an ultraperformance liquid chromatograph interfaced to a quadrupole time-of-flight mass spectrometer. Thirty-eight metabolites were detected. All of 34 identified metabolites were sulfate-conjugated. The mineralization of (14)C-metabolites, produced by C. elegans, was compared to mineralization of the parent (14)C-PAHs in soil slurries. It was hypothesized that the increased bioavailability and metabolic activation of the metabolites would increase mineralization in soil slurries compared to mineralization of the parent PAHs. Unexpectedly, the mineralization of the (14)C-metabolites was in all cases extremely slow compared to the mineralization of the parent (14)C-PAHs. Slow (14)C-metabolite mineralization was not caused by metabolite toxicity, neither was cometabolic mineralization of (14)C-metabolites stimulated by the presence of active PAH-degraders. High water solubility, low lipophilicity, and extremely slow mineralization of the metabolites indicate a potential problem of leaching of fungal PAH-metabolites to the groundwater.

Triclosan affects the microbial community in simulated sewage-drain-field soil and slows down xenobiotic degradation

Effects of the common antibacterial agent triclosan on microbial communities and degradation of domestic xenobiotics were studied in simulated sewage-drain-field soil. Cultivable microbial populations decreased 22-fold in the presence of 4 mg kg⁻¹ of triclosan, and triclosan-resistant Pseudomonas strains were strongly enriched. Exposure to triclosan also changed the general metabolic profile (Ecoplate substrate profiling) and the general profile (T-RFLP) of the microbial community. Triclosan degradation was slow at all concentrations tested (0.33-81 mg kg⁻¹) during 50-days of incubation. Mineralization experiments (¹⁴C-tracers) and chemical analyses (LC-MS/MS) showed that the persistence of a linear alkylbenzene sulfonate (LAS) and a common analgesic (ibuprofen) increased with increasing triclosan concentrations (0.16-100 mg kg⁻¹). The largest effect was seen for LAS mineralization which was severely reduced by 0.16 mg kg⁻¹ of triclosan. Our findings indicate that environmentally realistic concentrations of triclosan may affect the efficiency of biodegradation in percolation systems.

Quantification of centimeter-scale spatial variation in PAH, glucose and benzoic acid mineralization and soil organic matter in road-side soil.

The aim of the study was to determine centimeter-scale spatial variation in mineralization potential in diffusely polluted soil. To this end we employed a 96-well microplate method to measure the mineralization of 14C-labeled organic compounds in deep-well microplates and thereby compile mineralization curves for 348 soil samples of 0.2-cm3. Centimeter-scale spatial variation in organic matter and the mineralization of glucose, benzoic acid, and PAHs (phenanthrene and pyrene) was determined for urban road-side soil sampled as arrays (7×11cm) of 96 subsamples. The spatial variation in mineralization was visualized by means of 2-D contour maps and quantified by means of semivariograms. The geostatistical analysis showed that the easily degradable compounds (glucose and benzoic acid) exhibited little spatial variation in mineralization potential, whereas the mineralization was highly heterogeneous for the PAH compounds that require specialized degraders. The spatial heterogeneity should be taken into account when estimating natural attenuation rates.

A radiorespirometric method for measuring mineralization of [14C]-compounds in a 96-well microplate format

A radiorespirometric method was developed for quantifying mineralization of [14C]-labeled substrates in deep-well microplates. Ninety-six 14CO2-traps were placed on an adhesive microplate sealing tape which made it possible to trap 14CO2 simultaneously from all wells. Trapped radioactivity was quantified by digital autoradiography.

Univariate and multivariate characterization of heavy fuel oil weathering and biodegradation in soil

Polycyclic aromatic compounds (PACs) of petrogenic origin are widespread contaminants of the environment. We investigated the weathering and biodegradation of fuel oil PACs in microcosms that contained natural soil or natural soil primed by adding bioremediated soil to a 2% concentration. In the primed soil, lag-phases were shorter, and biodegradation was more pronounced. To describe PAC degradation, changes in the relative distribution of PACs and changes in isomer ratios, that are specific for biodegradation, were determined by univariate and multivariate techniques. A multivariate approach based on normalized areas or isomer ratios, in combination with principal component analysis, provided the most detailed description of weathering and biodegradation. In particular, the sequence of degradation of PACs was more transparent when using the multivariate approach. We also identified new diagnostic ratios of dimethylphenanthrenes for use in the study of moderately and severely biodegraded samples.