Ulrich Karlson

Aspects of phytoremediation of organic pollutants

Phytoremediation is a quite novel technique to clean polluted soils using plants. In theory, phytoremediation methods are cheap, are accepted by the public and, compared to physical or chemical approaches, are ecologically advantageous. Until today, however, there are only a few examples of successful applications. One reason is that the processes involved are complex, and a full clean up may require many years. Plants affect the water balance of a site, they change redox potential and pH, and stimulate microbial activity of the soil. These indirect influences may accelerate degradation in the root zone or reduce leaching of compounds to groundwater. Compounds taken up into plants may be metabolised, accumulated, or volatilised into air. Based on these processes, several phytoremediation methods have been developed: Phytoextraction, rhizofiltra-tion, phytostabilisation, rhizo and phytodegradation, pump and tree, land farming, phytovolatilisation, hydraulic control and more. Already in use are plants (and here willow, poplar and grass) for the degradation of petroleum products, aromatic hydrocarbons (BTEX), chlorinated solvents, explosives and cyanides. However, phytotoxicity and pollutant mass balances were rarely documented. Often, the success of the projects was not controlled, and only estimates can be made about the applicability and the potential of phytoremediation. This lack of experience about possibilities and limitations seems to be a hindrance for a broader use of these techniques.

Effects of C60 fullerene nanoparticles on soil bacteria and protozoans

Nanotechnology should produce numerous new materials in the coming years. Because of the novel design of nanomaterials with new physicochemical characteristics, their potential adverse impact on the environment and human health must be addressed. In the present study, agglomerates of pristine C60 fullerenes (50 nm to microm-size) were applied to soil at 0, 5, 25, and 50 mg/kg dry soil to assess their effect on the soil microbiota by measuring total respiration; biomass, number, and diversity of bacteria; and total number and diversity of protozoans during 14 d. Respiration and microbial biomass were unaffected by the fullerenes at any time, whereas the number of fast-growing bacteria was decreased by three- to fourfold just after incorporation of the nanomaterial. Protozoans seemed not to be very sensitive to C60, because their number decreased only slightly in the beginning of the experiment. With polymerase chain reaction and denaturing gradient gel electrophoresis analysis of eubacteria and kinetoplastids from the soil, however, a difference between the fullerene treatments and nonamended controls was demonstrated. The fullerenes did not induce more than 20 to 30% of relative dissimilarity (with both bacteria and protozoans) between treatments, but this effect was persistent throughout the experiment. It therefore is recommended that fullerene nanomaterial not be spread deliberately in the environment and that their ecotoxicology be further clarified.

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.

Hailstones: A Window into the Microbial and Chemical Inventory of a Storm Cloud

Storm clouds frequently form in the summer period in temperate climate zones. Studies on these inaccessible and short-lived atmospheric habitats have been scarce. We report here on the first comprehensive biogeochemical investigation of a storm cloud using hailstones as a natural stochastic sampling tool. A detailed molecular analysis of the dissolved organic matter in individual hailstones via ultra-high resolution mass spectrometry revealed the molecular formulae of almost 3000 different compounds. Only a small fraction of these compounds were rapidly biodegradable carbohydrates and lipids, suitable for microbial consumption during the lifetime of cloud droplets. However, as the cloud environment was characterized by a low bacterial density (Me = 1973 cells/ml) as well as high concentrations of both dissolved organic carbon (Me = 179 µM) and total dissolved nitrogen (Me = 30 µM), already trace amounts of easily degradable organic compounds suffice to support bacterial growth. The molecular fingerprints revealed a mainly soil origin of dissolved organic matter and a minor contribution of plant-surface compounds. In contrast, both the total and the cultivable bacterial community were skewed by bacterial groups (γ-Proteobacteria, Sphingobacteriales and Methylobacterium) that indicated the dominance of plant-surface bacteria. The enrichment of plant-associated bacterial groups points at a selection process of microbial genera in the course of cloud formation, which could affect the long-distance transport and spatial distribution of bacteria on Earth. Based on our results we hypothesize that plant-associated bacteria were more likely than soil bacteria (i) to survive the airborne state due to adaptations to life in the phyllosphere, which in many respects matches the demands encountered in the atmosphere and (ii) to grow on the suitable fraction of dissolved organic matter in clouds due to their ecological strategy. We conclude that storm clouds are among the most extreme habitats on Earth, where microbial life exists.

Response of fluoranthene-degrading bacteria to surfactants

Abstract A prerequisite for surfactant-enhanced biodegradation is that the microorganisms survive, take up substrate and degrade it in the presence of the surfactant. Two Mycobacterium and two Sphingomonas strains, degrading fluoranthene, were investigated for their sensitivity towards non-ionic chemical surfactants. The effect of Triton X-100 and Tween 80 above their critical micelle concentration on mineralization of [14C]-glucose and [14C]-fluoranthene was measured in shaker cultures. Tween 80 had no toxic effect on any of the tested strains. The surfactant inhibited fluoranthene mineralization by the hydrophobic Mycobacterium spp. slightly, but more than doubled that by the two less hydrophobic Sphingomonas strains. Triton X-100 inhibited fluoranthene mineralization by all strains, yet this was more pronounced for the Sphingomonas spp. Both surfactants caused cell wall permeabilization, as shown by transient colouring of surfactant-containing media. Inhibition of glucose mineralization, indicating non-specific toxic effects of Triton X-100, was observed only for the Sphingomonas strains and the toxicity was caused by micelle-to-cell interactions. These strains, however, appeared to recover from initial Triton X-100 toxicity within 50–500 h of exposure. The ratio of surfactant concentration to initial cell density was found to determine critically the bacterial response to surfactants. For both Sphingomonas and Mycobacterium strains, this work indicates that fluoranthene solubilized in surfactant micelles is only partially available for mineralization by the bacteria tested. However, our results suggest that optimal conditions for polycyclic aromatic hydrocarbon mineralization can be developed by selection of the proper surfactant, bacterial strains, cell density and incubation conditions.

Phytotoxicity of fresh and weathered diesel and gasoline to willow and poplar trees.

The toxicity of fresh and weathered gasoline and diesel fuel to willow and poplar trees was studied using a tree transpiration toxicity test. Soils were taken from an abandoned filling station. Concentrations in the samples were measured as the sum of hydrocarbons from C5 to C10 (gasoline) and C12 to C28 (diesel). Concentrations ranged from 145 to 921 mg/kg gasoline and 143 to 18231 mg/kg diesel. The correlation between log soil concentration and toxicity to willows(Salix viminalis x schwerinii) was highly significant for the diesel fraction (r2=0.81, n=19) and for the sum of hydrocarbons (r2=0.84, n=19). The EC50 (50% inhibition of transpiration) for the sum of hydrocarbons was determined at 3910 mg/kg (95% C.I., 2900 to 5270 mg/kg) and followed a log-normally distributed sigmoidal curve. The EC10 was 810 mg/kg (95% C.I., 396 to 1660 mg/kg). The results were verified with artificially mixed diesel and gasoline contaminated soils, and two willow and one poplar species(S. viminalis, S. alba and Populus nigra). Fresh diesel at about 1000 mg/kg showed no effect onS. alba, althoughP. nigra was more sensitive. 10000 mg/kg seriously affected the transpiration of all species, silver willow(S. alba) being the least sensitive. Free phase diesel killed all trees within six weeks. Fresh gasoline at 1000 mg/kg was deadly for all trees, hence was more toxic than weathered gasoline. Survival of poplars and willows planted at the abandoned filling station was compared to the laboratory findings. There was some correlation, but in the field, trees also suffered from other stress factors than fuel pollution.

Combined use of different Gfp reporters for monitoring single-cell activities of a genetically modified PCB degrader in the rhizosphere of alfalfa

Single-cell localization and activity of Pseudomonas fluorescens F113, colonizing alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1 ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of different stability. The monitoring systems permitted non-destructive in situ detection of F113rifpcb cells on the entire root system grown in both the presence and absence of 3-chlorobiphenyl (PCB-2). The root tip and sites of lateral root emergence were found to be hotspots for fast-growing cells. In addition, a reporter strain of P. fluorescens F113rifpcb for degradation of chlorinated biphenyl was constructed, using another gfp fusion with the meta-pathway Pm promoter from Pseudomonas putida (TOL plasmid). Expression of this promoter, which is strongly induced by the PCB-2 degradation product, 3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on alfalfa roots using the P. fluorescens F113rifpcb reporter. A small but distinct fraction of the introduced bacteria activated the Pm promoter and thus appeared to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading cells, which by design were identical to the sensing cells, were located in distinct microcolonies on the root surface or in intercellular crevices between the root epidermal cells. However, PCB-degrading cells were not observed in the root areas containing fast-growing cells, indicating that PCB degradation was not linked to high cellular activity.

Comparing the desorption and biodegradation of low concentrations of phenanthrene sorbed to activated carbon, biochar and compost

Carbonaceous soil amendments are applied to contaminated soils and sediments to strongly sorb hydrophobic organic contaminants (HOCs) and reduce their freely dissolved concentrations. This limits biouptake and toxicity, but also biodegradation. To investigate whether HOCs sorbed to such amendments can be degraded at all, the desorption and biodegradation of low concentrations of (14)C-labelled phenanthrene (≤5 μg L(-1)) freshly sorbed to suspensions of the pure soil amendments activated carbon (AC), biochar (charcoal) and compost were compared. Firstly, the maximum abiotic desorption of phenanthrene from soil amendment suspensions in water, minimal salts medium (MSM) or tryptic soy broth (TSB) into a dominating silicone sink were measured. Highest fractions remained sorbed to AC (84±2.3%, 87±4.1%, and 53±1.2% for water, MSM and TSB, respectively), followed by charcoal (35±2.2%, 32±1.7%, and 12±0.3%, respectively) and compost (1.3±0.21%, similar for all media). Secondly, the mineralization of phenanthrene sorbed to AC, charcoal and compost by Sphingomonas sp. 10-1 (DSM 12247) was determined. In contrast to the amounts desorbed, phenanthrene mineralization was similar for all the soil amendments at about 56±11% of the initially applied radioactivity. Furthermore, HPLC analyses showed only minor amounts (<5%) of residual phenanthrene remaining in the suspensions, indicating almost complete biodegradation. Fitting the data to a coupled desorption and biodegradation model revealed that desorption did not limit biodegradation for any of the amendments, and that degradation could proceed due to the high numbers of bacteria and/or the production of biosurfactants or biofilms. Therefore, reduced desorption of phenanthrene from AC or charcoal did not inhibit its biodegradation, which implies that under the experimental conditions these amendments can reduce freely dissolved concentration without hindering biodegradation. In contrast, phenanthrene sorbed to compost was fully desorbed and biodegraded.