Accumulation and degradation of dead-end metabolites during treatment of soil contaminated with polycyclic aromatic hydrocarbons with five strains of white-rot fungi

The white-rot fungi Trametes versicolor PRL 572, Trametes versicolor MUCL 28407, Pleurotus ostreatus MUCL 29527, Pleurotus sajor-caju MUCL 29757 and Phanerochaete chrysosporium DSM 1556 were investigated for their ability to degrade the polycyclic aromatic hydrocarbons (PAH) anthracene, benz[a]anthracene and dibenz[a,h]anthracene in soil. The fungi were grown on wheat straw and mixed with artificially contaminated soil. The results of this study show that, in a heterogeneous soil environment, the fungi have different abilities to degrade PAH, with Trametes showing little or no accumulation of dead-end metabolites and Phanerochaete and Pleurotus showing almost complete conversion of anthracene to 9,10-anthracenedione. In contrast to earlier studies, Phanerochaete showed the ability to degrade the accumulated 9,10-anthracenedione while Pleurotus did not. This proves that, in a heterogeneous soil system, the PAH degradation pattern for white-rot fungi can be quite different from that in a controlled liquid system. Received: 20 March 1996 / Received revision: 2 July 1996 / Accepted: 8 July 1996     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

Enhanced mineralization of pentachlorophenol by κ-carrageenan-encapsulated Pseudomonas sp. UG30

A pentachlorophenol(PCP)-degrading Pseudomonas sp. strain UG30 was encapsulated in κ-carrageenan for use in PCP degradation. Free and encapsulated cells were compared for their ability to dechlorinate and mineralize 100–800 μg/ml sodium pentachlorophenate in broth. Dechlorination was measured with a chloride ion electrode, and mineralization was measured by ¹⁴CO₂ evolution from radiolabelled [U-¹⁴C]PCP. Free and encapsulated Pseudomonas sp. UG30 cells mineralized up to 200 μg/ml and 600 μg/ml PCP, respectively, after 21 days. Encapsulation of UG30 cells provided a protective effect, allowing dechlorination and mineralization of high levels of PCP to occur. Received: 3 May 1996 / Received revision: 4 September 1996 / Accepted: 13 September 1996     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Bioremediation of diesel-oil-contaminated alpine soils at low temperatures

Bioremediation of two diesel-oil-contaminated alpine subsoils, differing in soil type and bedrock, was investigated in laboratory experiments at 10 °C after supplementation with an inorganic fertilizer. Initial diesel oil contamination of 4000 mg kg⁻¹ soil dry matter (dm) was reduced to 380–400 mg kg⁻¹ dm after 155 days of incubation. In both soils, about 30 % of the diesel oil contamination (1200 mg kg⁻¹ dm) was eliminated by abiotic processes. The residual decontamination (60 %–65 %) could be attributed to microbial degradation activities. In both soils, the addition of a cold-adapted diesel-oil-degrading inoculum enhanced biodegradation rates only slightly and temporarily. From C/N and N/P ratios (determined by measuring the contents of total hydrocarbons, NH₄ ⁺ N, NO₃ ⁻ N and PO₄ ³⁻ P) of soils␣it could be deduced that there was no nutrient deficiency during the whole incubation period. Soil biological activities (basal respiration and dehydrogenase activity) corresponded to the course of biodegradation activities in the soils. Received: 9 September 1996 / Accepted: 7 December 1996     

Response of fluoranthene-degrading bacteria to surfactants

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. Received: 6 February 1998 / Received revision: 19 June 1998 / Accepted: 19 June 1998     

Bioremediation of polychlorinated biphenyl-contaminated soil using carvone and surfactant-grown bacteria

Partial bioremediation of polychlorinated biphenyl (PCB)-contaminated soil was achieved by repeated applications of PCB-degrading bacteria and a surfactant applied 34 times over an 18-week period. Two bacterial species, Arthrobacter sp. strain B1B and Ralstonia eutrophus H850, were induced for PCB degradation by carvone and salicylic acid, respectively, and were complementary for the removal of different PCB congeners. A variety of application strategies was examined utilizing a surfactant, sorbitan trioleate, which served both as a carbon substrate for the inoculum and as a detergent for the mobilization of PCBs. In soil containing 100 μg Aroclor 1242 g⁻¹ soil, bioaugmentation resulted in 55–59% PCB removal after 34 applications. However, most PCB removal occurred within the first 9 weeks. In contrast, repeated addition of surfactant and carvone to non-inoculated soil resulted in 30–36% PCB removal by the indigenous soil bacteria. The results suggest that bioaugmentation with surfactant-grown, carvone-induced, PCB-degrading bacteria may provide an effective treatment for partial decontamination of PCB-contaminated soils. Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000     

Enhanced oily sludge biodegradation by a tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1

The biodegradation of an oily sludge is facilitated by a microbial tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1. The optimal oil-in-water dispersion conditions are as follows: pH 6.5, temperature 30 °C, agitation 150 rev/min. The total hydrocarbon content shows that the biodegradation of the oily substrate mediated by the biosurfactant or by the biosurfactant–P. aeruginosa USB-CS1 complex is significantly higher after 30 days of incubation than that in other experimental conditions, by a mean of 70%. Substrate fractionation by column chromatography reveals that, if biosurfactant is present, saturated and aromatic compounds are more susceptible to microbial degradation than they are in other biodegradation systems by an average of 55% and 40% respectively. These results suggest that the stimulatory effects of the biosurfactant on the biodegradation of the oily substrate are limited over time by the loss of surface activity of the biosurfactant after 30 days of incubation. Received : 7 August 1996 / Received revision : 6 December 1996 / Accepted : 4 January 1997     

Phycocyanin,a new elicitor for capsaicin and anthocyanin accumulation in plant cell cultures

  Elicitors of both fungal and bacterial origin that is, polysaccharides, proteins and fatty acids, are widely used for enhancement of secondary metabolites in plant cell cultures. In the present study, phycocyanin – a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis– was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in Capsicum frutescens and Daucus carota cell cultures respectively. Phycocyanin at 0.3, 0.6 and 1.2 mg% in capsicum cell cultures elicited a more than two-fold increase in capsaicin content with maximum productivity of 192 μg/g fresh weight. Similarly in Daucus carota cell cultures a two-fold increase in anthocyanin content was obtained at 0.3 mg% with a maximum productivity of 24.8 mg% on a dry-weight basis. In both the systems, phycocyanin showed an early elicitation of secondary metabolites. Received: 15 December 1995 / Received last revision: 15 July 1996 / Accepted: 18 July 1996     

Biodegradability of volatile hydrocarbons of gasoline

The biodegradability under aerobic conditions of volatile hydrocarbons (4–6 carbons) contained in gasoline and consisting of n-alkanes, iso-alkanes, cycloalkanes and alkenes, was investigated. Activated sludge was used as the reference microflora. The biodegradation test involved the degradation of the volatile fraction of gasoline in closed flasks under optimal conditions. The kinetics of biodegradation was monitored by CO₂ production. Final degradation was determined by gas chromatographic analysis of all measurable hydrocarbons (12 compounds) in the mixture after sampling the headspace of the flasks. The degradation of individual hydrocarbons was also studied with the same methodology. When incubated individually, all hydrocarbons used as carbon sources, except 2,2-dimethylbutane and 2,3-dimethylbutane, were completely consumed in 30 days or less with different velocities and initial lag periods. When incubated together as constituents of the light gasoline fraction, all hydrocarbons were metabolised, often with higher velocities than for individual compounds. Cometabolism was involved in the degradation of dimethyl isoalkanes. Received: 19 October 1999 / Received revision: 21 January 2000 / Accepted: 23 January 2000     

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 1998     

Influence of arbuscular mycorrhiza on clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons

 The effect of arbuscular mycorrhiza (AM) on white clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons (PAH) was assessed in a pot experiment. The soil was spiked with 500 mg kg^–1 anthracene, 500 mg kg^–1 chrysene and 50 mg kg^–1 dibenz(a,h)anthracene, representing common PAH compounds with three, four and five aromatic rings, respectively. Three treatments and two harvest times (8 and 16 weeks) were imposed on plants grown in spiked soil: no mycorrhizal inoculation, mycorrhizal inoculation (Glomus mosseae P2, BEG 69) and mycorrhizal inoculation and surfactant addition (Triton X-100). Pots without PAH were also included as a control of plant growth and mycorrhizal colonization as affected by PAH additions. The competitive ability of clover vis-à-vis ryegrass regarding shoot and root growth was enhanced by AM, but reduced by PAH and the added surfactant. This was reflected by mycorrhizal root colonization which was moderate for clover (20–40% of total root length) and very low for ryegrass (0.5–5% of total root length). Colonization of either plant was similar in spiked soil with and without the added surfactant, but the PAH reduced colonization of clover to half that in non-spiked soil. P uptake was maintained in mycorrhizal clover when PAH were added, but was reduced in non-mycorrhizal clover and in mycorrhizal clover that received surfactant. Similar effects were not observed on ryegrass. These results are discussed in the context of the natural attenuation of organic pollutants in soils. Accepted: 12 June 2000     

Transformation of tetrachloroethene in an upflow anaerobic sludgeblanket reactor

  Reductive dechlorination of tetrachloroethene was studied in a mesophilic upflow anaerobic sludge blanket reactor. Operating the reactor in batch mode the dynamic transformation of tetrachloroethene, trichloroethene and dichloroethene (DCE) was monitored. Tetrachloroethene was reductively dechlorinated to trichloroethene, which again was dechlorinated at the same rate as DCE was produced. DCE showed a lag period of 40 h before transformation was observed. During normal reactor operation trans-1,2-DCE was the major DCE isomer, followed by cis-1,2-DCE. Small amounts of 1,1-DCE but no vinyl chloride were detected. When the influent tetrachloroethene concentration was increased from 4.6 μM to 27 μM, the transformation rate increased, indicating that the system was not saturated with tetrachloroethene. The main organic component in the effluent was acetate, indicating that the aceticlastic methane-producing bacteria were inhibited by the chlorinated ethenes. Received: 29 July 1996 / Received revision: 13 September 1996 / Accepted: 13 September 1996     

Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998     

Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. Florida in solid wheat straw substrate

The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three ¹⁴C-labeled PAH by the white-rot fungus Pleurotus sp. Florida was investigated. Three concentrations containing 50, 250 or 1250 μg each unlabeled PAH/5 g straw were added to sterile sea sand. Selected treatments were added subsequently with ¹⁴C-labeled pyrene, benzo[a]anthracene or benzo[a]pyrene. The PAH-loaded sea sand was then mixed into straw substrate and incubated. The disappearance of the unlabeled four-to six-ring PAH: pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene, was determined by high-performance liquid chromatography. After 15 weeks of incubation, the recoveries were less than 25% for initial amounts of 50 μg (controls above 85%). The recoveries of unlabeled PAH increased in the inoculated samples with increasing concentrations applied. No correlation could be determined between the number of condensed rings of the PAH and the recoveries of added PAH. Pleurotus sp. Florida mineralized 53% [¹⁴C]pyrene, 25% [¹⁴C]benzo[a]anthracene and 39% [¹⁴C]benzo[a]pyrene to ¹⁴CO₂ in the presence of eight unlabeled PAH (50 μg applied) within 15 weeks. During the course of cultivation, Pleurotus sp. Florida degraded more than 40% of the wheat straw substrate. Variation of the initial concentration of PAH did not influence the extent of degradation of the organic matter. Received: 16 December 1996 / Received revision: 17 March 1997 / Accepted: 22 March 1997     

Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Effect of surfactant solubilization on biodegradation of polychlorinated biphenyl congeners by Pseudomonas LB400

A variety of commercial surfactants were tested to determine their effect on polychlorinated biphenyl (PCB) transformation by Pseudomonas LB400. Initial tests determined that most surfactants were fully or partially able to solubilize the PCB congeners 2,5,2′-chlorobiphenyl (CBP), 2,4,2′,4′-CBP, 2,3,5,2′,5′-CBP and 2,4,5,2′,4′,5′-CBP, at concentrations above the surfactants' critical micelle concentration (CMC). Surfactants were also found to have no negative effect on bacterial survival, as cell numbers were the same or higher after incubation in the presence of surfactants than after incubation without surfactants. A comparison of the extent of biotransformation of single PCB congeners by the bacterium revealed that, at surfactant concentrations above the CMC, the presence of an anionic surfactant promoted while nonionic surfactants inhibited PCB transformation, compared to a control with no surfactant. The rates of transformation of PCB congeners were also higher in the presence of the anionic surfactant compared to the control. The inhibitory effects of a nonionic surfactant, Igepal CO-630 at a concentration above its CMC, on transformation of 2,4,5,2′,5′-CBP could be eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant CMC. Received: 26 October 1998 / Received revision: 5 March 1999 / Accepted: 14 March 1999     

Humic acid effect on pyrene degradation: finding an optimal range for pyrene solubility and mineralization enhancement

The addition of humic acid (HA) to polycyclic aromatic hydrocarbon (PAH) contaminated systems has been shown to enhance, inhibit, or have no effect on the biodegradation of these PAHs. In this study, the surfactant effects of Elliott soil HA (ESHA) at two pH values were tested. At pH 7.0, ESHA did not behave as a surfactant. At pH 11.8, ESHA acted as a surfactant, as displayed by a decrease in surface tension with increasing concentrations of ESHA. The effect of ESHA on pyrene solubility was tested by adding 0 to 800 μg ESHA/g soil to soil-slurries. Enhancement of pyrene apparent solubility demonstrated a dose- and time-related effect. Broader doses from 0 to 10,080 μg ESHA/g soil and three higher doses from 3,360 to 10,080 μg ESHA/g soil were tested for their effects on pyrene mineralization by indigenous soil microorganisms and a novel PAH-degrading Mycobacterium sp. KMS in soil microcosms, respectively. ESHA amendments between 20 and 200 μg ESHA/g soil were found to consistently increase pyrene mineralization by indigenous microorganisms, while the 10,080 μg ESHA/g soil produced inhibition and all other doses presented no effects. Pyrene degradation by M. KMS was significantly inhibited by the addition of the highest dose of ESHA.