Phenanthrene Emulsification and Biodegradation Using Rhamnolipid Biosurfactants and Acinetobacter calcoaceticus In Vitro

The ability of biosurfactants and Acinetobacter calcoaceticus to enhance the emulsification and biodegradation of phenanthrene was investigated. Phenanthrene is a polycyclic aromatic hydrocarbon that may be derived from various sources, for example incomplete combustion of petroleum fuel, and thus it occurs ubiquitously throughout the environment. In order to assess the efficacy of a biosurfactant microparticle system, emulsification assays and in vitro biodegradation studies were conducted. Emulsification assays were carried out to assess the stability of phenanthrene emulsions. Emulsion stability was determined by the height of the emulsion layer (Emulsification Index) and turbidity. In vitro biodegradation tests were done to estimate phenanthrene degradation from an aqueous system by A. calcoaceticus supplemented with encapsulated (ERhBS) and nonencapsulated biosurfactants (NERhBS). Results show that phenanthrene emulsifications were stabilized after 48 h with NERhBS and remained stable for 72 additional hours. Phenanthrene emulsifications were stabilized with ERhBS after 216 h and remained stable for an additional 96 h. A. calcoaceticus alone and supplemented with rhamnolipid biosurfactant were able to biodegrade 10 to 50 mg L^?1 of phenanthrene within 250 h. When supplemented with NERhBS, A. calcoaceticus degraded phenanthrene significantly faster than when nonsupplemented or supplemented with ERhBS. Addition of exogenous biosurfactants was considered to be a major factor driving the direct correlation between decreasing phenanthrene concentration in the system and increasing bacterial biomass.     

Ammonium enhances the uptake, bioaccumulation, and tolerance of phenanthrene in cucumber seedlings

Aims

Phenanthrene is one of the ubiquitous, persistent organic pollutants commonly found in soil and sediments. The study will provide insight regarding the feasibility of nitrogen-assisted phytoremediation.

Methods

To study the effects of various nitrogen forms on cucumber seedling phenanthrene tolerance, hydroponic experiments were conducted in a greenhouse.

Results

Under phenanthrene stress, decreases in plant growth and biomass were more pronounced with a nitrate supply than with ammonium. In addition, phenanthrene concentrations in plants fed with ammonium were higher than those fed with nitrate. The reduction in plant protein and sugar, increases in nitrogen and phosphate concentrations, and increased activity of antioxidative enzymes may contribute to the phenanthrene stress response and adaptation. Higher peroxidase, superoxide dismutase, and catalase activities were found in ammonium-fed plants as compared to nitrate-fed plants under phenanthrene stress. Moreover, the reduction in soluble protein content and increases in phenanthrene transport and accumulation in non-photosynthetic organs may enable ammonium-fed plants to adapt more effectively to adverse conditions.

Conclusions

Overall, these results suggest that ammonium nutrition could provide a useful tool to improve the growth and adaption of plants under phenanthrene stress.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

The role of salicylate and biosurfactant in inducing phenanthrene degradation in batch soil slurries

The majority of polycyclic aromatic hydrocarbons (PAHs) sorb strongly to soil organic matter posing a complex barrier to biodegradation. Biosurfactants can increase soil-sorbed PAHs desorption, solubilisation, and dissolution into the aqueous phase, which increases the bioavailability of PAHs for microbial metabolism. In this study, biosurfactants, carbon sources, and metabolic pathway inducers were tested as stimulators of microorganism degradation. Phenanthrene served as a model PAH and Pseudomonas putida ATCC 17484 was used as the phenanthrene degrading microorganism for the liquid solutions and soil used in this investigation. Bench-scale trials demonstrated that the addition of rhamnolipid biosurfactant increases the apparent aqueous solubility of phenanthrene, and overall degradation by at least 20% when combined with salicylate or glucose in liquid solution, when compared to solutions that contained salicylate or glucose with no biosurfactant. However, salicylate addition, with no biosurfactant addition, increased the total degradation of phenanthrene 30% more than liquid systems with only biosurfactant addition. In soil slurries, small amounts of biosurfactant (0.25 g/L) showed a significant increase in total removal when only biosurfactant was added. In soil slurries containing salicylate, the effects of biosurfactant additions were negligible as there was greater than 90% removal, regardless of the biosurfactant concentration. The results of experiments performed in this study provide further evidence that an in situ enhancement strategy for phenanthrene degradation could focus on providing additional carbon substrates to induce metabolic pathway catabolic enzyme production, if degradation pathway intermediates are known.     

Adhesion to the hydrocarbon phase increases phenanthrene degradation by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> LP6a

Microbial adhesion is an important factor that can influence biodegradation of poorly water soluble hydrocarbons such as phenanthrene. This study examined how adhesion to an oil–water interface, as mediated by 1-dodecanol, enhanced phenanthrene biodegradation by Pseudomonas fluorescens LP6a. Phenanthrene was dissolved in heptamethylnonane and added to the aerobic aqueous growth medium to form a two phase mixture. 1-Dodecanol was non-toxic and furthermore could be biodegraded slowly by this strain. The alcohol promoted adhesion of the bacterial cells to the oil–water interface without significantly changing the interfacial or surface tension. Introducing 1-dodecanol at concentrations from 217 to 4,100 mg l⁻¹ increased phenanthrene biodegradation by about 30% after 120 h incubation. After 100 h incubation, cultures initially containing 120 or 160 mg l⁻¹ 1-dodecanol had mineralized >10% of the phenanthrene whereas those incubated without 1-dodecanol had mineralized only 4.5%. The production and accumulation of putative phenanthrene metabolites in the aqueous phase of cultures likewise increased in response to the addition of 1-dodecanol. The results suggest that enhanced adhesion of bacterial cells to the oil–water interface was the main factor responsible for enhanced biodegradation of phenanthrene to presumed polar metabolites and to CO₂.     

Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration

Air pollution is an environmental hazard that is associated with cardiovascular dysfunction. Phenanthrene is a three-ringed polyaromatic hydrocarbon that is a significant component of air pollution and crude oil and has been shown to cause cardiac dysfunction in marine fishes. We investigated the cardiotoxic effects of phenanthrene in zebrafish (Danio rerio), an animal model relevant to human cardiac electrophysiology, using whole-cell patch-clamp of ventricular cardiomyocytes. First, we show that phenanthrene significantly shortened action potential duration without altering resting membrane potential or upstroke velocity (dV/dt). L-type Ca²⁺ current was significantly decreased by phenanthrene, consistent with the decrease in action potential duration. Phenanthrene blocked the hERG orthologue (zfERG) native current, I_Kr, and accelerated I_Kr deactivation kinetics in a dose-dependent manner. Furthermore, we show that phenanthrene significantly inhibits the protective I_Kr current envelope, elicited by a paired ventricular AP-like command waveform protocol. Phenanthrene had no effect on other I_K. These findings demonstrate that exposure to phenanthrene shortens action potential duration, which may reduce refractoriness and increase susceptibility to certain arrhythmia triggers, such as premature ventricular contractions. These data also reveal a previously unrecognized mechanism of polyaromatic hydrocarbon cardiotoxicity on zfERG by accelerating deactivation and decreasing I_Kr protective current.     

Degradation of polycyclic aromatic hydrocarbons in soil by a two-step sequential treatment

The objectives of this work were to isolate the microorganisms responsible for a previously observed degradation of polycyclic aromatic hydrocarbons (PAH) in soil and to test a method for cleaning a PAH-contaminated soil. An efficient PAH degrader was isolated from an agricultural soil and designated as Mycobacterium LP1. In liquid culture, it degraded phenanthrene (58%), pyrene (24%), anthracene (21%) and benzo(a)pyrene (10%) present in mixture (initial concentration 50 μg ml⁻¹ each) and phenanthrene (92%) and pyrene (94%) as sole carbon sources after 14 days of incubation at 30°C. In soil, Mycobacterium LP1 mineralised ¹⁴C-phenanthrene (45%) and ¹⁴C-pyrene (65%) after 10 days. The good ability of this Mycobacterium was combined with the benzo(a)pyrene oxidation effect obtained by 1% w/w rapeseed oil in a sequential treatment of a PAH-spiked soil (total PAH concentration 200 mg kg⁻¹). The first step was incubation with the bacterium for 12 days and the second step was the addition of the rapeseed oil after this time and a further incubation of 22 days. Phenanthrene (99%), pyrene (95%) and anthracene (99%) were mainly degraded in the first 12 days and a total of 85% of benzo(a)pyrene was transformed during the whole process. The feasibility of the method is discussed.     

Optimization of Influencing Parameters on Phenanthrene Removal Efficiency in Soil Washing Process by Using Response Surface Methodology

Response surface methodology (RSM) under Box–Behnken design (BBD) was applied to evaluate the effect of the influencing parameters including surfactant concentration, liquid/soil ratio, Humic Acid concentration, and washing time on phenanthrene removal efficiency in soil washing process by using the nonionic surfactant Tween 80 and find an optimal operational conditions to achieve the highest removal efficiency. A polynomial quadratic model was used to correlate phenanthrene removal efficiency and four independent variables (R² = 0.9719). Based on the obtained results the most influential parameter on phenanthrene removal efficiency was surfactant concentration with an impact value of 69.519%. Liquid/soil ratio was also another factor that significantly influenced on removal efficiency with an impact value of 25.014%. The interaction between surfactant concentration and liquid/soil ratio was also shown to have a positive significant effect on removal efficiency (p_value = 0.0027). However, the other independent variables Humic Acid concentration and time were not significant in the ranges selected in this study. Based on the optimization results maximum removal efficiency of 70.692 ± 3.647% was achieved under the conditions of surfactant concentration 5000 mg L^?1, liquid/soil ratio 30 v/w, HA concentration 9.88 mg L^?1, and washing time 2 h, which was in good agreement with predicted value (66.643%).     

Phenanthrene toxicity and dissipation in rhizosphere of grassland plants (Lolium perenne L. and Trifolium pratense L.) in three spiked soils

Rhizodegradation is a technique involving plants that offers interesting potential to enhance biodegradation of persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs). Nevertheless, the behaviour of PAHs in plant rhizosphere, including micro-organisms and the physico-chemical soil properties, still needs to be clarified. The present work proposes to study the toxicity and the dissipation of phenanthrene in three artificially contaminated soils (1 g kg^-1 DW). Experiments were carried out after 2 months of soil aging. They consisted in using different systems with two plant species (Ryegrass—Lolium perenne L. var. Prana and red clover—Trifolium pratense L. var. fourragère Caillard), three kinds of soils (a silty-clay-loam soil “La Bouzule”, a coarse sandy-loam soil “Chenevières” and a fine sandy-loam soil “Maconcourt”). Phenanthrene was quantified by HPLC in the beginning (T ₀) and the end of the experiments (30 days). Plant biomass, microbial communities including mycorrhizal fungi, Rhizobium and PAH degraders were also recorded. Generally phenanthrene contamination did not affect plant biomass. Only the red clover biomass was enhanced in Chenevières and La Bouzule polluted soils. A stimulation of Rhizobium red clover colonisation was quantified in spiked soils whereas a drastic negative phenanthrene effect on the mycorrhization of ryegrass and red clover was recorded. The number of PAH degraders was stimulated by the presence of phenanthrene in all tested soils. Both in ryegrass and red clover planted soils, the highest phenanthrene dissipation due to the rhizosphere was measured in La Bouzule soils. On the contrary, in non-planted soils, La Bouzule soils had also the lowest pollutant dissipation. Thus, in rhizospheric and non-rhizospheric soils the phenanthrene dissipation was found to depend on soil clay content.     

Diversity and Activity of PAH-Degrading Bacteria in the Phyllosphere of Ornamental Plants

Phyllosphere bacteria on ornamental plants were characterized based on their diversity and activity towards the removal of polycyclic aromatic hydrocarbons (PAHs), the major air pollutants in urban area. The amounts of PAH-degrading bacteria were about 1–10% of the total heterotrophic phyllosphere populations and consisted of diverse bacterial species such as Acinetobacter, Pseudomonas, Pseudoxanthomonas, Mycobacterium, and uncultured bacteria. Bacterial community structures analyzed by polymerase chain reaction–denaturing gradient gel electrophoresis from each plant species showed distinct band patterns. The uniqueness of these phyllosphere bacterial communities was partly due to the variation in leaf morphology and chemical properties of ornamental plants. The PAH degradation activity of these bacteria was monitored in gas-tight systems containing sterilized or unsterilized leaves. The results indicated that phyllosphere bacteria on unsterilized leaves were able to enhance the activity of leaves for phenanthrene removal. When compared between plant species, phenanthrene removal efficiency corresponded to the size of phenanthrene-degrading bacteria. In addition, phyllosphere bacteria on Wrightia religiosa were able to reduce other PAHs such as acenaphthylene, acenaphthene, and fluorine in 60-ml glass vials and in a 14-l glass chamber. Thus, phyllosphere bacteria on ornamental plants may play an important role in natural attenuation of airborne PAHs in urban areas.     

两株多环芳烃降解菌协同对菲-镉污染的去除特性北大核心

【目的】从污染土壤中分离筛选一株多环芳烃降解菌,并探究其与Pseudomonas aeruginosa B6-2构建的混菌体系对菲-镉复合污染的修复效能,以及微生物代谢特性对不同镉浓度赋存的响应特性,以期为复合污染的生物修复提供优良菌株资源及应用技术参考。【方法】采用富集驯化、筛选纯化方法得到一株多环芳烃降解菌,通过生理生化特征和16S rRNA基因序列分析进行鉴定。利用高效液相色谱法和电感耦合等离子体质谱法评估不同镉浓度赋存下各反应体系对菲和镉的去除效能;通过菌体细胞形态的扫描电镜观测及菌株代谢活性检测,探讨镉胁迫对菲生物降解过程的影响机制。【结果】筛选得到一株具有重金属耐受性和多环芳烃高效降解菌SZ-3,经鉴定为节杆菌属;降解菌协同体系(M)具有良好的菲降解效能和抗镉胁迫优势。镉胁迫浓度为0.5、10 mg/L时,M对菲和镉的去除率分别高于85%、80%;镉胁迫浓度为25、50 mg/L时,2种污染物的去除率均大于65%。扫描电镜分析表明,镉胁迫导致菌体表面粗糙且出现不同程度变形,菌体间黏附性和聚集性提高。反应周期内,邻苯二酚1,2-双加氧酶活性与电子传递体系活性随镉浓度增加而降低,两者变化与菲降解速率变化一致。【结论】Arthrobacter sp.SZ-3是一株PAHs高效降解菌,能与Pseudomonas aeruginosa B6-2协同高效修复菲-镉复合污染,随着初始镉胁迫浓度增加,混菌协同对目标污染物去除的优势显著。     

Phenanthrene biodegradation by an algal-bacterial consortium in two-phase partitioning bioreactors

An algal-bacterial consortium formed by Chlorella sorokiniana and a phenanthrene-degrading Pseudomonas migulae strain was able to biodegrade 200-500 mg/l of phenanthrene dissolved in silicone oil or tetradecane under photosynthetic conditions and without any external supply of oxygen. Phenanthrene was only removed when provided in organic solvent, which confirms the potential of two-phase systems for toxicity reduction. Phenanthrene was degraded at highest rates when provided in silicone oil rather than in tetradecane since this solvent probably sequestered the PAH, reducing its mass transfer to the aqueous phase. The influence of phenanthrene concentration, amount of inoculum and light intensity on pollutant removal was also investigated and, under the best conditions, phenanthrene was degraded at 24.2 g m(-3).h(-1). In addition to being cost-effective and mitigating the release of greenhouse gases into the atmosphere, photosynthetic oxygenation was especially beneficial to the use of two-phase partitioning bioreactors since it prevented solvent emulsification and/or volatilization and evidence was found that the microalgae release biosurfactants that could further enhance phenanthrene degradation.     

Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established. Received: 25 May 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Biomarkers of toxicity in Clarias gariepinus exposed to sublethal concentrations of polycyclic aromatic hydrocarbons

Physiological, biochemical and histological indices in Clarias gariepinus broodstock, and teratogenic indices in embryos exposed to sublethal concentrations of naphthalene, phenanthrene and pyrene were investigated in 2014 using a static-renewal bioassay protocol. Phenanthrene (1.41 mg l^?1) was the most toxic, followed by pyrene (1.53 mg l^?1) and naphthalene (7.21 mg l^?1), based on 96 h LC50 values. Hepatosomatic indices were significantly higher in naphthalene- and pyrene-treated males compared with solvent controls, whereas fecundity in females was significantly lower by factors of 2.4 (naphthalene), 2.8 (phenanthrene) and 2.4 (pyrene), compared with controls. Catalase levels were lower in female phenanthrene-treated fish compared with controls. Histological alterations observed in PAH-treated fish include oedema, inflammatory cells, epithelial lifting and hyperplasia in the gills, vacuolation, haemosiderin pigments and sinusoidal congestion in the liver, and degenerated zona radiata in the ovary. Teratogenic effects were not observed, as evidenced by the lack of histological alterations in embryos spawned from pre-exposed broodstock. Sex-specific responses and the utility of biomarkers at cellular and individual levels of organisation are therefore demonstrated for holistic evaluations of polycyclic aromatic hydrocarbons in ecotoxicological studies.     

Biodegradation of Phenanthrene by the Green Alga Scenedesmus obliquus ES‐55

While the degradation of polycyclic aromatic hydrocarbons by bacteria and fungi has been broadly investigated, less is known about the metabolism of these compounds by algae. The goal of the experiments was to test the degradability of phenanthrene by the green alga Scenedesmus obliquus ES‐55 (Chlorophyceae) and to identify the metabolites. It was shown that S. obliquus ES‐55 metabolized phenanthrene. Under light conditions, phenanthrene (14 mg/L) inhibits cell division by more than twice. However, the metabolic processes in the cells affected by phenanthrene continued because the content of chlorophyll increased. In the exponential phase under phototrophic conditions the alga degraded phenanthrene. Phenanthrene was removed by algae up to 42 % in BBM medium and up to 24 % in Kuhl medium. Dihydroxy‐dihydro‐phenanthrene, a degradation metabolite in fungi, bacteria and cyanobacteria, could also be detected as a transformation product of S. obliquus ES‐55. Further detected common metabolites foster the assumption that both phototrophic and non‐photothrophic organisms metabolize phenanthrene via a similar pathway. The present study is the first evidence of the ability of an axenic culture of the green alga S. obliquus to biotransform phenanthrene into other metabolites.     

Relative role of eukaryotic and prokaryotic microorganisms in phenanthrene transformation in coastal sediments

THE RELATIVE ROLE OF EUKARYOTIC VERSUS PROKARYOTIC MICROORGANISMS IN PHENANTHRENE TRANSFORMATION WAS MEASURED IN SLURRIES OF COASTAL SEDIMENT BY TWO DIFFERENT APPROACHES: detection of marker metabolites and use of selective inhibitors on phenanthrene biotransformation. Phenanthrene biotransformation was measured by polar metabolite formation and CO(2) evolution from [9-C]phenanthrene. Radiolabeled metabolites were tentatively identified by high-performance liquid chromatography (HPLC) separation combined with UV/visible spectral analysis of HPLC peaks and comparison to authentic standards. Both yeasts and bacteria transformed phenanthrene in slurries of coastal sediment. Two products of phenanthrene oxidation by fungi, phenanthrene trans-3,4-dihydrodiol and 3-phenanthrol, were produced in yeast-inoculated sterile sediment. However, only products of phenanthrene oxidation typical of bacterial transformation, 1-hydroxy-2-naphthoic acid and phenanthrene cis-3,4-dihydrodiol, were isolated from slurries of coastal sediment with natural microbial populations. Phenanthrene trans-dihydrodiols or other products of fungal oxidation of phenanthrene were not detected in the slurry containing a natural microbial population. A predominant role for bacterial transformation of phenanthrene was also suggested from selective inhibitor experiments. Addition of streptomycin to slurries, at a concentration which suppressed bacterial viable counts and rates of [methyl-H]thymidine uptake, completely inhibited phenanthrene transformation. Treatment with colchicine, at a concentration which suppressed yeast viable counts, depressed phenanthrene transformation by 40%, and this was likely due to nontarget inhibition of bacterial activity. The relative contribution of eukaryotic microorganisms to phenanthrene transformation in inoculated sterile sediment was estimated to be less than 3% of the total activity. We conclude that the predominant degraders of phenanthrene in muddy coastal sediments are bacteria and not eukaryotic microorganisms.     

Effect of alginate encapsulation and selected disinfectants on survival of and phenanthrene mineralization byPseudomonas sp UG14Lr in creosote-contaminated soil

The survival and phenanthrene-mineralizing ability of free and alginate-encapsulatedPseudomonas sp UG14Lr cells were examined in a creosote-contaminated soil. Alginate encapsulation adversely affected both survival and phenanthrene mineralization. This was postulated to be due to concentration of water-soluble toxic compounds in the alginate beads. Toxicity studies showed that the concentrated water-soluble fraction of the creosote-contaminated soil may be toxic toPseudomonas sp UG14Lr in soil with a low moisture content. Survival of alginate-encapsulated cells improved with increasing soil moisture content. Free cells survived well at a steady population of 10⁸ CFU g^–1 dry soil for 28 days in the creosote-contaminated soil. However, phenanthrene mineralization was not improved compared to the uninoculated control. This was attributed to the existence of indigenous phenanthrene-mineralizing microorganisms already present in this contaminated soil. The effect of calcium hypochlorite and Germiphene on survival of and phenanthrene mineralization by free and alginate-encapsulatedPseudomonas sp UG14Lr cells in creosote-contaminated soil was also studied. Addition of 0.1% (w/w dry soil) calcium hypochlorite reduced the introduced free cells to below detection limits (10 CFU g^–1 dry soil) within 14 days, while Germiphene had no effect on cell numbers. Phenanthrene mineralization by free cells was not adversely affected by treatment with calcium hypochlorite or Germiphene. Survival of alginate-encapsulated cells after treatment with disinfectants was as poor as that without disinfection. The results show that alginate encapsulation may not be a suitable formulation for introduction ofPseudomonas sp UG14Lr into creosote-contaminated soils.     

Initial oxidative and subsequent conjugative metabolites produced during the metabolism of phenanthrene by fungi

Three filamentous fungi were examined for the ability to biotransform phenanthrene to oxidative (phase I) and conjugative (phase II) metabolites. Phenanthrene metabolites were purified by high-performance liquid chromatography (HPLC) and identified by UV/visible absorption, mass, and¹H NMR spectra.Aspergillus niger ATCC 6275,Syncephalastrum racemosum UT-70, andCunninghamella elegans ATCC 9245 initially transformed [9-¹⁴C]phenanthrene to produce metabolites at the 9,10-, 1,2-, and 3,4- positions. Subsequently, sulfate conjugates of phase I metabolites were formed byA. niger, S. racemosum, andC. elegans. Minor glucuronide conjugates of 9-phenanthrol and phenanthrenetrans-9,10-dihydrodiol were formed byS. racemosum andA. niger, respectively. In addition,C. elegans produced the glucose conjugates 1-phenanthryl -d-glucopyranoside and 2-hydroxy-1-phenanthryl -d-glucopyranoside, a novel metabolite. [9-¹⁴C]Phenanthrene metabolites were not detected in organic extracts from biotransformation experiments with the yeasts,Candida lipolytica 37-1,Candida tropicalis ATCC 32113, andCandida maltosa R-42.     

Effect of nitrogen and phosphorus addition on phenanthrene biodegradation in four soils

Phenanthrene mineralization rates were found to vary widely among four soils; differences in soil nutrient levels was one hypothesis to explain this variation. To test this hypothesis, phenanthrene mineralization rates were measured in these soils with, and without, added nitrogen and phosphorus. Mineralization rates either remained unchanged or were depressed by the addition of nitrogen and phosphorus. Phenanthrene degradation rates remained unchanged in the soil which had the highest indigenous levels of nitrogen and phosphorus and which showed the largest increase in phosphorus levels after nutrients were added. The soils in which degradation rates were depressed had lower initial phosphorus concentrations and showed much smaller or no measurable increase in phosphorus levels after nutrients were added to the soils. To understand the response of phenanthrene degradation rates to added nitrogen and phosphorus, it may be necessary to consider the bioavailability of added nutrients and nutrient induced changes in microbial metabolism and ecology.     

Combined effects of pH and biosurfactant addition on solubilization and biodegradation of phenanthrene

Phenanthrene solubilization and biodegradation with a biosurfactant (rhamnolipid) solution were investigated as a function of pH. Batch phenanthrene solubilization experiments were performed in the pH range 4–8 and the highest solubilities with the biosurfactant were detected around a pH of 4.5–5.5. The apparent solubility at pH 5.5 was 3.8 times greater than at pH 7 in the presence of 240 ppm rhamnolipid, probably due to the rhamnolipid—an anionic surfactant—forming different pH-dependent structures. Biodegradation experiments using Pseudomonas putida CRE 7 were performed in the absence and the presence of the rhamnolipid solution. Without the biosurfactant, the specific growth rate () at pH 6 was higher than at other pH values, and analysis for the total phenanthrene loss confirmed the trends in , with the greatest phenanthrene removal at pH 6. In presence of the rhamnolipid, the maximum value shifted to around pH 5, which showed maximum enhancement of solubility in the abiotic experiment. Although there was an increase in the observed specific growth rate with the biosurfactant, this increase was not as great as the increase in solubilization. For example, the 1.44 times increase in the value at pH 5 was lower than the 3.8 times enhancement in the solubility at the same pH. Thus, as observed by others, not all of the solubilized phenanthrene was bioavailable to the microorganisms. Interestingly, the results of a size distribution experiment showed that a large portion of the phenanthrene-rhamnolipid aggregates existed at a molecular weight of >300,000. Furthermore, this fraction appeared to be the most available for biodegradation, although not all the phenanthrene was bioavailable.