Biodegradation of phenanthrene in soil-slurry systems with different mass transfer regimes and soil contents

The effect of soil contents and mass transfer rates on soil bioremediation was investigated. Phenanthrene, a 3-ring polycyclic aromatic hydrocarbon (PAH), was chosen as a model target compound. The biodegradation tests were performed in soil-slurry systems at two distinct mass transfer rates: fast in flasks tests at 150 rpm and slow in roller-bottle tests at 2 rpm. The rate of phenanthrene biodegradation was similar at low soil content (2 wt.%) in both slurry systems, but the rates at high soil contents (6 and 18 wt.%) were higher in the roller-bottle tests. The maximum utilization rate constant for sorbed-phase biodegradation obtained from curve fitting using a mathematical model was decreased in the flask tests with increasing soil content, while not decreased in the roller-bottle tests.     

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.     

Enhanced biodegradation of phenanthrene by a marine bacterium in presence of a synthetic surfactant

The biodegradation of phenanthrene by the marine strain Sphingomonas sp. 2MPII (DSMZ 11572) was enhanced by the solubilizating properties of the nonionic surfactant Tween 80. After 197 h of incubation, 85 +/- 4% of the initial amount of phenanthrene (0.4 g l-1) was biodegraded in presence of Tween 80 (0.5 g l-1) as opposed to 52 +/- 5% without this synthetic surfactant. These results confirm that the activity of the strain 2MPII is limited by the bioavailability of the polycyclic aromatic hydrocarbon (PAH) substrate in the aqueous phase. Tween 80 appears to be efficient in increasing the bioavailability of hydrophobic compounds such as PAHs.     

Production of metabolites in the biodegradation of phenanthrene, fluoranthene and pyrene by the mixed culture of Mycobacterium sp. and Sphingomonas sp

The effects of the mixed culture of Mycobacterium sp. strain A1-PYR and Sphingomonas sp. strain PheB4 on the degradation characteristics of single polycyclic aromatic hydrocarbon were investigated. In the mixed bacterial culture, phenanthrene, fluoranthene and pyrene were degraded by 100% at Day 3, 71.2% and 50% at Day 7, respectively. Compared to their respective pure cultures, the degradation of phenanthrene and fluoranthene decreased, but that of pyrene increased significantly. Based on GC-MS analysis, eight and six new metabolites were produced from the biodegradation of phenanthrene and fluoranthene, respectively, while only two new metabolites were formed from pyrene. To our knowledge, this is the first report that the mixed bacterial culture could increase the diversity of metabolites from PAH, but the diverse metabolite pattern was not necessarily beneficial to the degradation of the recalcitrant PAH. The enhancement on pyrene degradation was possibly attributed to the rapid growth of strain PheB4.     

Quantifying the biodegradation of phenanthrene by Pseudomonas stutzeri P16 in the presence of a nonionic surfactant.

The low water solubility of polycyclic aromatic hydrocarbons is believed to limit their availability to microorganisms, which is a potential problem for bioremediation of polycyclic aromatic hydrocarbon-contaminated sites. Surfactants have been suggested to enhance the bioavailability of hydrophobic compounds, but both negative and positive effects of surfactants on biodegradation have been reported in the literature. Earlier, we presented mechanistic models of the effects of surfactants on phenanthrene dissolution and on the biodegradation kinetics of phenanthrene solubilized in surfactant micelles. In this study, we combined the biodegradation and dissolution models to quantify the influence of the surfactant Tergitol NP-10 on biodegradation of solid-phase phenanthrene by Pseudomonas stutzeri P16. Although micellized phenanthrene does not appear to be available directly to the bacterium, the ability of the surfactant to increase the phenanthrene dissolution rate resulted in an overall increase in bacterial growth rate in the presence of the surfactant. Experimental observations could be predicted well by the derived model with measured biokinetic and dissolution parameters. The proposed model therefore can serve as a base case for understanding the physical-chemical effects of surfactants on nonaqueous hydrocarbon bioavailability.     

Synergistic Effect of Yeast-Bacterial Co-Culture on Bioremediation of Oil-Contaminated Soil

The effects of various treatments, including biostimulation, bioaugmentation with bacterial consortium, yeast, and yeast-bacterial co-culture, on oil biodegradation were systematically compared. Synergistic effects were observed on the removal of total petroleum hydrocarbon and polycyclic aromatic hydrocarbons via the amendment of co-culture, with a 48-day degradation of 56% for total petroleum hydrocarbon and 32% for polycyclic aromatic hydrocarbons, respectively. Yeast played an important role in the removal of polycyclic aromatic hydrocarbons with 4–6 rings. The synergistic effect of yeast-bacteria was further evidenced by the increase of biomass and enzyme activities in soil. In comparison with the bacterial community, the yeast community was more sensitive to the inoculated cultures, which was indexed by the changes of diversity, abundance, and evenness in polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis.     

Microbial characterization and hydrocarbon biodegradation potential of natural bilge waste microflora

Shipping operations produce oily wastes that must be managed properly to avoid environmental pollution. The aim of this study was to characterize microorganisms occurring in ship bilge wastes placed in open lagoons and, particularly, to assess their potential to degrade polycyclic aromatic hydrocarbons (PAHs). A first-order kinetic was suitable for describing hydrocarbon biodegradation after 17 days of treatment. The calculated rate constants were 0.0668 and 0.0513 day^–1 with a corresponding half-life of 10.3 and 13.5 days for the aliphatic and aromatic hydrocarbon fractions, respectively. At day 17, PAH removal percentages were: acenaphtylene 100, fluorene 95.2, phenanthrene 93.6, anthracene 70.3, and pyrene 71.5. Methyl phenanthrene removals were lower than that of their parent compound (3-methyl phenanthrene 83.6, 2-methyl phenanthrene 80.8, 1-methyl phenanthrene 77.3, 9-methyl phenanthrene 75.1, and 2,7-dimethyl phenanthrene 76.6). Neither pure cultures nor the microbial community from these wastes showed extracellular biosurfactant production suggesting that the addition of an exogenously produced biosurfactant may be important in enhancing hydrocarbon bioavailability and biodegradation. DNA analysis of bilge waste samples revealed a ubiquitous distribution of the nahAc genotype in the dump pools. Although almost all of the isolates grew on naphthalene as sole carbon source, only some of them yielded nahAc amplification under the experimental conditions used. The variety of PAHs in bilge wastes could support bacteria with multiple degradation pathways and a diversity of catabolic genes divergent from the classical nah-like type.     

Cross-induction of pyrene and phenanthrene in a Mycobacterium sp. isolated from polycyclic aromatic hydrocarbon contaminated river sediments.

A polycyclic aromatic hydrocarbon (PAH)-degrading culture enriched from contaminated river sediments and a Mycobacterium sp. isolated from the enrichment were tested to investigate the possible synergistic and antagonistic interactions affecting the degradation of pyrene in the presence of low molecular weight PAHs. The Mycobacterium sp. was able to mineralize 63% of the added pyrene when it was present as a sole source of carbon and energy. When the enrichment culture and the isolated bacterium were exposed to phenanthrene, de novo protein synthesis was not required for the rapid mineralization of pyrene, which reached 52% in chloramphenicol-treated cultures and 44% in the absence of the protein inhibitor. In the presence of chloramphenicol, < 1% of the added pyrene was mineralized by the mixed culture after exposure to anthracene and naphthalene. These compounds did not inhibit pyrene utilization when present at the same time as pyrene. Concurrent mineralization of pyrene and phenanthrene after exposure to either compound was observed. Cross-acclimation between ring classes of PAHs may be a potentially important interaction influencing the biodegradation of aromatic compounds in contaminated environments.     

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ^? WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.     

乳白耙齿菌F17对单一和复合多环芳烃的降解差异解析

[目的] 针对菲、蒽、荧蒽多环芳烃（PAHs）污染物,利用乳白耙齿菌F17,研究单一和复合PAHs污染物的生物降解规律。[方法] 采用气相色谱-质谱法（GC-MS）分析降解过程中PAHs的浓度,并采用准一级反应动力学模型对降解结果进行拟合。[结果] 对于单一PAHs,第15天时菲、蒽、荧蒽的降解率由高到低依次为菲（97.8%） > 蒽（89.3%） > 荧蒽（81.5%）。菲、蒽和荧蒽的降解过程具有准一级反应动力学特征,菲的生物降解速率最快,其次是蒽,荧蒽的降解速率最慢。与单一PAHs的降解相比,在复合PAHs的降解过程中,乳白耙齿菌F17的生长和锰过氧化物酶的合成均表现出不同的特征。此外,水溶性极可能是复合污染物降解的重要控制因子,三者水溶性为：菲 > 荧蒽 > 蒽。因此,在菲或荧蒽加入条件下,微生物能优先降解这些污染物,抑制了污染物蒽的降解;同时,蒽或菲的存在对荧蒽的降解也有抑制作用;然而外源加入水溶性较差的蒽和荧蒽,则对菲的生物降解无显著影响。[结论] 复合PAHs的生物降解主要表现为相互竞争的特点,通过GC-MS分析了PAHs的生物降解途径。     

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways. The first, major pathway is through terminal aromatic ring cleavage (presumably at the C3–C4 bond) producing benzocoumarin and 1-hydroxy-2-naphthoic acid, whose further degradation with the formation of salicylic acid is difficult or is very slow. The second pathway is through the oxidation of the central aromatic ring at the C9–C10 bond, producing 9,10-dihydro-9,10-dihydroxyphenanthrene, 9,10-phenanthrenequinone, and 2,2′-diphenic acid. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites, has been reported for a bacterium of the Ensifer genus.     

Catabolic Gene Probe Analysis of an Aquifer Microbial Community Degrading Creosote-Related Polycyclic Aromatic and Heterocyclic Compounds

Abstract The biodegradation of a mixture of several creosote-related compounds, p-cresol, phenanthrene, fluorene, and carbazole was examined in columns containing aquifer sands. The aquifer material, itself, had an effect on the migration of the test compounds, with p-cresol being retarded the least, followed by carbazole, then fluorene, and finally phenanthrene. The biodegradation of all the compounds was greatly enhanced by the inclusion of p-cresol (10 ppm) in the substrate mixture. Associated with this enhanced degradation was a 100-fold increase in the total culturable bacterial population, and increases in the xylE- and ndoB-positive bacterial populations of more than three orders of magnitude. The products of these two genes are involved in the degradation of monocyclic and polycyclic aromatic compounds, respectively. In columns that did not receive p-cresol, there was no significant change in either the total culturable bacterial population density or the xylE-positive bacterial population, but there were significant increases of one to two orders of magnitude in the ndoB-positive bacterial populations. The results suggest that the ndoB gene probe can detect bacteria capable of utilizing phenanthrene, carbazole, and possibly fluorene. Received: 26 January 1996; Accepted: 20 June 1996     

Biodegradation of phenanthrene by the indigenous microbial biomass in a zinc amended soil

AIMS: To study the effect of zinc on the biodegradation of phenanthrene by the microbial biomass in soil. METHODS AND RESULTS: Uncontaminated soil was amended with zinc and phenanthrene as single or co-contaminants, and microbial metabolic activity was measured using an intracellular dehydrogenase enzyme bioassay over 37 days. Contaminants were amended at optimum, action and double the action level specified in 'The New Dutch List' (Ministry of Housing, Spatial Planning and Environment, the Netherlands, 2000). Microbial activity in soils with zinc or phenanthrene alone indicated the presence of tolerant, albeit inhibited soil micro-organisms. A zinc concentration at the optimum level of 140 mg kg(-1) in the co-contaminated soil (phenanthrene at 40 mg kg(-1)) resulted in marginal stimulation of the rate of phenanthrene biodegradation. However, Zn2+ concentrations at the action and double the action level of zinc (720 and 1440 mg kg(-1)) inhibited phenanthrene degradation. CONCLUSIONS: Biodegradation of phenanthrene in soils co-contaminated with zinc at concentrations above the action value is impeded. SIGNIFICANCE AND IMPACT OF THE STUDY: Bioremediation efforts to remove polycyclic aromatic hydrocarbon in zinc co-contaminated soils are likely to be constrained.     

Two-Stage Mineralization of Phenanthrene by Estuarine Enrichment Cultures

The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring ¹⁴CO₂ production from low concentrations (0.5 mg liter⁻¹) of radiolabeled phenanthrene. Here, mineralization of ¹⁴C-labeled 1H2NA could explain the incremental ¹⁴CO₂ produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.     

Kinetics and key enzyme activities of phenanthrene degradation by Pseudomonas mendocina

Phenanthrene degradation by Pseudomonas mendocina CGMCC 1.766, a new phenanthrene-degrading strain, was investigated in this work. When cells were grown on 20, 50, 100 and 200 mg l⁻¹ of phenanthrene, the doubling time was 18.3, 19.8, 21.0 and 20.3 h and the growth yield during exponential phase was 242, 271, 221 and 206 mg protein (g phenanthrene)⁻¹, respectively. High level accumulation of the intermediate metabolite 1-hydroxy-2-naphthoic acid (1H2N) up to ≈94% of its theoretical yield was observed. Dynamic profiles of the activities of two key enzymes, i.e. polycyclic aromatic hydrocarbon (PAH) dioxygenase (PDO) and catechol-2,3-oxygenase (C23O), during the biodegradation were revealed and the results suggest a delicate mechanism in the regulation of these phenanthrene-degrading enzymes in this strain.     

Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants.

The biodegradation of polycyclic aromatic hydrocarbons (PAH) often is limited by low water solubility and dissolution rate. Nonionic surfactants and sodium dodecyl sulfate increased the concentration of PAH in the water phase because of solubilization. The degradation of PAH was inhibited by sodium dodecyl sulfate because this surfactant was preferred as a growth substrate. Growth of mixed cultures with phenanthrene and fluoranthene solubilized by a nonionic surfactant prior to inoculation was exponential, indicating a high bioavailability of the solubilized hydrocarbons. Nonionic surfactants of the alkylethoxylate type and the alkylphenolethoxylate type with an average ethoxylate chain length of 9 to 12 monomers were toxic to a PAH-degrading Mycobacterium sp. and to several PAH-degrading mixed cultures. Toxicity of the surfactants decreased with increasing hydrophilicity, i.e., with increasing ethoxylate chain length. Nontoxic surfactants enhanced the degradation of fluorene, phenanthrene, anthracene, fluoranthene, and pyrene.     

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.     

Degradation of phenanthrene by Phanerochaete chrysosporium occurs under ligninolytic as well as nonligninolytic conditions.

In order to delineate the roles of lignin and manganese peroxidases in the degradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium, the biodegradation of phenanthrene (chosen as a model for polycyclic aromatic hydrocarbons) was investigated. The disappearance of phenanthrene from the extracellular medium and mycelia was determined by using gas chromatography. The disappearance of phenanthrene from cultures of wild-type strains BKM-F1767 (ATCC 24725) and ME446 (ATCC 34541) under ligninolytic (low-nitrogen) as well as nonligninolytic (high-nitrogen) conditions was observed. The study was extended to two homokaryotic (basidiospore-derived) isolates of strain ME446. Both homokaryotic isolates, ME446-B19 (which produces lignin and manganese peroxidases only in low-nitrogen medium) and ME446-B5 (which totally lacks lignin and manganese peroxidase activities), caused the disappearance of phenanthrene when grown in low- as well as high-nitrogen media. Moreover, lignin and manganese peroxidase activities were not detected in any of the cultures incubated in the presence of phenanthrene. Additionally, the mineralization of phenanthrene was observed even under nonligninolytic conditions. The results collectively indicate that lignin and manganese peroxidases are not essential for the degradation of phenanthrene by P. chrysosporium. The observation that phenanthrene degradation occurs under nonligninolytic conditions suggests that the potential of P. chrysosporium for degradation of certain environmental pollutants is not limited to nutrient starvation conditions.     

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.