Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.     

Characterization of a Polycyclic Aromatic Hydrocarbon-Degrading Microbial Consortium from a Petrochemical Sludge Landfarming Site

Anthracene, phenanthrene, and pyrene are polycyclic aromatic hydrocarbon (PAHs) that display both mutagenic and carcinogenic properties. They are recalcitrant to microbial degradation in soil and water due to their complex molecular structure and low solubility in water. This study presents the characterization of an efficient PAH (anthracene, phenanthrene, and pyrene)-degrading microbial consortium, isolated from a petrochemical sludge landfarming site. Soil samples collected at the landfarming area were used as inoculum in Warburg flasks containing soil spiked with 250 mg kg^-1 of anthracene. The soil sample with the highest production of CO₂-C in 176 days was used in liquid mineral medium for further enrichment of anthracene degraders. The microbial consortium degraded 48%, 67%, and 22% of the anthracene, phenanthrene, and pyrene in the mineral medium, respectively, after 30 days of incubation. Six bacteria, identified by 16S rRNA sequencing as Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, two Microbacteriaceae bacteria, and a fungus identified as Fusarium oxysporum were isolated from the enrichment culture. The consortium and its monoculture isolates utilized a variety of hydrocarbons including PAHs (pyrene, anthracene, phenanthrene, and naftalene), monoaromatics hydrocarbons (benzene, ethylbenzene, toluene, and xylene), aliphatic hydrocarbons (1-decene, 1-octene, and hexane), hydrocarbon mixtures (gasoline and diesel oil), intermediary metabolites of PAHs degradation (catechol, gentisic acid, salicylic acid, and dihydroxybenzoic acid) and ethanol for growth. Biosurfactant production by the isolates was assessed by an emulsification index and reduction of the surface tension in the mineral medium. Significant emulsification was observed with the isolates, indicating production of high-molecular-weigh surfactants. The high PAH degradation rates, the wide spectrum of hydrocarbons utilization, and emulsification capacities of the microbial consortium and its member microbes indicate that they can be used for biotreatment and bioaugumentation of soils contaminated with PAHs.     

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。     

表面活性剂TW-80对土壤中多环芳烃生物降解的影响

以表面活性剂TW80为供试物,进行了为期150d的实验研究,并分别在30、60和150d间隔采样监测PAHs降解率。结果表明,30d后,土壤中PAHs的降解率达90%,比对照提高约30%.60d后,浓度为10000mg·kg^-1表面活性剂的土壤和对照中,PAHs降解率从65.1%和60%迅速提高到93.8%和79.2%.其它处理中,PAHs的平均降解率仅比30d的结果提高4%.150d后,所有处理中PAHs的降解率均达到90%以上。可以认为,表面活性剂能提高PAHs的生物可利用性,加快PAHs的降解速率,从而减少污染暴露时间。但表面活性剂浓度过高可抑制微生物活性。研究还发现,TW80土壤中含有优势真菌。经鉴定为常见青霉、蠕形青霉、淡紫青霉和顶孢头孢霉。它们是土壤PAHs迅速降解的动因.     

Anthracene biodegradation under nitrate-reducing condition and associated microbial community changes

Anaerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) and degraders in the subsurface environment have aroused increasing attention. Molecular techniques are especially useful when isolates are hard to obtain. Nitrate-reducing microcosms inoculated with aquifer sediment were constructed to investigate anthracene biodegradation. The associated microbial community changes were characterized using terminal restriction fragment length polymorphism analysis (TRFLP) in combination with 16S rRNA gene clone library analysis. A nearly complete removal of anthracene was achieved after an eighty day incubation under the nitrate-reducing condition. The two molecular techniques revealed a significant shift of microbial community structure, coupled with anthracene biodegradation. Species of genera Paracoccus, Herbaspirillum, Azotobacter, and Rhodococcus were grouped into four major operational taxonomic units (OTUs) in the library that was constructed with the microcosm sample on day 80. The enrichment of these genera might have links to anthracene biodegradation under the nitrate-reducing condition. Microbial consortia likely played a part in anthracene degradation.     

Effects of carbon substrate enrichment and DOC concentration on biodegradation of PAHs in soil

AIMS: Two common reasons to explain slow environmental biodegradation of polycyclic aromatic hydrocarbons (PAHs), namely lack of appropriate carbon sources for microbial growth and limited bioavailability of PAHs, were tested in a laboratory bioassay using a creosote-contaminated soil. METHODS AND RESULTS: The soil, containing a total of 8 mg g-1 of 16 PAHs, was sieved and incubated in bottles for 45 days. The first explanation was tested by enrichment with the analogue anthracene and the non-analogue myristic acid, and both failed to stimulate degradation of all PAHs except anthracene. The second explanation was tested by addition of different concentrations of dissolved organic carbon (DOC), with effects depending on the DOC concentration and the molecular size of the PAH. The degradation was enhanced from 10 to 35% for 12 PAHs when the soil was saturated. The degraded amounts of individual PAHs were proportional to their concentration in the soil. CONCLUSIONS: The slow in situ degradation of PAHs was enhanced by more than three times by adding water as a solvent. Addition of DOC facilitated the degradation of four- to six-ring PAHs. SIGNIFICANCE AND IMPACT OF STUDY: Bioremediation of PAH-contaminated sites may be facilitated by creating water-saturated conditions but retarded by addition of other carbon substrates, such as analogue compounds.     

Enhanced Biodegradation of Casablanca Crude Oil by A Microbial Consortium in Presence of a Rhamnolipid Produced by Pseudomonas Aeruginosa AT10

The biodegradation of oil products in the environment is often limited by their low water solubility and dissolution rate. Rhamnolipids produced by Pseudomonas aeruginosa AT10 were investigated for their potential to enhance bioavailability and hence the biodegradation of crude oil by a microbial consortium in liquid medium. The characterization of the rhamnolipids produced by strain AT10 showed the effectiveness of emulsification of complex mixtures. The addition of rhamnolipids accelerates the biodegradation of total petroleum hydrocarbons from 32% to 61% at 10 days of incubation. Nevertheless, the enhancement of biosurfactant addition was more noticeable in the case of the group of isoprenoids from the aliphatic fraction and the alkylated polycyclic aromatic hydrocarbons (PHAS) from the aromatic fraction. The biodegradation of some targeted isoprenoids increased from 16% to 70% and for some alkylated PAHs from 9% to 44%.     

Screening of bacteria, isolated from PAH-contaminated soils, for production of biosurfactants and bioemulsifiers

Fifty-seven bacterial strains were isolated from PAH-contaminated soils using PAH-amended minimal medium. The isolates were screened for their production of biosurfactants and bioemulsifiers when grown in liquid media containing selected PAHs. The results suggest that many, but not all, of the isolates are able to produce biosurfactants or bioemulsifiers under the experimental conditions. The majority of the strains isolated on phenanthrene, pyrene, and fluoranthene were better emulsifiers than surface tension reducers and the stability of the formed emulsions was in general high. The strains isolated on anthracene were in general better in lowering the surface tension than in forming emulsions. In all strains, reduction of surface tension and emulsion formation did not correlate. However, in the majority of strains the two factors were associated with the bacterial cell surfaces, rather than the culture supernatants. Nevertheless, supernatants from selected surfactant-producing anthracene isolates increased the aqueous solubility of anthracene. Although a significant potential for surfactant and emulsifier production in the microbiota of the PAH-contaminated soils was found in this study, the ability of individual strains to mineralize PAHs did not coincide with production of surface-active compounds.     

微生物降解多环芳烃(PAHs)的研究进展

从多环芳烃(PAHs)的降解菌株的筛选、降解机制以及PAHs污染的生物修复等方面介绍了微生物降解PAHs的最新研究进展。     

糖脂类生物表面活性剂的特性及其对白腐真菌降解蒽的强化作用

【目的】对铜绿假单胞菌(Pseudomonas aeruginosa)所产生物表面活性剂的稳定性进行分析,考察该生物表面活性剂对乳白耙齿菌F17(Irpex lacteus F17)降解蒽的强化作用。【方法】采用三氯甲烷萃取的方法从铜绿假单胞菌的发酵液中提取生物表面活性剂,采用表/界面张力仪测定该生物表面活性剂在不同条件下的表面张力值,对其进行稳定性研究。在乳白耙齿菌F17降解蒽的过程中加入适量的生物表面活性剂,测定蒽的降解率,探讨其对蒽生物降解的强化作用。【结果】铜绿假单胞菌所产生物表面活性剂的临界胶束浓度为40 mg/L,在15-150°C及pH 6.0-13.0范围内表现出优良的稳定性,对盐浓度的耐受性也很高。在蒽的生物降解过程中,生物表面活性剂能极大地促进蒽的降解,在生物表面活性剂浓度为50 mg/L时,第15天蒽的降解率达到了82.9%。生物表面活性剂在接种乳白耙齿菌F17前1天加入培养基中,能更好地促进蒽的降解。与化学表面活性剂相比,生物表面活性剂对蒽降解的强化作用更显著。【结论】该生物表面活性剂性能优良、稳定性好,能够显著强化乳白耙齿菌F17对蒽的降解,具有良好的应用前景。     

Integrated Approach for Polycyclic Aromatic Hydrocarbon Solubilization from the Soil Matrix to Enhance Bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are known to be toxic to living organisms and have been identified as carcinogenic. In this study, a pathway of surfactant flushing, chemical oxidation, and biological treatment is proposed to remediate the soils polluted with the hydrophobic PAHs. Different surfactants such as Tween 80, Brij 35, sodium dodecyl sulfate (SDS), and polyethylene glycol (PEG) 6000 were tested in order to increase the PAH solubilization from the soil matrix. The maximum desorption efficiency of naphthalene and anthracene were found to be 56.5% and 59%, respectively, when Brij and SDS were used. The soluble PAH in the aqueous phase was amended with sodium thiosulfate (3%) to oxidize the PAH into a more bioavailable form. The chemical oxidation with subsequent biodegradation by Pseudomonas aeruginosa exhibited the relatively high PAH degradation rate (1.24 times higher) when compared with chemical oxidation alone. These results display the efficiency of chemical pretreatment of PAH-contaminated soil for improved bioremediation.     

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.     

表面活性剂影响微生物降解多环芳烃的研究进展

多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)的强疏水性是阻止其在土壤和水环境中微生物降解的主要因素.表面活性剂由于能够提高PAHs的表观溶解度而在PAHs的微生物降解中得到了广泛研究.截至目前,有关化学或生物表面活性剂促进PAHs的微生物降解已有大量报道,然而也有学者发现了表面...     

Chemical characterization and physical and biological activities of rhamnolipids produced by Pseudomonas aeruginosa BN10

Pseudomonas aeruginosa BN10 isolated from hydrocarbon-polluted soil was found to produce rhamnolipids when cultivated on 2% glycerol, glucose, n-hexadecane, and n-alkanes. The rhamnolipids were partially purified on silica gel columns and their chemical structures elucidated by combination of one- and two-dimensional 1H and 13C NMR techniques and ESI-MS analysis. Eight structural rhamnolipid homologues were identified: Rha-C10-C8, Rha-C10-C10, Rha-C10-C12:1, Rha-C10-C12, Rha2-C10-C8, Rha2-C10-C10, Rha2-C10-C12:1, and Rha2-C10-C12. The chemical composition of the rhamnolipid mixtures produced on different carbon sources did not vary with the type of carbon source used. The rhamnolipid mixture produced by Pseudomonas aeruginosa BN10 on glycerol reduced the surface tension of pure water from 72 to 29 mN m(-1) at a critical micellar concentration of 40 mg 1(-1), and the interfacial tension was 0.9 mN m(-1). The new surfactant product formed stable emulsions with hydrocarbons and showed high antimicrobial activity against Gram-positive bacteria. The present study shows that the new strain Pseudomonas aeruginosa BN10 demonstrates enhanced production of the di-rhamnolipid Rha2-C10-C10 on all carbon sources used. Due to its excellent surface and good antimicrobial activities the rhamnolipid homologue mixture from Pseudomonas aeruginosa BN10 can be exploited for use in bioremediation, petroleum and pharmaceutical industries.     

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.     

Biodegradation of selected UV-irradiated and non-irradiated polycyclic aromatic hydrocarbons (PAHs)

Biodegradation of UV-irradiated anthracene, pyrene,benz[a]anthracene,and dibenz[a,h]anthracene was comparedto that of the non-irradiated samples, individuallyand in synthetic mixtures with enrichment cultures.Combined treatment was repeated for individual anthraceneand for the PAH mixture with Sphingomonas sp.strain EPA 505 and Sphingomonas yanoikuyae.Enrichment culture studies were performed on the PAHmixtures in the presence of the main photoproduct ofanthracene, pure 9,10-anthracenedione. Photochemicallypretreated creosote solutions were also subjected tobiodegradation and the results were compared tothose of the non-irradiated solutions. The primaryinterest was on 16 polycyclic aromatic hydrocarbons(PAHs) listed as priority pollutants by European Union(EU) and the United States Environmental ProtectionAgency (USEPA). Irradiation accelerated thebiodegradation onset for anthracene, pyrene, andbenz[a]anthracene when they were treatedindividually. The biodegradation of irradiatedpyrene started with no lag phase andwas complete by 122 h whereas biodegradation of thenon-irradiated sample had a lag of 280 h andresulted in complete degradation by 720 h. Biodegradation ofPAHs was accelerated in synthetic mixtures, especiallyin the presence of pure 9,10-anthracenedione.In general, irradiation had no effect on the biodegradation of PAHsincubated in synthetic mixtures or with pure cultures. Undercurrent experimental conditions, the UV-irradiation invariablyreduced the biodegradation of PAHs in creosote. Based onthe results of the present and previous photochemical-biologicalstudies of PAHs, the influence of the photochemical pretreatmenton the biodegradation is highly dependent on the compoundsbeing treated and other process parameters.     

A strain of Pseudomonas aeruginosa growing on petroleum hydrocarbons

The strain Pseudomonas aeruginosa BS313 was used to isolate mutants that are capable to utilize octane as the sole carbon source. By means of conjugation plasmids of camphor (CAM) and naphthalene (pBS2) biodegradation were inserted into one of the mutant strains P. aeruginosa BS316. The resultant strain P. aeruginosa BS315 shows the capacity to degrade aliphatic, aromatic and cyclic oil hydrocarbons.     

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.     

Polycyclic aromatic hydrocarbon biodegradation in extracellular fluids and static batch cultures of selected sub-tropical white rot fungi

Four sub-tropical white rot fungi, Trametes versicolor, Trametes pocas, Trametes cingulata and isolate DSPM95 were studied alongside the well studied white rot fungus, Phanerochaete chrysosporium, for their ability to remove polycyclic aromatic hydrocarbons (PAHs) from culture media. Both static shallow cultures and extracellular fluids were studied using media contaminated with a defined mixture of the PAHs; fluorene, phenanthrene, anthracene, pyrene and benzo(a)anthracene. With all isolates, the total loss of the parent compound in 31 days was high for fluorene, at +60%, phenanthrene at +40% and anthracene at +42%. Biotransformation of pyrene and benzo(a)anthracene by all the isolates was low, with the highest reduction of pyrene of 15.2% and benzo(a)anthracene of 15.8% being achieved with P. chrysosporium. Disappearance of the more condensed PAHs, pyrene and benzo(a)anthracene, increased in shallow static cultures with the addition of glucose and glucose oxidase as a source of additional H2O2. The addition of Mn2+ and ABTS (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) to culture supernatants was associated with higher levels of biotransformation. Comparison of the isolates T. versicolor, T. pocas, T. cingulata and isolate DSPM95 with P. chrysosporium showed that these strains were competitive in the reduction of the PAHs, reducing the PAHs by more or less the same magnitude. Also these sub-tropical isolates did not accumulate a lot of HPLC detectable metabolites as much as P. chrysosporium.     

Enhanced Biodegradation of Anthracene by Bacillus Cereus Strain JMG-01 Isolated from Hydrocarbon Contaminated Soils

The present study displays the biodegradation capacities of native bacteria toward polycyclic aromatic hydrocarbons with particular emphasis to anthracene. A total of 23 bacterial strains were isolated from hydrocarbon-contaminated sites of Guwahati city, using anthracene as the carbon source. Among all these isolates, one Gram-positive strain (JMG-01) was selected as an efficient anthracene degrader, based on its maximum growth ability in anthracene enriched medium (100 ppm–700 ppm). At 500 ppm concentration, strain JMG-01 showed the maximum growth rate with 98% of anthracene degradation within 21 days of observation. The strain also demonstrated its potentiality by utilizing naphthalene and higher molecular hydrocarbons like pyrene, and benzo(a)pyrene at 500 ppm. The morphological, biochemical and molecular characterization identified the strain as Bacillus cereus. Surface morphology of the biomass, captured by Atomic Force Microscope, showed a distinctive modification, during the process of degradation. Study revealed that the effect of hydrocarbon exhibited the alteration, which concurrently enhanced the metabolic activity. Further, Gas chromatography-mass spectrometer analysis elucidates the possible metabolic pathway of anthracene degradation, depending on the intermediate metabolites produced. The finding thus suggests the essence of Bacillus cereus strain JMG-01 in enhanced anthracene degradation along the utilization of other hydrocarbons.