1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds that have accumulated in the environment due to a variety of anthropogenic activities and their persistence is chiefly due their low water solubility. PAHs are often mutagenic and carcinogenic which emphasizes the importance of their removal from the environment [1,2]. Since the 1970s, research on the biological degradation of PAHs has demonstrated that bacteria, fungi and algae possess catabolic abilities that may be utilized for the remediation of PAH-contaminated soil and water. Phenanthrene (Phe) is one of several PAHs that are commonly found as pollutants in soils [3], estuarine waters, sediments and other terrestrial and aquatic sites [4] and has been shown to be toxic to marine diatoms, gastropods, mussels, and fish [5,6].
Solid culture systems have shown great effectiveness in the removal of toxic compounds from soil. In this method, agroindustrial wastes are used such as wheat straw, corn stalks, sugarcane bagasse and pine wood chips [7], among others. When small amounts of agroindustrial residues are added to contaminated soil they confer soil apparent bulk density and porosity, help to diffuse oxygen between the particles and increase water retention. They are also used to support the growth of exogenous microorganisms, which are bioaugmented in soil to accelerate the degradation process, and, because of their nature, serve as carbon, phosphorus and nitrogen sources which are potentially important for the growth of organic pollutant degrading microorganisms [8]. Agroindustrial waste also contributes microorganisms with the ability to degrade toxic compounds; some studies, for example, have demonstrated that microbial biostimulation in a soil/sugarcane bagasse system at a ratio of 85:15 could remove 74% of total petroleum hydrocarbons (TPH) from the soil at 16 days [9, 10].
Several ligninolytic fungi have been grown on sugarcane bagasse and used as inoculum for the bioremediation of soil contaminated with polychlorinated biphenyls [10], phenanthrene [11], and benzo(a)pyrene [12]; such lignocellulosic materials are the natural habitat of the fungi. Previous work [13] has reported that non-ligninolytic filamentous fungi, such as
Filamentous fungi offer certain advantages over bacteria for bioremediation in solid culture because of their rapid colonization of solid substrates, such as soil or agroindustrial residues. In addition, they secrete large numbers of extracellular enzymes in solid culture and tolerate high concentrations of toxic compounds [14].
The most extensive studies have focused on white-rot basidiomycetes species such as
Also, non-ligninolytic fungi, such as
The efficient application in bioremediation of contaminated soils is dependent, then, on having fungal strains with the ability to grow in contaminated soil without being displaced by indigenous microflora and which also produce efficient PAH-degrading enzymes such as lignin and manganese peroxidases or phenoloxidases which allow the mineralization of toxic compounds (figure 1).
To achieve this goal, genetic engineering has been an important tool to generate genetically modified microorganisms (GEMs) through the expression of gene clusters encoding the degradation of a wide variety of pollutants. For example, simple aromatics, nitro aromatics, chloroaromatics, polycyclic aromatics, biphenyls, polychlorinated biphenyls, oil components etc., have been cloned and characterized for an increased degradation potential compared to their naturally occurring counterparts. Studies have focused primarily on bacteria and obtained good results for bioremediation systems [30, 31]. Knowledge of similar activities in fungi is limited to some white-rot fungi and a few species of non-ligninolytic fungi; however some studies have focused on toxic compound degradation, where recombinant strains were more efficient in the removal of PAHs from soil than wild-type strain [32]. It is therefore hypothesized that heterologous expression of genes codifying MnP and LiP in non-ligninolytic fungi will complement the degradation pathway of cytochrome P450 to obtain complete mineralization of the hydrocarbon without leaving more toxic intermediary compounds which accumulate in the soil (figure 1).
Some studies on the homologous expression of peroxidases in ligninolytic fungi in submerged culture, have shown that a transformed
A study on the heterologous expression of these genes has been done on the baculovirus expression system [36]. In the
The expression of the lignin peroxidase gene of
We have studied the possibility of producing these peroxidases in non-ligninolytic fungi isolated from contaminated soil because of their capacity to remove PAHs in soil; a number of filamentous fungal species are capable of secreting large amounts of proteins into the medium.
In our laboratory, one fungal strain was isolated from sugarcane bagasse using Mexican “Mayan” crude oil as carbon source [9]. This strain was identified by the sequence of ITS (Internal Transcription Spacer) fragments as:
Although the transformant SCB2-T3 strain presented MnP enzymatic activity and production was maintained for 5 d, production levels of the recombinant proteins still remained lower than the control strain of
In this study we obtained an effective heterologous expression of the mnp1 cassette controlled by the gpdA constitutive promoter in A. niger SCB2 strain. The MnP+7 transformant strain was selected due to its mayor MnP enzymatic activity after 48 h culture and up to 7 d, this important result shows that the new promoter favors protein production with catalytic activity from growth to idiophase, in comparison with SCB2-T3 strain, which shows only recombinant enzyme production while there was maltose in the culture medium, the compound that induces mnp1 gene expression in this strain. It is important for bioremediation systems that the oxidation involved PAHs enzymes are produced while stay in the soil. The longer the time in soil the increased enzyme production and higher removal of toxic compounds. On the other hand, the MnP+7 strain, was able to grow, tolerates and efficiently removed high Phe concentrations in contaminated soil as compared with the wild-type strain. After heterologous expression and the acquisition of these characteristics the MnP+7 strain, is a viable and important alternative for application in bioremediation of PAHs contaminated soils. This strain may have some potential as a bioaugmentation agent: is an efficient degrader of PAHs in high concentrations compared to other non-ligninolytic fungal strains which produce more toxic intermediaries than the original compound and tolerate lower PAH concentrations, and also compared to ligninolytic fungi not grown in soil and which are displaced by native soil microflora.
2. Methods
2.1. Heterologous expression of a P. chrysosporium
mnp 1 gene in A. niger SCB2
Fungal transformation was done through a biolistic transformation protocol previously described for
2.2. Evaluation of enzymatic activity of recombinant MnP
Colony transformants were assayed for MnP activity using a modified plate assay method [34]. The spores of transformants obtained with HygB were inoculated onto disks (0.5 mm in diameter) of MM agar medium [47] supplemented with hemoglobin (1 g/l). The disks were incubated at 30°C for 2 d; when fungal growth began, the disks were inoculated in Petri dishes with MM agar medium, in addition to o-anisidine. The plates were incubated at 30°C for 24 h and then flooded with a solution of 50 mM Na-phosphate buffer (pH 4.5) and 0.04% H2O2 on the surface of the plate and incubated at 30°C for 10 d in the dark. Positive controls were prepared with
Incorporation of the recombinant
The MnP extracellular activity was determined spectrophotometrically by a modification of the method previously described using phenol red oxidation [49]. Absorbance was read at 610 nm. One unit of MnP activity was defined as 1 µmol product formed per minute.
The kinetics of wild-type and transformant strains of
2.3. Phenanthrene removal by A. niger Mnp+7 and wild-type strain in solid culture
The ability of wild-type and transformant strains to remove Phe was determined at several times in the solid-state microcosm system, using the same culture conditions. Sugarcane bagasse was used as a fungal growth support and carbon source. The sterile material was moistened with MM medium and inoculated with 2 × 108 fungal spores/ml; all cultures were incubated for 2 d at 30°C. Uncontaminated soil obtained from a zone near a contaminated region in Coatzacoalcos, Veracruz, Mexico, was sterilized and contaminated with 600 ppm of Phe. The newly contaminated soil was mixed with the inoculated sugarcane bagasse and incubated at 30°C for 14 d, as well as a control (sterile bagasse and contaminated soil without fungi) to determine abiotic Phe removal. Evolution of CO2 was measured daily to quantify the heterotrophic activity. After this period, Phe removal for both strains was determined by HPLC.
Heterotrophic activity was determined by Gas Chromatography (GC). Headspace samples were taken from the flasks and analyzed for CO2 evolution. The headspace in each flask was flushed out daily for 15 min with sterile and moistened air. This allowed the preservation of aerobic conditions and avoided carbon dioxide accumulation. CO2 quantification was reported as milligrams of CO2 per gram of initial dry matter (IDM). Phenanthrene was extracted with microwave assisted extraction, according to EPA method 3546. Analysis of Phe was based on EPA method 3540 for the HPLC system.
3. Results
3.1. Heterologous expression of a P. chrysosporium
mnp 1 gene in A. niger SCB2
The heterologous expression of genes coding for different isoforms of MnP from
Expression plasmid pGMG-Hyg was introduced into wild-type
3.2. Evaluation of enzymatic activity of recombinant MnP
The transformants were evaluated for MnP activity by the o-anisidine coloration method in MM medium with hemoglobin plates. Transformants that developed a purple halo were selected. Four transformants (MnP+1, MnP+4, MnP+7 and MnP+8) formed purple halos around the agar disk after 8 d of incubation, indicating extracellular peroxidase activity, as show in figure 4. The wild-type strain showed no coloration; however, the control strain of
The MnP+7 transformant strain showed higher Phe tolerance than wild-type strain when inoculated into Cove's medium in Petri dishes at different Phe concentrations. At concentrations above 600 ppm, both strains showed a decrease in growth rate compared to their respective controls without Phe; however, the wild-type strain showed an inhibition in sporulation while the transformant strain was able to sporulate (figure 5). This coincides with the results in reference [53], which reported that fluorene at concentrations above 100 ppm caused growth inhibition of fungal strains isolated from a contaminated soil. In contrast, reference [54] reported that 100 ppm of anthracene had no inhibitory effect on the growth of fungi isolated from soil. This fact suggests that due to the production of MnP by the transformant strain for
MnP productivity of the four selected strains was quantified in liquid culture using MM medium with hemoglobin. The activity was measured every 24 h for 7 days. As shown in figure 6, the wild-type strain did not present MnP activity. Although all transformant strains present different MnP activity, maximum activity was obtained by
After 48 h, all transformant strains showed MnP activity in liquid medium. Because
The specific activity obtained by recombinant MnP with
3.3. Phenanthrene removal by A. niger Mnp+7 and wild-type strain in solid culture
In order to evaluate the growth of the microorganism in solid culture, CO2 evolution was quantified. Two tested strains showed different profiles and the ANOVA test indicated a significant (p<0.05) difference in the accumulated CO2 production: the transformant strain produced more CO2 than the wild-type, both in the presence and absence of Phe, and was around 15-18 mg CO2 accumulated per gram of initial dry matter (IDM), whereas the wild-type strain produced only around 5-7 mg CO2 accumulated/g IDM (figure 7A). This result demonstrated that
The residual Phe extracted from treated soil was quantified by HPLC and the results of two strains are presented in figure 8. The wild-type strain had the lowest Phe removal capacity (approximately 7%) compared with the transformant MnP+7 strain which was able to remove approximately 44% of the initial Phe (0.6 mg/g IMD) in 14 d. The Phe extraction efficiency of the abiotic controls was 98%.
The increase in the removal percentage of Phe by the MnP+7 transformant strain in solid culture suggests that it is due to the production of MnP enzyme by the transformant strain which showed the ability to express the
The increase in Phe concentration in solid culture showed a higher toxic effect on the wild-type strain. Compared to cultures carried out using soil contaminated with 0.4 mg/g IMD, the wild-type strain SCB2 was able to remove 75% of Phe, while
With respect to the intermediaries formed during Phe oxidation in solid culture for
1-phenanthrol, 2-phenanthrol, and phenanthrene trans-9,10-dihydrodiol have been reported as major metabolites from the metabolism of Phe by
Bioaugmentation with an
The results from this study also show that non-ligninolytic fungal strains are a viable alternative for application in bioremediation systems; moreover, bioaugmentation with genetically modified exogenous fungal strains for heterologous protein production in solid culture accelerates the process of removal and biodegradation of toxic compounds in contaminated soils.
4. Conclusion
The action of genetically modified non-ligninolytic fungal strains in bioremediation systems has not been reported, so that the results obtained in this investigation suggest that these microorganisms may have some potential as a bioaugmentation agent: they are efficient degraders of PAHs in high concentrations compared to other non-ligninolytic fungal strains which produce more toxic intermediaries than the original compound and tolerate lower PAH concentrations, and also compared to ligninolytic fungi not grown in soil and which are displaced by native soil microflora.
Acknowledgement
This work was supported by SEP-CONACYT, project CB2008-105643 and Instituto Politécnico Nacional, project SIP20121707.
References
- 1.
IARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans.92 Some Non-heterocyclic Polycyclic Aromatic Hydrocarbons and Some Related Exposures. Lyon France.2010 - 2.
Sudip K. S andSingh O. V R. K Jain 2002 Polycyclic aromatic hydrocarbons: environmental pollution and bioremediation Trend. Biotechnol.20 243 248 - 3.
Chen B X Xuan L Zhu J Wang Y Gao K Yang andX Shen B Lou 2004 Distributions of polycyclic aromatic hydrocarbons in surface waters, sediments and soils of Hangzhou city China, Water Res.38 3558 3568 - 4.
Shiaris M. P 1989 Seasonal biotransformation of naphthalene, phenanthrene and benzo(a)pyrene in surficial estuarines sediments Appl. Environ. Microbiol.55 1391 1399 - 5.
Black J. A W. J Birge andA. G Westerman P. C Francis 1983 Comparative aquatic toxicology of aromatic hydrocarbons. Fundamental and Appl. Toxicol,3 353 358 - 6.
White K. L 1986 An overview of immunotoxicology and carcinogenic polycyclic aromatic hydrocarbons Environ. Carcin. Re. C4 163 202 - 7.
Sample K. T Reid B. J Fermor T. R 2001 Review of composting strategies to treat organic pollutants in contaminated soils. Environ. Pollut.112 269 283 - 8.
Pandey A Soccol C Nigam P Soccol V 2000 Biotechnological potential of agroindustrial residues I: Sugarcane bagasse. Bioresource Technol.74 69 80 - 9.
Pérez-armendáriz B Loera-corral O Fernández-linares L Esparza-garcía F Rodríguez-vázquez R 2004 Biostimulation of micro-organisms from sugarcane bagasse pith for the removal of weathered hydrocarbon from soil Lett Appl Microbiol;38 373 377 - 10.
Fernández-sánchez J. M R Rodríguez-vázquez andG Ruiz-aguilar P. J. J Alvarez 2001 PCB biodegradation in aged contaminated soil: interactions between exogenous Phanerochaete chrysosporium and indigenous microorganisms. J. Environ. Sci. Health, part. A.36 7 1145 1162 - 11.
R (Chávez-gómez B Quintero R Esparza-garcía F Mesta-howard A De La Serna F. J. Z. D Hernández-rodríguez C Gillen T Poggi-varaldo H Barrera-corte s J Rodríguez-vázquez 2003 Removal of phenanthrene from soil by co-cultures of bacteria and fungi pregrown on sugarcane bagasse pith. Bioresource Technol.89 177 183 - 12.
Dzul-puc J Esparza-garcía F Barajas-aceves M Rodríguez-vázquez R 2004 Benzo[a]pyrene removal from soil by Phanerochaete chrysosporium grown on sugarcane bagasse and pine sawdust 58 1 7 - 13.
Cortés-espinosa D. V Fernández-perrino F. J Arana-cuenca A Esparza-garcía J. F Loera O Rodríguez-vázquez R 2006 Selection and identification of fungi isolated from sugarcane bagasse and their application for phenanthrene removal from soil J. Environ. Sci. Health., Part A.41 3 475 486 - 14.
Cerniglia C. E andG. L White R. H Helflich 1985 Fungal metabolism and detoxification of polycyclic aromatic hydrocarbons. Arch. Microbiol.143 105 110 - 15.
andBarr D. P S. D Aust 1994 Mechanisms white rot fungi use to degrade pollutants. Environ Sci. technol.28 79 87 - 16.
Bezalel L Y Hadar P. P Fu andJ. P Freeman C. E Cerniglia 1996 Initial oxidation products in the metabolism of pyrene, anthracene, fluorene and dibenzothiophene by the white rot fungus Pleurotus ostreatus. Appl Environ Microbiol.62 2554 2559 - 17.
Bezalel L Y Hadar P. P Fu andJ. P Freeman C. E Cerniglia 1996 Metabolism of phenantrene by the white rot fungus . Appl. Environ. Microbiol.62 2547 2553 - 18.
D Annibale A. , M Ricc V Leonardi D Quaratino andE Mincione M Petruccioli 2005 Degradation of aromatic hydrocarbons by white-rot fungi in a historically contaminated soil. Biotechnol. Bioeng.90 723 731 - 19.
Boyle C. D 1995 Development of a practical method for inducing white rot fungi to grow into and degrade organopollutants in soil Can. J. Microbiol.41 345 353 - 20.
Capotorti G P Digianvincenzo P Cesti andA Bernardi G Guglielmetti 2004 Pyrene and benzo(a)pyrene metabolism by an strain isolated from a polycyclic aromatic hydrocarbons polluted soil. Biodegradation15 79 85 - 21.
andMeysami P H Baheri 2003 Pre-screening of fungi and bulking agents for contaminated soil bioremediation Adv Environ Res.7 881 887 - 22.
andOkeke R. C H. U Agbo 1996 Influence of environmental parameters on pentachlorophenol biotransformation in soil by and Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol.45 263 266 - 23.
Launen L Pinto L Wiebe C andKielmann E Moore M 1995 The oxidation of pyrene and benzo(a)pyrene by nonbasidiomycete soil fungi. Can. J. Microbiol.41 477 488 - 24.
Boonchan S Britz M. L Stanley G. A 2000 Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures Appl Environ Microbiol.66 1007 1019 - 25.
Voigt KD & Kirsche B (Gramss G 1999 Degradation of polycyclic aromatic hydrocarbons with three to seven aromatic rings by higher fungi in sterile and unsterile soils. 10 51 62 - 26.
Shimada T 2006 Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons. Drug. Met. Pharmacokinet.21 4 257 276 - 27.
andShimada T Y Fujii-kuriyama 2004 Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes Cancer Sci. 95(1):1-6450 A1 and 1B1. - 28.
Sutherland J. B 1992 Detoxification of polycyclic aromatic hydrocarbons by fungi. J. of Ind. Microbiol.9 1 53 62 - 29.
andJuhasz A. L R Naidu 2000 Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: A Review of the Microbial Degradation of Benzo[a]pyrene Int. Biodeterior. Biodegrad.45 57 88 - 30.
andPieper D. H W Reineke 2000 Engineering bacteria for bioremediation. Curr. Opin. Biotechnol.11 262 270 - 31.
andSayler G. S S Ripp 2000 Field applications of genetically engineered microorganisms for bioremediation processes Curr. Opin. Biotechnol.11 286 289 - 32.
Cortés-Espinosa, Ángel E. Absalón, Noé Sánchez, Octavio Loera, Refugio Rodríguez-Vázquez and Francisco J. Fernández (Diana V 2011 Heterologous expression of manganese peroxidase in Aspergillus niger and its effect on phenanthrene removal from soil J Mol Microbiol Biotechnol.21 120 129 - 33.
Tsukihara T Y Honda andT Watanabe T Watanabe 2006 Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2 Appl Microbiol Biotechnol.71 114 120 - 34.
Mayfield M. B K Kishi andM Alic M. H Gold 1994 Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium. Appl. Environ. Microbiol.60 4303 4309 - 35.
Sollewijn Gelpke M. D., M. Mayfield-Gambill, G. P. Lin Cereghino, and M. H. Gold (1999 Homologous expression of recombinant lignin peroxidase in . Appl. Environ. Microbiol.65 1670 1674 - 36.
andJohnson T. M J. K Li 1991 Heterologous expression and characterization of an active lignin peroxidase from Phanerochaete chrysosporium using recombinant baculovirus. Arch Biochem Biophys. 291(2):371 EOF 8 EOF - 37.
andDoyle W A. T Smith 1996 Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem. Biochem J.315 15 19 - 38.
andWhitwam R M Tien 1996 Heterologous of fungal Mn peroxidase in and refolding to yield active enzyme. Biochem Biophys Res Commun216 1013 1017 - 39.
Saloheimo M V Barajas andM. -L Niku-paavola J. K. C Knowles 1989 A lignin peroxidase-encoding cDNA from the white fungus Phlebia radiata: characterization and expression in Trichoderma reesei. 85 343 351 - 40.
Stewart P R Whitwam P Kersten andD Cullen M Tien 1996 Efficient expression of Phanerochaete chrysosporium manganese peroxidase in Aspergillus oryzae. Appl. Environ. Microbiol.62 860 864 - 41.
Aifa M. S andS Sayadi A Gargouri 1999 Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Aspergillus niger Biotechnol. lett.21 849 853 - 42.
Hondel, and P. J. Put (Conesa A C. A Van Den 2000 Studies on the production of fungal peroxidases in . Appl. Environ. Microbiol.66 7 3016 3023 - 43.
September (Andersen H. D andE. B Jensen K. G Welinder 1992 A process for producing heme proteins. European Patent Application EP 0 505 311 A2. - 44.
Fowler T M. W Rey P Vaha-vahe andS. D Power R. M Berka 1993 The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter. Mol. Microbiol.9 989 998 - 45.
Fungaro M. H. P Rech E Muhlen G. S Vainstein M. H Pascon R. C Dequeiroz M. V andPizzirani-kleiner A. A De Azevedo J. L 1995 Transformation of by microprojectile bombardment on intact conidia, FEMS Microbiol. Lett.125 293 298 - 46.
Herzog R. W Daniell H ySingh N. K Lemke P. A 1996 A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol. Appl. Microbiol. Biotechnol.45 333 337 - 47.
Bennett J. W Lasure L. L 1991 Growth media. In: Bennett, J.W., Lasure, L.L. (Eds.), More Gene Manipulations in Fungi. Academic Press, San Diego,441 458 - 48.
Reader U Broda P 1985 Rapid preparation of DNA from filamentous fungi. Lett. Appl. Microbiol.1 17 20 - 49.
Kuwahara M Glenn J. K Morgan M. A Gold M. H 1984 Separation and characterization of two extracellular H2O2 dependent oxidases from ligninolytic culture of . FEBS Lett.169 247 250 - 50.
Larrondo L. F S Lobo P Stewart andD Cullen R Vicuña 2001 Isoenzyme multiplicity and characterization of recombinant manganese peroxidase and Phanerochaete chrysosporium. Appl. Environ. Microbiol.67 2070 2075 - 51.
andSingh D S Chen 2008 The white-rot fungus Phanerochaete chrysosporium: conditions for the production of lignin-degrading enzymes Appl. Microbiol Biotechnol.81 399 417 - 52.
van den Hondel (Lokman B. C Joosten V Hovenkamp J Gouka R. J Verrips C. T 2003 Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori. J Biotechnol.103 183 190 - 53.
Garon D Krivobok S Wouessidjewe D Seigle-murandi F 2002 Influence of surfactants on solubilization and fungal degradation of fluorene 47 303 309 - 54.
Krivobok S E Miriouchkine F Seigle-murandi J. -L Benoit-guyod 1998 Biodegradation of Anthracene by soil fungi 37 3 523 530 - 55.
Nevalainen K. M. H V. S. J Te o a. n. d P. L Bergquist 2005 Heterologous protein expression in filamentous fungi. Trends Biotechnol.23 9 469 474 - 56.
andGlenn J. K M. H Gold 1985 Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete . Arch. Biochem. Biophys.242 329 341 - 57.
Elrod S Zelson S 1999 Methods for increasing hemoprotein production in fungal mutants. Word Intellectual Property Organization. WO/1999/029874. - 58.
Capotorti G P Digianvincenzo P Cesti andA Bernardi G Guglielmetti 2004 Pyrene and benzo(a)pyrene metabolism by an strain isolated from a polycyclic aromatic hydrocarbons polluted soil. Biodegradation15 79 85 - 59.
Chulalaksananukul S Gadd G. M Sangvanich P Sihanonth P Piapukiew J Vangnai A. S 2006 Biodegradation of benzo(a)pyrene by a newly isolated . FEMS Microbiol Lett.262 99 106 - 60.
Alvarez PJJ, Rodríguez-Vázquez R (Meléndez-estrada J Amezcua-allieri M. A 2006 Phenanthrene removal by Penicillium frequentans grown on a solid-state culture: effect of oxygen concentration Environ Technol.27 1073 1080 - 61.
Crow Jr. SA, Heinze TM, Deck J, Cerniglia CE (Casillas R. P 1996 Initial oxidative and subsequent conjugative metabolites produced during the metabolism of phenanthrene by fungi. J Ind Microbiol.16 205 215 - 62.
Sack U Heinze T. M Deck J Cerniglia C. E Cazau M. C Fritsche W 1997 Novel metabolites in phenantrene and pyrene transformation by . Appl Environ Microbiol.63 2906 2909 - 63.
Hammel K. E Gai W. Z Green B Moen M. A 1992 Oxidative degradation of phenanthrene by the ligninolytic fungus Phanerochaete chrysosporium. Appl Environ Microbiol.58 1832 1838 - 64.
Sutherland J. B Selby A. L Freeman J. P Evans F. E Cerniglia C. E 1991 Metabolism of phenanthrene by Phanerochaete chrysosporium. Appl. Environ. Microbiol.57 3310 3316 - 65.
Serrano Silva I da Costa dos Santos E, Ragagnin de Menezes C, Fonseca de Faria A, Franciscon E, Grossman M, Durrant LR (2009 Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia. Biores Technol.100 4669 4675 - 66.
Potin O Veignie E Rafin C 2004 Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by Cladosporium sphaerospermum isolated from an aged PAH contaminated soil. FEMS Microbiol Ecol.51 71 78