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BIOREMEDIATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN WATER AND SOIL IN AQUEOUS, SLURRY AND SOLID PHASE Bardi L.*, Fabbian R.^, Belviso S.°, Biasizzo M.°, Marzona M.° *C.R.A. Istituto Sperimentale per la Nutrizione delle Piante, Via Pianezza, 115, 10151 Turin, Italy. E-mail laura.bardi@entecra.it ^Biosearch Ambiente Srl, Turin, Italy °Dip. Chimica Generale Organica Applicata, Università di Torino, Turin, Italy

INTRODUCTION INTRODUCTION

MATERIALS MATERIALS AND AND METHODS METHODS

Fig.2. Biodegradation of phenanthrene in a slurry and in a solid phase in absence and in presence of β -cyclodextrin

100 s lurry phas e s olid phas e

Phenanthrenedegradation(% )

90 80 70 60 50 40 30 20 10 0 Without betacyclodextrin

With betacyclodextrin

REFERENCES

Biodegradation Biodegradation assays assays Microcosms Microcosms were were prepared prepared in in 250 250 ml ml flasks flasks and and inoculated inoculated with with 11 ml ml microbial microbial consortium. consortium. Aqueous Aqueous phase: phase: 50 50 ml ml BHB BHB with with 1000 1000 ppm ppm phenanthrene phenanthrene were were added added with with 500 500 mg mg αα−−,, β− ββ− or γγ−−cyclodextrin cyclodextrin and and sterilized sterilized at at 120°C 120°C xx 20’; 20’; after after the the inoculum inoculum they they β−− or were were incubated, incubated, at at 28°C 28°C or or 37°C 37°C for for 26 26 days. days. Slurry Slurry phase: phase: 50 50 gg of of unsteril unsteril soil soil was was added added with with 1000 1000 ppm ppm phenanthrene, phenanthrene, 80% 80% water water and and 500 500 mg mg ββ-cyclodextrin; -cyclodextrin; itit was was balanced balanced to to C:N:P=100:15:1 C:N:P=100:15:1 with with urea urea and and DAP, DAP, then then inoculated inoculated and and incubated incubated in in an an Infors Infors shaker shaker at at 150 150 rpm rpm at at 37°C 37°C for for 80 80 days. days. Solid Solid phase: phase: as as slurry slurry phase phase but but with with 25% 25% water water and and static static incubation incubation at at room room temperature temperature for for 90 90 days.. days..

28 °C 37 °C

50 40 30 20 10 0

(1) no CD

(2) beta-CD

(5) alfa-CD

(6) gammaCD

RESULTS

Slurry Slurry and and solid solid phase phase The The biodegradation biodegradation in in soil soil was was studied studied in in slurry slurry and and solid solid phase. phase. In In spite spite of of the fact that slurry phase, the fact that slurry phase, due due to to shaking, shaking, makes makes itit possible possible aa better better contact contact between between hydrocarbon hydrocarbon and and microbial microbial cells cells and and allows allows the the temperature temperature control, control, itit was was observed observed that that the the phenanthrene phenanthrene biodegradation biodegradation was was higher higher than than in in slurry slurry phase phase (Figure (Figure 2). 2). In In slurry slurry phase phase the the positive positive effect effect of of the the higher higher temperature temperature (37°C) (37°C) associated associated to to the the presence presence of of ββ-cyclodextrin -cyclodextrin was was confirmed. confirmed. The The presence presence of of ββ-cyclodextrin -cyclodextrin increased increased the the degradation degradation in in both both solid solid and and slurry slurry phase, phase, as as aa confirmation confirmation of of the the fact fact that that bioavailability bioavailability has has aa crucial crucial role role in in bioremediation bioremediation of of phenanthrene. phenanthrene. The The better better performance performance obtained obtained in in solid solid phase phase can can be be explained explained by by the the higher higher oxygen oxygen availability availability respect respect to to the the slurry slurry phase. phase. Microbial Microbial consortium consortium The The heterothrophic heterothrophic microbial microbial population population in in microcosmos microcosmos at at the the end end of of degradation degradation processes processes shown shown to to be be different different in in aqueous aqueous phase phase and and in in soil. soil. In In aqueous aqueous phase phase almost almost one one single single strain strain was was found, found, that that after after the the molecular molecular analysis analysis shown shown aa similarity similarity >99% >99% to to aa β-Proteobacteria β-Proteobacteria (closely (closely related related to to Achromobacter Achromobacter xylosoxidans) xylosoxidans) already already described described in in aa benzopyrene-degrading benzopyrene-degrading microbial microbial consortium consortium [18]. [18]. In In soil soil microcosmos microcosmos aa more more variable variable microbial microbial population population was was observed, observed, in in which which nine nine strains strains were were chosen chosen as as different different on on the the basis basis of of the the different different morphological morphological aspect aspect of of colonies colonies grown grown in in solid solid medium medium and and of of cells cells observed observed in in optical optical microscopy. microscopy. These These strains strains were were purified purified and and analyzed analyzed for for the the 16S 16S and and ITS ITS sequences. sequences. They They shown shown to to be be three three Streptomyces Streptomyces spp., spp., two two Bacillus Bacillus spp. spp. (Bacillus (Bacillus sp. sp. and and Bacillus Bacillus subtilis subtilis ),), two two Aspergillus Aspergillus spp spp (Aspergillus (Aspergillus terreus terreus and and Aspergillus Aspergillus sp.) sp.) and and Penicillium Penicillium restrictum. restrictum. Penicillium Penicillium ssp., ssp., Aspergillus Aspergillus ssp. ssp. ee Streptomyces Streptomyces ssp. ssp. are are already already known known for for their their capability capability to to degrade degrade PAHs. PAHs. The The presence presence of of Bacillus Bacillus sp. sp. and and Bacillus Bacillus subtilis subtilis is is very very interesting interesting for for the the degradative degradative microbial microbial consortium consortium as as these these microrganisms microrganisms can can produce produce bioemulsifier bioemulsifier compounds, compounds, so so they they could could contribute contribute both both directly directly and and indirectly indirectly to to PAH PAH degradation, degradation, by by increasing increasing bioavailability bioavailability also also to to other other degrading degrading strains. strains. Anyway, Anyway, these these results results support support the the hypothesis hypothesis that that even even in in the the case case of of aa recent recent pollution pollution event event the the indigenous indigenous microorganisms microorganisms of of aa soil soil can can be be effective effective in in bioremediation bioremediation processes processes ifif adequately adequately supported supported with with technical technical interventions interventions mainly mainly aimed aimed to to stimulate stimulate the the microbial microbial activity activity and and to to increase increase biovailability biovailability of of hydrophobic hydrophobic xenobiotics. xenobiotics.

1500

1500

850

850

400

400

A = Bacillus sp. D = Streptomyces sp.

Phenanthrene Phenanthrene analysis analysis The The residual residual phenanthrene phenanthrene concentration concentration was was analysed analysed at at the the end end of of degradation degradation process process by by extraction extraction in in ethyl ethyl acetate acetate and and HPLC HPLC analysis analysis ina ina Jasco Jasco HPLC HPLC PU-980 PU-980 equipped equipped with with Jasco Jasco UV-2075 UV-2075 Plus Plus UV/V UV/V detector detector in in aa Supelcosil Supelcosil LC18 LC18 15 15 cm cm xx 4.6 4.6 mm mm column. column. Operating Operating conditions: conditions: elution elution with with acetonitrile:water acetonitrile:water 60:40, 60:40, flux flux 22 ml/min, λ 254 nm. ml/min, λ 254 nm. Molecular Molecular characterization characterization of of pure pure strains strains DNA DNA from from fungal fungal isolates isolates was was extracted extracted and and purified; purified; primers primers ITS1 ITS1 and and ITS4 ITS4 were were used used to to amplify amplify aa region region of of rDNA rDNA including including ITS1 ITS1 and and ITS2 ITS2 and and the the 5.8S 5.8S rDNA rDNA gene. gene. PCR PCR amplifications amplifications of of genomic genomic DNA DNA from from bacterial bacterial isolates isolates were were performed performed using using universal universal primers primers 27F 27F and and 1492R. 1492R. For For strain strain isolated isolated from from the the consortium, consortium, sequence sequence determination determination was was carried carried out out by by using using internal internal 16S 16S rRNA rRNA primers primers 27F, 27F, 531R, 531R, COM1 COM1 and and 1378R. 1378R. PCR PCR products products were were checked checked by by agarose agarose gel gel electrophoresis electrophoresis and and were were purified purified with with the the GenElute GenElute PCR PCR Clean-up Clean-up Kit Kit (SIGMA). (SIGMA). Sequence Sequence determination determination was was performed performed using using the the automated automated sequencer sequencer model model ABI ABI PRISM PRISM 3100 3100 (Applied (Applied Biosystems). Biosystems). Database Database searches searches were were conducted conducted by by using using the the BLAST BLAST program program with with the the GenBank GenBank database. database. 16S 16S rDNA rDNA sequences sequences retrieved retrieved from from the the database database were were aligned aligned by by using using ClustalX ClustalX software, software, version version 1.81. 1.81.

Fig.3. Fig.3. Biodegradation Biodegradation kinetic kinetic of of phenanthrene phenanthrene with with ββ-cyclodextrin cyclodextrin at at different different temperatures temperatures

120 100 80 60 40 20

28°C biodegradation 37°C biodegradation

B = Streptomyces sp. E = Streptomyces sp.

A

B

C

D

E

R

28°C blank 37°C blank

0 0

5

10

15 Days

20

25

CONCLUSIONS CONCLUSIONS

G

H

I

C = Bacillus subtilis

R = Benzo [a] pyrene-degrading bacterium BPC7 (Achromobacter xylosoxidans).

30

The The bioavailability bioavailability of of phenanthrene phenanthrene can can be be improved improved by by the the addition addition of of ββ-cyclodextrin -cyclodextrin both both in in soils soils and and in in waters, waters, causing causing aa significant significant biodegradation biodegradation increase. increase. A A temperature temperature increase increase gives gives rise rise to to aa better better degradation, degradation, in in particular particular in in water, water, probably probably due due to to to to aa higher higher inclusion inclusion complex complex solubility. solubility. At At lower lower temperature temperature α α-cyclodextrin -cyclodextrin shown shown to to be be the the most most effective effective among among the the tested tested cyclodextrins, cyclodextrins, but but the the higher higher price, price, makes makes itit not not interesting interesting for for practical practical applications. applications. In In soil, soil, an an in in situ situ bioremediation bioremediation strategy strategy can can be be considered considered as as probably probably more more effective effective than than an an ex ex situ situ process process carried carried out out in in slurry slurry phase. phase. The The degradative degradative microbial microbial consortium consortium selected selected from from aa natural natural soil soil is is effective effective in in phenanthrene phenanthrene biodegradation; biodegradation; moreover, moreover, the the heterothrophic heterothrophic microbial microbial population population at at the the end end of of degradative degradative process process was was composed composed by by several several microorgansims microorgansims (more (more than than those those found found in in the the enrichment enrichment culture),. culture),. This This means means that that even even in in case case of of aa recent recent pollution event the indigenous microorganisms of a soil pollution event the indigenous microorganisms of a soil can can be be effective effective in in bioremediation bioremediation process. process.

MARKER

R (COM1/1378R)

R (27F/531R)

E (27F/1492R)

D (27F/1492R)

C (27F/1492R)

B (27F/1492R)

MARKER

A (27F/1492R)

Finlayson-Pitts B.J. and Pitts, J.N., Jr (1997). Science 276: 1045-1052 Shennan. J.L. (1984). In Petroleum Microbiology Atlas. Macmillan R.M..ed. pp. 643683. Mastrangela. G. (1997) Health Perspect. 104: 1166-1170. Weis L.M. (1998) Environ. Health Perspect. 106, 17-22 Fritsche, W. (1994) : Proceeding of the 6th European Congress of Biotechnology. Ed. Alberghina L., Frontal. L., Sensi P.: 1213-1221 Atlas, R.M.(1995) 31, 178-182 Hammel K.E., Gai Z.G., Green B. & Moen M.A. (1992) Appl. Environ. Microbiol. 58: 1831-1838. Dagley S. (1971). Adv. Microbiol. Physiol. 6:1-76 Wilson SC, Jones KC (1993). Environ Pollut, 81:229-249. Brusseau ML. (1998) In: Biotechnology for soil remediation. Scientific bases and practical applications. R.Serra. C.I.P.A. S.r.l. Milan, Italy, 81-98. Szejtli J (1988). Kluwer Academic Publishers, Dordrecht, The Netherland, 1-393 Olah J, Cserhati T, Szejtli J. (1988). Water Res; 22:1345-52. Wang J.M., Marlowe E.M., Miller-Maier R.M., and Brusseau M.L. (1998). Environ. Sci. Technol., 32 : 1907-1912. Bardi, L., Mattei, A., Steffan, S. and Marzona, M. (2000) ‘, Enzyme and Microbial Technology 27(9) : 709--713. Henrion, B., Chevalier, G. & Martin, F. (1994).. Mycological Research 98: 37-43 White, T. J., T. Bruns, S. Lee, and J. Taylor. (1990). In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, San Diego, Calif. Altschul, X., F. Stephen, L. Thomas, X. Madden, A. Alejandro, X. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402. Kanaly R.A., Harayama S.and Watanabe K. (2002) Rhodanobacter sp. Strain BPC1 in a Benzo[a]pyrene-mineralizing bacterial consortium . Appl. Environ. Microbiol. 68: 5826-5833.

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Microorganisms Microorganisms The The microbial microbial consortium consortium was was selected selected from from aa natural, natural, grass-covered grass-covered soil soil in in aa liquid liquid selective selective medium medium (BHB (BHB with with 250 250 ppm ppm phenanthrene phenanthrene as as the the sole sole energy energy and and carbon carbon source). source). Single Single colonies colonies were were obtained obtained from from microcosms microcosms by by plating plating cell cell suspension suspension on on Tryptic Tryptic Soy Soy Agar Agar (TSA), (TSA), then then plated plated on on BHA BHA added added with with phenanthrene phenanthrene to to ascertain ascertain their their ability ability to to grow grow with with phenanthrene phenanthrene as as the the sole sole energy energy and and carbon carbon source. source.

% residual phenanthrene

Aqueous Aqueous phase phase The The phenanthrene phenanthrene biodegradation biodegradation assays assays in in aqueous aqueous phase phase at at either either 28°C 28°C or or 37°C 37°C in in presence presence of of 1% 1% αα−−,, ββ−−,, or or γγ−−cyclodextrin cyclodextrin and and without without cyclodextrin (Fig. 1) shown that at 28°C the addition of cyclodextrins cyclodextrin (Fig. 1) shown that at 28°C the addition of cyclodextrins did did not not induced induced significant significant increases increases of of degradation. degradation. The The formation formation of of the the inclusion inclusion complexes, complexes, even even ifif they they increases increases water water solubility solubility of of phenanthrene, could cause a shield that reduces its bioavailability when the phenanthrene, could cause a shield that reduces its bioavailability when the diameter γγ-CD), diameter of of the the internal internal cavity cavity of of the the cyclodextrin cyclodextrin is is higher higher (γ (γ -CD), while while when αα-CD) when the the cavity cavity is is smaller smaller (α (α -CD) part part of of the the molecule molecule could could be be exposed, exposed, making making itit available available to to the the microbial microbial attack. attack. At At 37°C 37°C the the behaviour behaviour was was completely completely different. different. Without Without cyclodextrin cyclodextrin itit was was observed observed aa decrease decrease of of degradation degradation activity activity (16.4%), (16.4%), while while in in presence presence of ββ-CD: of cyclodextrins cyclodextrins itit was was always always observed observed an an increase increase (β (β -CD: 56.8%; 56.8%; αα-CD: -CD: 36,8%; 36,8%; γγ-CD: -CD: 18.9%). 18.9%). This This indicates indicates that that an an increase increase of of temperature temperature did did not not increased, increased, the the microbial microbial metabolic metabolic activity; activity; then then the the increase increase of of degradation degradation in in presence presence of of CDs CDs is is not not due due to to biological, biological, but but probably probably to to physico-chemical physico-chemical effects effects on on the the inclusion inclusion constant constant of of the the phenanthrene phenanthrene complex. complex. This This result result was was confirmed confirmed by by aa degradation degradation kinetic kinetic assay assay (Fig. (Fig. 3). 3). The The degradation degradation increase increase higher higher for for ββ-CD -CD than than for for other other CD CD could could be be due due to to higher higher effect effect of of temperature temperature on on its its solubility solubility..

Figure 1 - Biodegradation of phenanthrene in an aqueous phase at different temperatures and with different cyclodextrins

Phenanthrene degradation (%)

Polycyclic Polycyclic aromatic aromatic hydrocarbons hydrocarbons (PAHs) (PAHs) are are widely widely spread spread as as pollutants pollutants of of soils soils and and waters. waters. PAHs PAHs are are toxic, toxic, mutagenic mutagenic and/or and/or carcinogenic. carcinogenic. Phenanthrene Phenanthrene is is known known as as photosensitizing photosensitizing for for human human skin skin and and potent potent inhibitor inhibitor of of gap gap junctional junctional intercellular intercellular communication. communication. Several Several studies studies have have proved proved that that biodegradation biodegradation of of PAH PAH by by aa single single microorganism microorganism or or aa microbial microbial consortium consortium is is an an effective effective way way to to reach reach decontamination decontamination of of soils soils and and sediments. The complete biodegradation of hydrocarbons lead to sediments. The complete biodegradation of hydrocarbons lead to final final non-toxic non-toxic products water and and cellular cellular biomass. biomass. Several Several microorganisms microorganisms have have been been products like like CO CO22,, water characterised characterised for for their their ability ability to to degrade degrade phenanthrene: phenanthrene: i.e., i.e., Phanaerochaete Phanaerochaete chrysosporium chrysosporium oxidise oxidise the the molecule molecule in in C9 C9 and and C10; C10; marine marine cyanobacteria cyanobacteria like like Agmellum Agmellum quadruplicatum quadruplicatum PR-6 PR-6 and and Synechoccus Synechoccus oxidise oxidise phenanthrene phenanthrene by by aa monoxygenase. monoxygenase. Bioremediation Bioremediation of of PAHs PAHs is is not not frequently frequently applied. applied. PAHs PAHs are are recalcitrant recalcitrant to to biodegradation: biodegradation: due due to to their their hydrophobicity hydrophobicity and and low low water water solubility solubility they they pass pass very very slowly slowly to to the the aqueous aqueous phase phase in in which which they they are are metabolized metabolized by by microorganisms; microorganisms; moreover moreover in in soil soil they they are are adsorbed adsorbed to to clay clay or or humus humus fractions. fractions. Bioavailability Bioavailability can can be be increased increased by by cyclodextrins, cyclodextrins, cyclic cyclic oligosaccharides oligosaccharides formed formed by by 6, 6, 77 or or 88 a-1,4-linked a-1,4-linked glucose glucose units units that that form form water water soluble soluble inclusion inclusion complexes. complexes. In In aa previous previous work work we we demonstrated demonstrated that that ββ-cyclodextrin -cyclodextrin accelerates accelerates degradation degradation kinetics kinetics of of anthracene anthracene and and naphthalene naphthalene in in aqueous aqueous phase. phase. The The aim aim of of this this work work was was to to investigate investigate the the effect effect of of the the temperature temperature and and of of the the addition addition of of different different kind kind of of cyclodextrins cyclodextrins on on the the microbial microbial degradation degradation of of PAHs PAHs in in water water and and soil; soil; phenanthrene phenanthrene as as one one of of the the most most recalcitrant recalcitrant PAHs PAHs was was used used as as aa model model compound, compound, and and aa phenanthrene–degrading phenanthrene–degrading microbial microbial consortium consortium was was selected selected from ββ− from aa natural natural soil. soil. αα−−,, β− and γγ-- cyclodextrin cyclodextrin were were tested tested in in an an aqueous aqueous phase phase at at β−− and 28°C 28°C and and 37°C; 37°C; ββ-cyclodextrin -cyclodextrin in in aa slurry slurry phase phase at at 37°C, 37°C, and and ββ-cyclodextrin -cyclodextrin in in solid solid phase phase at at room room temperature. temperature. The The heterothrophic heterothrophic microbial microbial population population of of microcosms microcosms at at the the end end of of biodegradation biodegradation process process was was examined. examined.

G = Aspergillus terreus

H = Penicillium restrictum

I = Aspergillus sp

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