a cromului 3,6 pe celule de pseudomonas aeruginosa

8/9/2019 a Cromului 3,6 Pe Celule de Pseudomonas Aeruginosa

1/5

Biochemical Engineering Journal 36 (2007) 5458

Biosorption of Cr(III) and Cr(VI) onto the cell surfaceofPseudomonas aeruginosa

So-Young Kang a, Jong-Un Lee b, Kyoung-Woong Kim a,

a Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST),

Gwangju 500-712, South KoreabDepartment of Civil, Geosystem and Environmental Engineering, Chonnam National University,

Gwangju 500-757, South Korea

Received 6 September 2005; received in revised form 16 May 2006; accepted 12 June 2006

Abstract

Biosorption of the chromium ions Cr(III) and Cr(VI) onto the cell surface of Pseudomonas aeruginosa was investigated. Batch experiments

were conducted with various initial concentrations of chromium ions to obtain the sorption capacity and isotherms. It was found that the sorption

isotherms of P. aeruginosa for Cr(III) were described well by Langmuir isotherm models, while Cr(VI) appeared to fit Freundlich models. The

results of FT-IR analysis suggested that the chromium binding sites on the bacterial cell surface were most likely carboxyl and amine groups. The

bacterial surface ofP. aeruginosa seemed to engage in reductive and adsorptive reactions with respect to Cr(VI) biosorption.

2006 Elsevier B.V. All rights reserved.

Keywords: Biosorption; Pseudomonas aeruginosa; Chromium; FT-IR spectroscopy; Bioremediation; Wastewater treatment

1. Introduction

Toxic heavy metals are frequently contained in wastewaters

produced by many industrial processes, such as those employed

in the electroplating, metal finishing, metallurgical, tannery,

chemical manufacturing, mining, and battery manufacturing

industries [1,2]. The existence of heavy metals in the environ-

ment represents a very significant and long-term environmental

hazard. Even at low concentrations these metals can be toxic to

organisms, including humans. In particular, chromium is a con-

taminant that is a known mutagen, teratogen and carcinogen [3].

Chromium is generally found in electroplating and metal finish-

ing industrial effluents, as well as sewage and wastewater treat-

ment plant discharges [4]. Among the several oxidation states

(di, tri, penta and hexa), trivalent chromium, Cr(III), together

with the hexavalent state, Cr(VI), can be the main forms present

in aquatic environments [5]. Chromate (CrO42) is the prevalent

species of Cr(VI) in natural aqueous environments, and is the

major pollutant from chromium-related industries [6]. Although

Cr(III) is less toxic than Cr(VI), long-term exposure to Cr(III)

Corresponding author. Tel.: +82 62 970 2442; fax: +82 62 970 2434.

E-mail address: [email protected] (K.-W. Kim).

is known to cause allergic skin reactions and cancer [7]. As a

result, the total chromium level in effluent is strictly regulatedin many countries. In the USA, the concentration of chromium

in drinking water has been regulated with a maximum level of

0.1 mg/l for total chromium [8].

The removal of heavy metals from aqueous solutions has

therefore received considerable attention in recent years. How-

ever, the practical application of physicochemical technology

such as chemical precipitation, membrane filtration and ion

exchange is sometimes restricted due to technical or economical

constraints. For example, the ion exchange process is very effec-

tive but requires expensive adsorbent materials [9,10]. The use

of low-costwastematerials as adsorbents of dissolved metal ions

provides economic solutions to this global problem and can be

considered an eco-friendly complementary [11,12]. At present,

emphasis is given to the utilization of biological adsorbents for

the removal and recovery of heavy metal contaminants.

Biomass involving pure microbial strains has shown high

capacities for the selective uptake of metals from dilute metal-

bearing solutions. Several investigations have reported that

Pseudomonas aeruginosa displays efficiency for metal uptake

[1315]. Chang and Hong [16] found that the amount of mer-

cury adsorbed by a P. aeruginosa biomass sample (180 mg Hg/g

dry cells) was higher than that bound to a cation exchange

1369-703X/$ see front matter 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.bej.2006.06.005
mailto:[email protected]://dx.doi.org/10.1016/j.bej.2006.06.005http://dx.doi.org/10.1016/j.bej.2006.06.005mailto:[email protected]


2/5

S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 5458 55

resin (100 mg Hg/g dry resin). Hu et al. [17] reported that P.

aeruginosa strain CSU showed the highest affinity and maximal

capacity for uranium (100 mg U/g dry weight), and that it was

alsocompetitive when compared to commercialcation exchange

resins.

Previous studies on biosorption using microorganisms have

generally focused on the removal of metal ions from aqueous

solutions. However, a few studies were undertaken to interpret

and establish the mechanisms involved in metal ion binding.

Furthermore, the binding sites for chromium have not been

specifically identified.

The objective of the present work was to assess the potential

of P. aeruginosa for the biosorption of chromium. The func-

tional groups involved in chromium biosorption were identified

using FT-IR analysis. These results would contribute to a better

understanding of biosorption phenomena and aid in the devel-

opment of potential biosorbents that possess high capacities for

heavy metal uptake from aqueous environments.

2. Materials and methods

2.1. Preparation of the biomass

P. aeruginosa PAO1 (courtesy of Dr. Beveridge, University

of Guelph, Canada) was used as a biosorbent in these exper-

iments. P. aeruginosa is a bacterium commonly isolated from

various environmental sources such as soil, water and plant sur-

faces [18]. The bacteria were cultured following the procedure

outlined in Kang et al. [19]. In brief, the cells were grown aer-

obically with agitation in a growth medium (TSB; Trypticase

Soy Broth, Difco) at 37 C and set at 180 rpm in a shaking incu-

bator. After reaching the mid-exponential growth phase of cells(OD600nm = 1.0), the cells were harvested by centrifugation at

6000 rpm for 15 min and washed three times with deionized

water. Prepared biomass was used as adsorbent in batch experi-

ments within 1 h under nutrient-absent conditions, and therefore

metabolic processes were minimized or negligible [19,20].

2.2. Surface characterization

To determine the nature of functional groups on the cell

wall for metal sorption, potentiometric titration of an aqueous

cellular suspension and IR spectrum analysis of the solid phase

biomass were performed. For the potentiometric titration, the

biomass was washed three times with deionized water, and thebiomass suspension (3 g dryweight/l) was then potentiometri-

cally titrated with prepared 0.05N NaOH using potentiometric

titrator (Metrohm Ltd., Switzerland). To identify functional

groups using the IR spectrum procedure, the bacteria were

pelleted by centrifugation at 6000 rpm for 15 min and dried

overnight at 50 C in an oven. One milligram of finely crushed

particles of biomass was encapsulated in 300 mg of KBr. The

infrared spectra of the biomass were recorded in KBr disks

using an FT-IR spectrophotometer (Jasco, Japan) from 900

to 4000 cm1 under ambient conditions. Infrared spectra of

P. aeruginosa with or without adsorbed chromium ions were

obtained.

2.3. Biosorption experiments

Thirty-milliliter solutions of chromium were prepared with

Cr(NO3)39H2OandK2Cr2O7 (SigmaAldrich Co.) at different

initial concentrations without adjusting pH. A biomass sample

of 75 mg (dry-weight basis) was added separately into the each

flask and the solutions were then agitated in a shaking incubator

at 180 rpm and 25 C. Five-milliliter samples were obtained and

filtered through a syringe filter (0.2m pore size) after 10 h

(well above the adsorption equilibrium time of about 5 h [19]),

and dissolved metals in the filtrates were determined. Specific

metal uptake by the cells was then calculated from the initial

and final concentrations of chromium ions.

2.4. Analysis of chromium ions

Total concentrations of chromium in the samples were mea-

sured by an inductively coupled plasma atomic emission spec-

trometer (ICP-AES, Thermo Jarrell Ash, USA). The analyses

of Cr(VI) in aqueous samples were performed using a UVvisspectrophotometer (Shimadzu, Japan) at 540 nm after complex-

ation with 1,5-diphenylcarbazide. The analytical method for

chromium is outlined in Standard Methods [21].

3. Results and discussion

3.1. Surface characterization of P. aeruginosa

The bacterial species used in this study was P. aeruginosa,

a gram-negative aerobic species that is commonly found in

near-surface systems [18]. The cell wall of this bacterium is

composed of peptidoglycan and teichoic acids, and possessescarboxyl, phosphoryl, hydroxyl, and amino functional groups at

the surface [22]. Before a study of the biosorption of metal, an

investigation of the surface characteristics ofP. aeruginosa was

required to understand the mechanisms of metal biosorption.

The titration curve of protonated P. aeruginosa shows that the

bacteria provide a substantial buffering capacity that is depen-

dent on the types of functional groups present in the biomass

(Fig. 1). The biomass titration results revealed that there are at

least two main functional groups on the surface of bacteria. To

evaluate the properties of functional groups, we can consider a

chemical model incorporating the reactions between the groups

of bacteria and the protons in the solution. The reactions and

equilibrium constants (Ka, Kb) of certain functional groups maybe defined as follows:

AH A +H+, Ka =[A][H+]

[AH](1)

BH B +H+, Kb =[B][H+]

[BH](2)

where [A],[B] and[AH], [BH] represent theconcentration of

deprotonated and protonated surface species, respectively, and

[H+] represents the activity of protons in solution. We solved

the equilibrium constants of each surface functional group using

the surface equilibrium program FITEQL 4.0 [23]. The negative


3/5

56 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 5458

Fig. 1. Biomass potentiometric titration: P. aeruginosa was washed by distilled

ionized water, and then potentiometrically titrated with 0.05N NaOH. Symbols

are experimental data and the curve is the prediction of the model.

logarithm of the equilibrium constants pKa and pKb were found

to be 5.2 and 9.5, respectively, which correspond to carboxyl

( 4 < pKa < 6) and hydroxyl (9 < pKa < 11) groups [24,25].

An FT-IR analysis was carried out to confirm the type of

functional groups. As shown in Fig. 2, the FT-IR spectrum ofP.

aeruginosa displays a number of absorption peaks, indicating

the complex nature of the biomass examined. The broad absorp-

tion peak around 3430 cm1 is indicative of the existence of

OHand NHstretching, thus showing the presence of hydroxyl

and amine groups on the bacteria. The band at 2938 cm1 can be

assigned to the CH stretch. The absorption bands at 1660 cm1

(mainly C O stretch) and 1551 cm1 (mainly NH stretch) can

be attributed to the amide I and amide II bands of amide bond

due to the protein peptide bond. The moderately strong bands

at 1080 cm1 could be assigned to the CN stretching vibration

of the protein fractions. It was clear that the carboxylate ions

gave rise to two bands: C O stretch at 1468 and 1414 cm1.

A band at about 1246 cm1, representing SO3 stretching, was

observed in the FT-IR spectrum ofP. aeruginosa.

Fig. 2. IR spectrum in solid phase of the lyophilized P. aeruginosa in KBr disk.

Fig. 3. Biosorption isotherms of Cr(III) onto P. aeruginosa. The biomass was

contactedwithmetal solutionfor 10h at 25C and180rpm inshakingincubator.

The line was produced by using the MINEQL+.

3.2. Biosorption isotherms of chromium

Experimental biosorption isotherms of chromium ions were

obtained to evaluate sorption capacity and understandthe pattern

of chromium biosorption by P. aeruginosa.

Sorption experiments of Cr(III) were carried out at a vari-

ety of initial concentrations, and revealed that the amount of

specific Cr(III) uptake increased in response to augmentation

of the initial metal concentration of each metal for the range

05 mmol/l of equilibrium concentration (Fig. 3). However, the

amount of Cr(III) adsorbed onto the cells remained constant

beyond 150mol/l of equilibrium concentration and was unaf-

fected by increases in the initial input metal concentration, with

the sorption profile showing a clear plateau. Sorption isotherms

displaying such a pattern indicated that Cr(III) uptake by P.

aeruginosa cells reached equilibrium, with the mechanisms of

uptake being saturated. The isotherms derived from these exper-

iments fit the Langmuir isotherm model to a reasonable degree.

The linearized forms of Langmuir adsorption isotherms were

used to evaluate the sorption data and are represented as follows

[26]:

1

=

1

max+

1

Kadsmax

1

[A](3)

where is the amount of adsorbed metal ion per mass of

adsorbent (mol/g)and max is the maximum adsorption capac-ity of metal ion (mol/g); A is the equilibrium concentration

of metal ions in solution (mol/l) and Kads the equilibrium

adsorption constant (l/mol). Linear transformation of the plots

in Fig. 3 using the Langmuir model showed that the maxi-

mum amount of Cr(III) adsorbed by P. aeruginosa cells was

136mol/g of dry biomass weight and that Kads was equivalent

to 1.1 106 l/mol.

The biosorption of Cr(VI) ions from aqueous solution by P.

aeruginosa was also studied in a batch system (Fig. 4), with

results showing that biosorption of Cr(VI) by P. aeruginosa

includes two processes. The first process is the reduction of

Cr(VI) to Cr(III) by reductive functional groups according to


4/5

S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 5458 57

Fig. 4. Biosorption isotherms of Cr(VI) onto P. aeruginosa. The biomass was

contactedwithmetal solutionfor 10h at 25C and180rpm inshakingincubator.

The lines were produced by using the MINEQL+.

the following reaction:

Cr2O72+ 16e+14H+ 2Cr3++7H2O (4)

A number of previous experimental studies of bacterial Cr(VI)

reduction reported the enzymatic reduction as a product of

metabolic activity [27,28], and most of these focused on the

requirement of external electron donors for reduction to occur

[29,30]. However, Fein et al. [31] suggested that the Cr(VI)

reduction is not dependent on cell metabolism and that some

component of the cell wall serves as the electron donor for the

reduction reaction. We have also investigated the reduction of

Cr(VI) to Cr(III) in the absence of externally supplied electron

donors.In the second process, chromium ions are removed from

wastewater using the adsorptive functional groups ofP. aerug-

inosa. The adsorptive property is due to the electrostatic inter-

action between the charged surfaces of bacteria and chromium

ions. The experimental sorption isotherms of Cr(VI) are rep-

resented by the Freundlich sorption isotherm in Fig. 4. The

linearized form of Freundlich is represented by the following

equation [26]:

log = log m + n log[A] (5)

where m represents the Freundlich constant and n is the measure

of the nonlinearity involved. Values of m and n were, respec-tively, found to be 80.8 and 1.03 as the total adsorbed chromium

ions; 38.6 and 1.02 as the adsorbed Cr(III) in Cr(VI) biosorp-

tion to P. aeruginosa. The difference of concentration between

total and hexavalent chromium wastaken as the concentration of

trivalent chromium. These results show that the bacterial func-

tionalgroups ofP. aeruginosa can act as reductive and adsorptive

sites in metal biosorption.

3.3. FT-IR spectra of chromium-loaded P. aeruginosa

To confirm the difference between functional groups in rela-

tion to biosorption of Cr(III) and Cr(VI), the FT-IR study was

Fig. 5. FT-IR spectra ofP. aeruginosa prepared in KBr disks: (a) pristine; (b)

Cr(III)-loaded; (c) Cr(VI)-loaded bacteria.

carried out using chromium-loaded P. aeruginosa. The absorp-

tion spectrum of chromium-loaded biomass (at pH 5) was

compared with that of pristine biomass. The chromium-loaded

biomass was washed, dried and powdered after biosorption of

chromium ions under thesameconditions used in thepreparation

of pristine biomass. A change of absorption bands can be seen

when comparing the FT-IR spectra of pristine and chromium-

loaded biomass (Fig. 5).

Fig. 5(b) shows the changes in the spectrum of the biomass

after sorption of Cr(III) by P. aeruginosa. An interesting phe-

nomenon was the sharp decrease in the band intensity at

1414 cm1 corresponding to C O stretching after metal bind-

ing. On the basis of the change of the band, it was reasonable

to assume that the peak value suggested the chelating (biden-

tate) character of the Cr(III) biosorption onto carboxyl groups

[32]. The structure of the metal bound to carboxyl ligands on

the bacteria is likely to take the following form [33]:

In the case of Cr(VI)-loaded bacteria, the spectral analysis

of P. aeruginosa before and after metal binding indicated that

NH is involved in Cr(VI) biosorption (Fig. 5(c)). There is a

substantial decrease in the absorption intensity of NH bands

at 1660 and 1551 cm1. The broad overlapping range for NH

and OH stretching in the range 32003600 cm1 also presents

some changes, but it is difficult to determinethe group that causes

the shift. These amino groups are protonated at pH 3 [34] and

the negatively charged chromate ions become electrostatically

attracted to the positively charged amines of the biomass cell

wall. Similar to Cr(III)-loaded bacteria, the characteristic peak


5/5

58 S.-Y. Kang et al. / Biochemical Engineering Journal 36 (2007) 5458

of C O stretching at 1414cm1 decreased and indicated Cr(III)

binding after reduction of Cr(VI) to Cr(III).

4. Conclusions

This study shows that P. aeruginosa can be applied to

chromium-contaminated wastewater. The sorption of chromium

ions by P. aeruginosa was modeled well by the Langmuir and

Freundlich sorption isotherms. The data of potentiometric titra-

tion indicated thepresenceof twomajor functional groupson the

cell wall,corresponding to pKa values of 5.2and 9.5. FT-IRspec-

trometry showed bindings of chromium ions were dominated by

complexation to the carboxyl and amine groups on the biomass

surface. In the case of Cr(VI) biosorption of P. aeruginosa, the

reduction and adsorption of Cr(VI) occurred coincidently in an

abiotic process. This phenomenonis environmentally significant

because most bacteria in the subsurface exist in nutrient-poor

or -absent conditions under natural conditions [31]. This study

shows the potential for the use of P. aeruginosa for chromium

recoveryin various water and wastewater treatmentapplications,and highlights the efficacy of using biological agents for the

remediation of polluted aqueous environments.

Acknowledgements

This research was supported by the Gwangju Institute of

Science and Technology (GIST) Research Fund and National

Research Laboratory Project (Arsenic Geoenvironment Lab.) to

K.-W. Kim.

References

[1] B.J.Alloway, Heavy Metals in Soils, Kluwer Academic/Plenum Publishers,

New York, 1994.

[2] C. Polprasert, L.R.J. Liyanage,Hazardous waste generation andprocessing,

Resour. Conserv. Recycl. 16 (1996) 213226.

[3] L.W. Chang, Toxicology of Metals, CRC Press, Boca Raton, FL, 1996.

[4] E. Merian, Metals and their Compounds in the Environment. Occurrence,

Analysis and Biological Relevance, VCH, Weinheim, 1991.

[5] Agency for Toxic Substances and Disease Registry (ATSDR), Toxicologi-

cal Profile for Chromium, U.S. Public Health Service, U.S. Department of

Health and Human Services, Atlanta, GA, 1998.

[6] U.S. Environmental Protection Agency, Toxicological Review of Hexava-

lent Chromium, National Center for Environmental Assessment, Office of

Research and Development, Washington, DC, 1998.

[7] U.S. Environmental Protection Agency, Toxicological Review of Triva-

lent Chromium, National Center for Environmental Assessment, Office ofResearch and Development, Washington, DC, 1998.

[8] U.S. Environmental Protection Agency, List of Drinking Water Contam-

inants and MCLs, EPA 816-F-03-016, Office of Water, Washington, DC,

2003.

[9] M. Lehmann, A.I. Zouboulis, K.A. Matis, Removal of metal ions from

dilute aqueous solutions: a comparative study of inorganic sorbent materi-

als, Chemosphere 39 (1999) 881892.

[10] B. Volesky, Detoxification of metal-bearing effluents: biosorption for the

next century, Hydrometallurgy 59 (2001) 203216.

[11] M.D. Mullen, D.C. Wolf, F.G. Ferris, T.J. Beveridge, C.A. Flemming, G.W.

Bailey, Bacterial sorption of heavy metals, Appl. Environ. Microbiol. 55

(1989) 31433149.

[12] B. Volesky, Z.R. Holan, Biosorption of heavy metals, Biotechnol. Prog. 11

(1995) 235250.

[13] G.M. Strandberg, S.E. Shumate II, J.R. Parrott, Microbial cells as biosor-

bents for heavy metals: accumulation of uranium by Saccharomyces sere-

visiae and Pseudomonas aeruginosa, Appl. Environ. Microbiol. 41 (1981)

237245.

[14] A.-C. Texier, Y. Andres, P. Le Cloirec, Selecive biosorption of lanthanide

(La, Eu, Yb) ions by Pseudomonas aeruginosa, Environ. Sci. Technol. 33

(1999) 489495.[15] A.-C. Texier, Y. Andres, M. Illemassene, P. Le Cloirec, Characterization of

lanthanide ions binding sites in the cell wall ofPseudomonas aeruginosa,

Environ. Sci. Technol. 34 (2000) 610615.

[16] J.-S. Chang, J. Hong, Biosorption of mercury by the inactivated cells of

Pseudomonas aeruginosa PU21(Rip64), Biotechnol. Bioeng. 44 (1994)

9991006.

[17] M.Z.-C. Hu, J.M. Norman, B.D. Faison, M.E. Reeves, Biosorption of

uranium by Pseudomonas aeruginosa strain CSU: characterization and

comparison studies, Biotechnol. Bioeng. 51 (1996) 237247.

[18] J.-L. Ramos, Pseudomonas, Kluwer Academic/Plenum Publishers, New

York, 2004.

[19] S.Y. Kang, J.U.Lee, K.W. Kim,Metalremoval fromwastewaterby bacterial

biosorption: kinetics and competition studies, Environ. Technol. 26 (2005)

615624.

[20] S.Y. Kang, J.U. Lee, K.W. Kim, A study of the biosorption characteristicsof Co2+ in wastewater using Pseudomonas aeruginosa, Key Eng. Mater.

277279 (2005) 418423.

[21] A.D. Eaton, L.S. Clesceri, A.E. Greenberg, Standard Methods for

the Examination of Water and Wastewater, 17th ed., American Pub-

lic Health Association (APHA), American Water Works Association

(AWWA), Water Pollution Control Federation (WPCF), Washington, DC,

2000.

[22] T.J. Beveridge, Role of cellular design in bacterial metal accumulation and

mineralization, Annu. Rev. Microbiol. 43 (1989) 147171.

[23] A. Herbelin, J. Westall, FITEQL, A computational program for determina-

tionof chemicalequilibriumconstants fromexperimentaldata, Version 4.0,

Report 99-01, Department of Chemistry, Oregon St. University, Corvallis,

OR, USA, 1999.

[24] J.B. Fein, C.J. Daughney, N. Yee, T.A. Davis, A chemical equilibrium

model for metal adsorption onto bacterial surfaces, Geochim. Cosmochim.

Acta 61 (1997) 33193328.

[25] C.J. Daughney, J.B. Fein, N. Yee, A comparison of the thermodynamics

of metal adsorption onto two common bacteria, Chem. Geol. 144 (1998)

161176.

[26] W. Stumm, J.J.Morgan, Aquatic Chemistry: ChemicalEquilibria andRates

in Natural Waters, Wiley/Interscience, New York, 1996.

[27] P.C. DeLeo, H.L. Ehrlich, Reduction of hexavalent chromium by Pseu-

domonasfluorescens LB300 in batch andcontinuouscultures, Appl. Micro-

biol. Biotechnol. 40 (1994) 756759.

[28] L. Philip, L. Iyengar, C. Venkobachar, Cr(VI) reduction by Bacillus coag-

ulans isolated from contaminated soils, J. Environ. Eng. 124 (1998)

11651170.

[29] L.H. Bopp, H.L. Ehrlich, Chromate resistance and reduction in Pseu-

domonas fluorescens strain LB 300, Arch. Microbiol. 150 (1988) 426

431.

[30] H. Shen, Y.-T. Wang, Biological reduction of chromium by E. coli, J. Env-

iron. Eng. 120 (1994) 560572.

[31] J.B. Fein, D.A. Fowle, J. Cahill, K. Kemner, M. Boyanov, B. Bunker, Non-

metabolic reduction of Cr(VI) by bacterial surfaces under nutrient-absent

conditions, Geomicobiol. J. 19 (2002) 369382.

[32] N. Yee, J.B.Fein, Cd adsorption ontobacterialsurfaces: a universal adsorp-

tion edge? Geochim. Cosmochim. Acta 65 (2001) 20372042.

[33] M.M. Figueira, B. Volesky, H.J. Mathieu, Instrumental analysis study of

iron species biosorption by Sargassum biomass, Environ. Sci. Technol. 33

(1999) 18401846.

[34] B. Volesky, Sorption and Biosorption, BV Sorbex, Inc., Canada, 2003.

a cromului 3,6 pe celule de pseudomonas aeruginosa

Documents