Aromatic hydrocarbons removal by immobilized bacteria (Pseudomonas sp.; Staphylococcus sp.) in fluidized bed bioreactor

(1)

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/241641402

Aromatic hydrocarbons removal by

immobilized bacteria (Pseudomonas sp.,

Staphylococcus sp.) in fluidized bed bioreactor

Article in Annals of Microbiology · January 2004

CITATIONS

5

READS

110 11 authors, including:

Some of the authors of this publication are also working on these related projects:

Dans quelle mesure une approche de l’enseignement /apprentissage par le serious games design serait- elle pédagogiquement plus efficace que les approches traditionnelles ? Cas de l’enseignement des sciences expérimentales View project

The engineering andragogical in the device “blended learning”: Case of the academic formation courses professionalizing View project

J. Taoufik

Université Mohammed V Souissi (UM5S)

80PUBLICATIONS

223CITATIONS

SEE PROFILE

Adnane Moutaouakkil

National Center for Energy Sciences and Nuc…

16PUBLICATIONS

322CITATIONS

SEE PROFILE

Mohammed Talbi

Université Hassan II de Casablanca

193PUBLICATIONS

722CITATIONS

SEE PROFILE

Mohamed Blaghen

Université Hassan II de Casablanca

64PUBLICATIONS

817CITATIONS

SEE PROFILE

All content following this page was uploaded by Mohammed Talbi on 01 June 2014.

(2)

Annals of Microbiology, 54 (2), 189-200 (2004)

Aromatic hydrocarbons removal by immobilized bacteria (Pseudomonas sp., Staphylococcus sp.)

in fluidized bed bioreactor

J. TAOUFIK¹, Y. ZEROUAL¹, A. MOUTAOUAKKIL¹, S. MOUSSAID¹, F.Z. DZAIRI², M. TALBI², A. HAMMOUMI¹, K. BELGHMI¹, K. LEE³,

M. LOUTFI¹, M. BLAGHEN^1*

1Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences Aïn Chock, University Hassan II, Aïn Chock, km 8 Route d’El Jadida, B.P. 5366 Mâarif,

Casablanca, Marocco; ²Laboratory of Analytical Chemistry, Faculty of Sciences Ben M’sik, University Hassan II, Mohammedia, Casablanca, Morocco;

3Laboratory of Enzyme Technology, Chonbuk National University, Chonju 561-576, Korea

Abstract– Pseudomonassp. and Staphylococcussp. isolated from hydrocarbons- con- taminated river in Morocco appeared to be able to use benzene, toluene, and naphthalene as sole source of carbon and energy. These bacterial strains were immobilized in vermiculite and used for removing hydrocarbons from synthetic water polluted by benzene, toluene or naphthalene by using a fluidized bed bioreactor. Immobilized bacterial cells of Pseudomonassp. and of Staphylococcussp. could completely eliminate the tested hydrocarbons. Moreover, the efficiency of treatment was much greater than with the native cells.

Key words:aromatic hydrocarbons, bacteria, cell-immobilization, fluidized bed bioreactor.

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) include a group of organic priority pollutants of critical environmental and public health concern due to the following characteristics: (1) chronic health effects (carcinogenicity); (2) microbial recal- citrance; (3) high bioaccumulation potential; and (4) low removal efficiencies in traditional treatment processes (Herbes et al., 1976; Stapleton et al., 1998).

The microbial degradation of these PAHs is an important way for their elimination especially when it is compared to the other physicochemical treatments (Cernigla, 1984; Kuritz and Wolk, 1995; Coates et al., 2002; Pruden et al.,

* Corresponding Author. E-mail: [email protected]

(3)

2003). Several algae, bacteria and fungi species appeared to be able to degrade PAHs (Trzesicka-Mlynarz and Ward, 1995; Whyte et al., 1997; Dojka et al., 1998; Macnaughton et al., 1999). This degradation was achieved by a complex series of biochemical reactions, which varies according to the implied substrates and micro-organisms (Field et al., 1992; Lovanh et al., 2002).

Immobilization of living microorganisms has been described by several investigators (Chibata, 1983; Hyde et al., 1991; Zeroual et al., 2001) as being useful in biological wastewater treatment. Immobilization of bacteria, yeast cells, and fungi has been done in a variety of ways. Conventional immobilization methods are generally classified into three categories: covalent binding, physical adsorption and entrapment processes (Monsan, 1982). It is widely known that immobilized cells offer many advantages: reusability of the same biocatalyst, control of reactions, and the noncontamination of products (Engasser, 1988).

On the basis of the bacterial degradation of PAHS, and exploiting advantages that offer immobilization’s techniques, we have planned to remove hydrocarbons from a synthetic water using a bacterial strain that has been isolated, identified. This bacterial strain has been immobilized by physical absorption on vermiculite and used for removal some aromatic hydrocarbons like benzene, toluene and naphthalene in fluidized bed bioreactor.

MATERIAL AND METHODS

Biological material. Water samples collected aseptically from the river “Oued El Maleh” in Mohammedia receptacle of industrial wastes water (in particular contaminated by hydrocarbons), were diluted in a sterile solution of sodium chloride 0.85% (w/v).

Each dilution (0.1 ml) was spread on plates of agarized minimum medium composed by: MgSO₄, 0.1 g, KH₂PO₄, 1.36 g, (NH₄)₂SO₄, 0.6 g, CaCl₂, 0.02 g, MnSO₄, 1.1 mg, CuSO₄, 0.2 mg, ZnSO₄, 0.2 mg, FeSO₄, 0.14 mg, NaCl, 0.5 g, agar, 15 g, H₂O, 1 liter. Naphthalene, 30 mg/l, was added as sole carbon source. All plates were then incubated at 37 °C for 24 h. A pure culture was obtained by successive isolation of colonies in the same medium.

Bacterial identification was done by biochemical analysis according to the standardized micromethod: API 20 E, 20 NE and Staph (Biomerieux), after car- rying out Gram coloration test, test of mobility, catalase test, coagulase test, oxydase test, and cultures on selective media (Kligler, King A, Chapman).

Bacterial growth in the presence of hydrocarbons as sole source of car- bon: benzene, toluene and naphthalene. Test tubes, containing 5 ml of liquid minimum medium in the presence of an aromatic hydrocarbon (2.5 mM benzene, 2 mM toluene or 2 mM naphthalene) were inoculated with isolated strains Pseudomonassp., Klebsiella pneumoniae, Proteus mirabilisand Staphylococ- cussp. precultivated overnight at 37 °C in a liquid minimum medium containing 2% of glucose. The tubes were then incubated for 24 h at 37 °C. These precultures were inoculated (1/100) in Erlenmeyer flasks containing each one the minimum medium and a hydrocarbon at the above-mentioned concentration.

The Erlenmeyer flasks were placed at 37 °C under stirring (100 rpm). The bac-

(4)

terial growth was followed by the measurement of the optical density at 600 nm during one week using a spectrophotometer (Cecil 300, England).

Immobilization of bacterial cells on vermiculite. One liter of nutrient broth containing 125 g of sterile vermiculite (Labon, Canada) was inoculated with 5 ml of precultures of each isolated strains and incubated overnight at 37 °C under stirring (80 rpm); the supernatant was removed and the activated vermiculite was washed three times with sterile distilled water.

Elimination of hydrocarbons in fluidized bed bioreactor using immobi- lized and free cells. The fluidized bed bioreactor is composed of a cylindrical Plexiglas flask (30 cm i.d.) having a capacity of 15 liters (Fig. 1). The bioreactor was equipped with a stirrer system (Bicasa BE 5/2000, Italy) regulated at 60 rpm. In the bioreactor was suspended activated vermiculite (125 g) in 5 l of minimum medium enriched by vitamins [A (2500 U.I.), D2 (750 U.I.), B1 (4 mg), B2 (4 mg), B6 (0.5 mg), B12 (1.5 mg), B5 (6 mg), PP (25 mg), C (60 mg), E (5 mg), B9 (1 mg)] and hydrocarbon (benzene 2.5 mM, toluene 2 mM or naphthalene 2 mM). At the end of every 12 h and during 30 days, aliquots of 1 ml were picked from the bioreactor and centrifuged at 15000 rpm. The hydrocarbon concentration in the supernatant was straightly determined by spectrofluorometer (Jasco, Japan). The excitation wavelengths were 254 nm whereas the emission wavelengths were 402.5, 282.5, 347.5 nm for benzene, toluene and naphthalene respectively. All experiments were done in triplicate and the average val- ues obtained are represented in the figures. The elimination of aromatic hydrocarbons in a fluidized bed bioreactor was also studied by using simultaneously Pseudomonas sp. and Staphyloccocus sp. (isolated strains having given the best epurifying performance) under the same conditions. To evaluate the hydrocarbons fixation capacity on the support, an experiment was carried out under the same conditions except that the vermiculite was not activated (absence of bacteria). Also, the elimination of tested hydrocarbons was studied using free suspended bacterial cells.

Enumeration of the immobilized bacteria. An enumeration of the bacteria fixed on vermiculite was carried out at the beginning and the end of each experiment. One g of activated support was vortexed in 1 ml of sterile distilled water.

The supernatant was recovered and the support added to 1 ml of sterile distilled water. This operation was repeated five times, until no more bacteria release FIG. 1 – Scheme of the used fluidized bed bioreactor.

Activated vermiculite Plexiglass flask

Stirrer system (60 rpm)

(5)

was observed. Thereafter different volumes were regrouped to carry out a bacterial enumeration after spreading on solid medium (TSA) by the technique of dilutions.

Elimination of hydrocarbons on a semi-industrial scale using immobilized bacterial cells.The biodegradation of benzene and toluene was carried out on a semi-industrial scale. These tests were carried out in a fluidized bed bioreactor similar to that described above but of a great capacity (250 liters). The quan- tity of activated vermiculite used was 2500 g suspended in 100 liters of minimum medium enriched by vitamins [A (50000 U.I.), D2 (15000 U.I.), B1 (80 mg), B2 (80 mg), B6 (10 mg), B12 (30 mg), B5 (6 mg), PP (500 mg), C (1200 mg), E (100 mg), B9 (20 mg)] and benzene 2.5 mM or toluene 2 mM. The isolated strain tested was Staphyloccocus sp.

RESULTS AND DISCUSSION

The analysis of the samples collected from the river “Oued El Maleh” in Moham- media revealed the presence of 4 bacterial strains able to grow in the presence of naphthalene as sole source of carbon. These bacteria were subjected to Gram stain, three of them were Gram negative bacilli identified as Pseudomonas sp.,Klebsiella pneumoniaeand Proteus mirabilisand the fourth bacterial strain was Gram positive cocci identified asStaphylococcus sp. Isola- tion of Staphylococcus sp.strain able to degrade PAHs constitutes an interest observation because a few organisms of this genus were associated to the depollution.

Bacterial growth in the presence of hydrocarbon as sole carbon source Bacterial growth in the presence of hydrocarbons as sole source of carbon represented in figure 2 shows the same general profile, but with a latency times and growth rates varying according to the bacterial strain and the hydrocarbon tested. In the presence of benzene and toluene (Fig. 2a and 2b), Staphylococ- cus sp. and Pseudomonas sp. present a latency time of approximately 18 h whereas in the case of Proteus mirabilisand Klebsiella pneumoniaethe latency time was about 30 h. The longest latency times were recorded in the case of naphthalene (Fig. 2c); they were 30 h for Staphylococcus sp. and Pseudomonas sp. and 48 h for the other strains. The comparison of the time growth rates (Table 1), in the presence of various hydrocarbons tested, revealed that the bacteria metabolize faster benzene and toluene than naph- TABLE 1 – Growth rate, expressed in h^-1, in the presence of hydrocarbon as sole carbon

source

Tested Pseudomonas Staphylococcus Klebsiella Proteus

hydrocarbons sp. sp. pneumoniae mirabilis

Benzene 0.018 0.018 0.017 0.012

Toluene 0.019 0.018 0.012 0.017

Naphtalene 0.012 0.015 0.008 0.012

(6)

FIG. 2 – Bacterial growth in the presence of hydrocarbons as sole carbon source.

–––– Pseudomonassp., ––O–– Klebsiella pneumoniae, –––– Proteus mirabilis, ––x–– Staphylococcussp. A: benzene 2.5 mM; B: toluene 2 mM;

C: naphtalene 4 mM.

C B A

Time (h)

O.D. at 600 nm

Time (h)

O.D. at 600 nm

Time (h)

O.D. at 600 nm

thalene. This result is in favor to the assumption that the polycyclic aromatic hydrocarbons are more resistant to microbial degradation than monoaromatic (Sepic et al., 1997).

Elimination of hydrocarbons in a fluidized bed bioreactor

Staphylococcus sp. and Pseudomonas sp., which showed a great ability to grow in the presence of the tested hydrocarbon, were selected to study the

(7)

FIG. 3 – Hydrocarbon residuals rate in the fluidized bed bioreactor.–––– Without bacteria, ––O–– Pseudomonas sp., –––– Staphylococcus sp., ––––

Pseudomonassp. and Staphylococcussp.

Time (days)

nM Naphtalene

Time (days)

nM Toluene

Time (days)

nM Benzene

(8)

elimination of benzene, toluene and naphthalene in fluidized bed bioreactor.

The evolution of the tested hydrocarbons concentration according to time in the fluidized bed bioreactor was represented in figure 3. The use of these two bacterial strains allowed the elimination of the polluting loads with yields around the 100% (Fig. 4). The recorded elimination speeds vary according to the used bacterial strain and tested hydrocarbon (Table 2). Similar results with elimination yields around the 100% were obtained in the case of toluene and benzene biodegradation using Pseudomonas sp. However, Kelley et al.(1990) reported that Mycobacterium sp. a strain isolated from a sediment contaminated by hydrocarbons and used for a biodegradation of naphthalene leads only to the elimination of 55%. When Pseudomonas sp. and Staphylococcus sp. were combined, the expected synergy effect in the biodegradation was not observed.

Indeed the analysis occurring in the medium showed the presence of only Pseudomonas sp., which suggests that Staphylococcus sp. was depleted under the followed conditions or inhibited by the presence of Pseudomonas sp. This phenomenon is probably due to the fact that these two bacterial strains belong- ing to two different classes. Indeed Kuritz and Wolk (1995) reported that Pseudomonas aeruginosaand Pseudomonas putida were able to degrade flu- oranthene with 5mg/l in perfect synergy with an elimination yield of 90% at the end of 10 days. In absence of bacteria the concentrations of benzene and FIG. 4 – Elimination percentages of hydrocarbons in the fluidized bed bioreactor.

Without bacteria, Pseudomonassp., Staphylococcussp., Pseudomonas sp. and Staphylococcussp.

TABLE 2 – Elimination speed of hydrocarbons in the fluidized bed bioreactor expressed in mM/d/10⁹bacterial cells for Pseudomonassp. and Staphylococcussp.

Tested Pseudomonassp. Staphylococcussp. Pseudomonassp. and

hydrocarbons Staphylococcussp.

Benzene 0.0120 0.0123 0.0090

Toluene 0.0081 0.0077 0.0110

Naphtalene 0.0170 0.0140 0.0150

Benzene Toluene Naphtalene

Elimination percentages (%)

(9)

toluene decrease approximately to 70% and 53% respectively, this would be mainly due to their adsorption and their volatility accentuated by the stirring which allows the homogenization of the reactional medium.

Elimination of hydrocarbons by free bacteria

The evolution of the concentration of hydrocarbons tested according to time by using free by suspended bacteria was represented in figure 5. The elimination rates of hydrocarbon recorded under these conditions (Fig. 6) were almost identical to those obtained with bacteria used in a fluidized bed bioreactor.

Pseudomonas sp. for example has elimination rates of 93.6%, 88% and 94%

for benzene, toluene and naphthalene and approximately 94% respectively for three hydrocarbons by using bacteria fixed on vermiculite.

In absence and in the presence of non-activated vermiculite, the elimination rates of benzene are respectively 70% and 50%. This result suggests that the vermiculite fixed approximately 20% of benzene. The same manner we noted that the vermiculite fixed approximately 14% of toluene and 18% of naphthalene. Previous studies using pieces of wood as support for fixation of bacteria for aromatic hydrocarbons biodegradation revealed that wood fixed the totality of the hydrocarbons (Taoufik, 1998). Dwyer et al.,obtained very weak decom- position rates of aromatic hydrocarbons by using bacteria entrapped in gel.

Elimination speeds of aromatic hydrocarbons obtained with free bacteria (Table 3) are clearly lower than those obtained with immobilized bacteria. For example, those of Pseudomonas sp. fixed are of 12 µM/d/10⁹bacteria for benzene, 8.1 µM/d/10⁹bacteria for toluene and 17 µM/d/10⁹bacteria for naphthalene. These speeds, decreased when free cells were used: 1.1, 0.73 and 1.5 µM/d/10⁹bacteria of hydrocarbons respectively. These results show clearly the interest of the immobilization of the bacteria in such a process. The immobilization of the bacteria stabilizes the activity of the bioreactor and conserves the strains allowing the design of continuous processes for treatment of waste water (Hamilton et al., 1985; Engasser, 1988). Indeed using immobilized bacterial strains in a fluidized bed bioreactor offers a great stability of the bacterial activity for more than 10 cycles of traitement without loss of activity.

Elimination of hydrocarbons on a semi-industrial scale

In order to confirm the effectiveness of these bacterial strains in the treatment of water contaminated by aromatic hydrocarbons, tests of hydrocarbons biodegradation were carried out on a semi-industrial scale and which consisted in treat- ing volumes of about 100 liters of water contaminated by benzene and toluene in a fluidized bed bioreactor. The passage on a semi-industrial scale in a fluidized bed bioreactor decreases the incubation time necessary to eliminate the whole of hydrocarbons at 4 days. At the semi-industrial scale the elimination rates were around 100% for the two tested hydrocarbons (Fig. 7) while those obtained without bacteria were 40% for toluene and 70% for benzene (data not shown). Elimination speeds obtained are 0.034 mM/d/10⁹ bacteria and 0.02 mM/d/10⁹bacteria respectively for benzene and toluene (Table 4). These speeds are about 3 times higher than those obtained with a bioreactor on the laboratory scale.

(10)

FIG. 5 – Hydrocarbon residual rates using free suspended bacterial cells. –––– With- out bacteria, ––O–– Pseudomonassp., –––– Staphylococcussp.

Time (days)

nM Naphtalene

Time (days)

nM Toluene

Time (days)

nM Benzene

(11)

CONCLUSION

Pseudomonas sp. andStaphylococcus sp. isolated from the river “Oued El Maleh” in Mohammedia, contaminated by hydrocarbons, appeared to be able to grow in the presence of benzene, toluene and naphthalene as sole source of carbon with very high elimination rates. The epurifying performance of these strains in a fluidized bed bioreactor testifies their competence in the field of biotreatment of water contaminated by hydrocarbon. The use of these bacteria TABLE 3 – Elimination speed of hydrocarbons recorded with free suspended bacterial cells expressed in mM/d/10⁹bacterial cells for Pseudomonassp. and Staphy- loccussp.

Tested

hydrocarbons Pseudomonassp. Staphylococcussp.

Benzene 0.00110 0.00100

Toluene 0.00073 0.00070

Naphtalene 0.00150 0.00150

TABLE 4 – Elimination speed of hydrocarbons in the fluidized bed bioreactor. Semi-indu- trial scale expressed in mM/10⁹bacterial cells

Tested hydrocarbons Elimination speed

(mM/10⁹bacterial cells)

Benzene 0.034

Toluene 0.020

FIG. 6 – Elimination percentages of hydrocarbons obtained using free suspended bacterial cells. OWithout bacteria, OPseudomonassp., Staphylococcussp.

Benzene Toluene Naphtalene

Elimination percentages (%)

(12)

in a fluidized bed bioreactor could be an interesting method for the decontami- nation of the effluents polluted by aromatic hydrocarbons.

Acknowledgements

This work was supported by the Moroccan CNRST and forms part of the post- doctoral project of Dr. Youssef Zeroual (Chonbuk National University, Chonju, Republic of Korea).

REFERENCES

Cernigla C.E. (1984). Microbial metabolism of polycyclic aromatic hydrocarbons. Adv.

Appl. Microbiol., 30: 31-71.

Chibata I. (1983). In: Allaender A.H., Rogers P., Eds, Basic Biology of New Develop- ments in Biotechnology. Plenum Press, New York, pp. 465-496.

Coates J.D., Chakraborty R., McInerney M.J. (2002). Anaerobic benzene biodegradation - a new era. Res. Microbiol., 153: 621-628.

Dojka M.A., Hugenholtz P., Maack S.K., Pace N.R. (1998). Microbial diversity in a hydrocarbon and chlorinated solvent contaminated aquifer undergoing intrinsic bioremediation. Appl. Microbiol. Biotechnol., 64: 3869-3877.

Dwyer D.F., Krmne M.L., Boyd S.A., Tiedji J.M. (1986). Kinetics of phenol biodegradation by an immobilized methanogenic consortium. Appl. Environ. Microbiol., 52:

345-351.

Engasser J.M. (1988). Reacteurs à enzymes et cellules immobilisées. Biotechnologie.

Tec et Doc, pp. 468-468.

Field J.A., De Jong E., Feijoo C.G., De Bont J.A. (1992). Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white fungi. Appl. Environ. Microbiol., 58: 2219-2226.

Hamilton B.K., Hsiao H.Y., Swamn W.E. (1985). Manifacture of L-amino acids with bioreactors. Biotechnol., 3: 64-68.

FIG. 7 – Hydrocarbon residual rates in fluidized bed bioreactor in semi-industrial scale.

–––– Benzene, ––O–– Toluene.

Time (days)

Hydrocarbons concentration mM

(13)

Herbes S.E., Southworth G.R., Gehra C.W. (1976). Organic contamination in aqueous coal conversion effluents: environmental consequences and research priorities.

In: Hemphill D.D., Ed., Trace Substances in the Environment.

Hyde F.W., Hunt G.R., Errede L.A. (1991). Immobilization of bacteria and Saccha- romyces cerevisiaein poly (tetrafluoroethylene) membranes. Appl. Environ.

Microbiol., 57: 219-222.

Kelley I., Freeman J.P., Cerniglia C.E. (1990). Identification of metabolites from degradation of naphthalene by Mycobacterium sp. Biodegradation, 1: 283-290.

Kuritz T., Wolk C.P. (1995). Use of filamentous cyanobacteria for biodegradation of organic pollutants. Appl. Environ. Microbiol., 61: 234-238.

Leahy J.G., Colwell R.R. (1990). Microbial degradation of hydrocarbons in the environment. Microbiol. Rev., 14: 305-315.

Lovanh N., Hunt C.S., Alvarez P.J. (2002). Effect of ethanol on BTEX biodegradation kinetics: aerobic continuous culture experiments. Water Res., 36: 3739-3746.

Macnaughton S.J., Stephen J.R., Venosa A.D., White D.C. (1999). Microbial popula- tion changes during bioremediation of an experimental oil spill. Appl. Environ.

Microbiol., 65: 3566-3574.

Monsan P. (1982). Les enzymes production et utilisations industrielles. In: Durand G., Monsan P., Eds, Gauthier-villars, pp. 81-118.

Pruden A., Sedran M., Suidan M., Venosa A. (2003). Biodegradation of MTBE and BTEX in an aerobic fluidized bed reactor. Water Sci. Technol., 47: 123-128.

Sepic E., Bricelj M., Leskovsek H. (1997). Biodegradation studies of polyaromatic hydrocarbons in aqueous media. J. Appl. Microbiol., 83: 561-568.

Stapleton R.D., Savage D.C. Sayler G.S., Stacey G. (1998). Biodegradation of aromatic hydrocarbons in extremely acidic environment. Appl. Environ. Microbiol., 64: 4180-4184.

Taoufik J. (1998). Epuration biologique des eaux polluées par le mercure et biodégra- dation des hydrocarbures aromatiques en bioreacteurs à lits fixe et fluidisé. Thèse de 3éme cycle, Faculté des Sciences, Université Hassan II, Aïn Chock, Casablan- ca.

Trzesicka-Mlynarz D., Ward O.P. (1995). Degradation of polycyclic aromatic hydrocarbons by a mixed culture and its component pure cultures, obtained from PAH-contaminated soil. Can. J. Microbiol., 41: 470-476.

Whyte L.G., Bourbonnière L., Greer C.W. (1997). Biodegradation of petroleum hydrocarbons by psychrotrophic Pseudomonas strains possessing both alkane (alk) and naphthalène (nah) catabolic pathways. Appl. Environ. Microbiol., 63: 3719- 3723.

Zeroual Y., Moutaouakkil A., Blaghen M. (2001). Volatilization of mercury by immobilized bacteria (Klebsiella pneumoniae) in different supports by using fluidized bed bioreactor. Curr. Microbiol., 43: 322-327.

200 J. TAOUFIK et al.