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Bacterial Volatilization of Mercury by
Immobilization Bacteria in Fixed and Fluidized Bed Bioreactors
Article in Annals of Microbiology · January 2004
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Annals of Microbiology, 54 (4), 353-364 (2004)
Bacterial volatilization of mercury by immobilized bacteria in fixed and fluidized bed bioreactors
F.Z. DZAIRI1, Y. ZEROUAL2,3, A. MOUTAOUAKKIL2, J. TAOUFIK2, M. TALBI1, M. LOUTFI2, K. LEE3, M. BLAGHEN2*
1Laboratory of Analytical Chemistry, Faculty of Sciences Ben M’sik, University Hassan II – Mohammedia, Casablanca, Morocco; 2Laboratory 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, Morocco; 3Laboratory of Enzyme Technology, Chonbuk National
University, Chonju 562-576, Republic of Korea
Abstract -Pseudomonas aeruginosaand Klebsiella pneumoniae, mercury resistant bac- terial strains, which are able to grow at 1200 µM HgCl2and to reduce mercuric ion to volatile metal mercury, were isolated from hydrocarbons-contaminated river in Morocco.
These bacteria were used for removing mercury from synthetic water polluted by mercu- ry using fixed bioreactor and fluidized bed bioreactor. This mercury bio-decontamination system has permitted to obtain cleanup rates bordering on 100% in both of bioreactors.
Key words:mercury, bacteria, immobilization, fixed bed bioreactor, fluidized bed biore- actor.
INTRODUCTION
Industrial use of mercury, a highly toxic metal, has led to significant mercury pol- lution of the environment (Bryan and Lanston, 1992; Zilloux et al., 1993). To prevent the detrimental effects of this heavy metals toxin, many bacterial species have evolved a sophisticated and highly regulated detoxification sys- tem in which mercurials and Hg(II) are actively transported into the intracellular space, where ultimate reduction of Hg(II) to the much less toxic Hg(0) leads to elimination of the toxin from the cell (Silver and Misra, 1988; Walsh et al., 1988;
Silver and Walderhaug, 1992). This detoxification mechanism is governed by synthesis of flavoprotein mercuric ion reductase (Ghosh et al., 1996a; Essa et al., 2002; Zeroual et al., 2003). Bacterial detoxification of organomercurials is governed by another enzyme called organomercurial lyase that cleaves the C- Hg bound and releases Hg(II) in addition to respective organic compound (Be- gley et al., 1986).
* Corresponding Author. E-mail: [email protected].
Recently, an increased application of immobilized cells has occurred be- cause of their numerous advantages compared with free cells: reuse of the same biocatalyst, stability of bioreactors, great purity and control of reactions (Engasser, 1988). Thus, Immobilization of living microorganisms has been de- scribed by several investigators as being useful in biological wastewater treat- ment (Sumino et al., 1985; Sumino et al., 1992, Von Canstein et al., 1999).
On based of the bacterial mercury volatilization mechanism, and by exploit- ing advantages that offer immobilization techniques, we planned to remove mercury from a synthetic mercurial water solution using bacterial strains that had been isolated, identified and appeared to be resistant to high mercury con- centrations, compared to those reported in literature. These bacterial strains have been immobilized by physical absorption on either vermiculite or cooper beech. The mercury volatilization was studied in both fluidized and fixed bed bioreactors. Reduction and volatilizing rates obtained were compared.
MATERIALS AND METHODS
Isolation of mercury resistant bacteria. Mercury contaminated sludge was collected from the river “Oued El Maleh” in Mohammedia receptacle of industri- al wastes water (in particular contaminated by hydrocarbons). One gram of sludge was suspended in 10 ml of sterile sodium chloride solution 0.85% (w/v) and mixed thoroughly. The mixture was serially diluted with sterile sodium chlo- ride solution 0.85% (w/v). Aliquots of 0.1 ml of 10-1, 10-2and 10-3 dilutions were spread onto nutritive agar plates containing 200 µM HgCl2. All plates were in- cubated at 37 °C for 2 days. Colonies were picked and sowed onto nutritive agar plates containing 200 µM HgCl2. The plates were again incubated at 37 °C for 2 days to confirm their resistance to mercury.
Bacterial identification was done by biochemical analysis according to the standardized micromethod API 20E, 20NE, and Staph (Biomerieux), after Gram staining tests and cultures on selective media (Kligler, King A and Chapman).
Yersinia enterocolotica 138A14 used in this study is mercury resistant bac- terial strain of which the mechanism of the resistance to mercury has been studied and reported in the literature (Vidon et al., 1981; Blaghen et al., 1983).
Determination of minimal inhibitory concentrations (MICs).Either the solid media TSA, or liquid media none amended (controls) or amended with the re- spective metal element at different concentrations (75, 150, 300, 600, 1200, and 2400 µM) from stock solutions were inoculated with 100 µl of cell suspen- sions from precultures grown overnight diluted to 1% (v/v). Heavy metals and organomercurials tested were mercury (HgCl2, Panreac), silver (AgNO3, Rec- tapur), cadmium (CdCl2, Rectapur), nickel (NiCl2, Panreac), CMB (4-chloromer- curil-benzoic acid, Riedel de Haene), HMB (4-hydroxymercuril-benzoic acid, Riedel de Haene) and merthiolate (sodium ethylmercurithiosalicylate, Riedel de Haene).
The minimal inhibitory concentration is defined as the lowest concentration that causes no visible growth.
Immobilization of bacterial cells.Five hundred milliliters of sterile nutrient broth (NB), containing 50 µM HgCl2and 25 g of sterile vermiculite (Labon, Canada) or 50 g of pieces of copper beech (length: 0.5 cm, width: 0.2 cm) were inoculated with 5 ml of cell suspensions from precultures of one night of each isolate. After 16 h of incubation at 37 °C under stirring conditions (80 rpm), 20 µM HgCl2was added to the culture medium and growth was stopped 2 h later. The supernatant was removed and the activated supports were washed three times with sterile distilled water.
Mercury volatilization in fixed bed bioreactor.Mercurial solutions tested in fixed bed bioreactor were at 125, 250 and 500 µM of Hg2+. These synthetic mercurial solutions were prepared by dissolving appropriate amount of HgCl2 in distilled water. The bioreactor used was schematized in Fig. 1. It is composed of a glass column (6 cm i.d.) filled with pieces of activated copper beech, through which circulates in a closed system the mercurial solution (1 liter) using a peristaltic pump with a flow rate of 5 ml/min. The mercury vapors are con- veyed by a current air generated on the bottom by compressor distributing 150 l/h of air, and directed towards a mercury trap flask containing 500 ml of an FIG. 1 – Scheme of fixed bed bioreactor.
Peristaltic pump
Oxidizing solution
Actived support (copper beech)
Peristaltic
pump Air
compressor Mercurial
solution
Activated carbon
oxidizing solution containing of 1.5 N nitric acid, 4 N sulfuric acid and 1 ml of potassium permanganate (5%, w/v).
Mercury’s reduction rate was followed according to time in both mercurial and oxidizing solutions. The mercury contents were determined by flameless atomic absorption spectrophotometer M.A.S. 50 (Mercury Analyzer System, Bacharach, USA): ionic mercury was reduced with SnCl2(5 g/l) to metallic mer- cury, which was volatilized by a vector gas (air) and detected at 253.7 nm by the atomic absorption spectrometer.
Mercury volatilization in fluidized bed reactor.Mercurial solutions tested in fixed bed bioreactor were at 125, 250 and 500 µM of Hg2+. These synthetic mercurial solutions were prepared by dissolving appropriate amount of HgCl2 in distilled water. Two Erlenmeyer flasks constitute the fluidized bed bioreactor (Fig. 2): the first one contains the mercurial solution (1 liter) and activated ver- miculite (25 g). The mercury vapors are conveyed by a current air generated on the bottom by compressor distributing 150 l/h of air from mercurial solution Er- lenmeyer flask and directed towards a mercury trap flask containing 500 ml of an oxidizing solution consisting of 1.5 N nitric acid, 4 N sulfuric acid and 1 ml of potassium permanganate (5%, w/v). Mercury’s reduction rate was followed ac- cording time in both mercurial and oxidizing solutions. The mercury contents were determined as described above.
Enumeration of the immobilized bacteria. An enumeration of the bacteria fixed on vermiculite and on cooper beech 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 was observed. Thereafter, different volumes were re- grouped to carry out a bacterial enumeration after spreading on solid medium (TSA) by the technique of dilutions.
FIG. 2 – Scheme of fluidized bed bioreactor.
Air compressor
Mercurial solution + immobilized bacteria
Oxidizing solution
Activated carbon
RESULTS AND DISCUSSION Identification of isolated bacterial strains
The analysis of the samples collected from the river “Oued El Maleh” in Mo- hammedia revealed the presence of 4 bacterial strains able to grow on mercu- rial plates. Three of these bacteria were Gram-negative bacilli and the fourth bacterial strain was Gram-positive coccum. The API identification system re- vealed that the strains were: Pseudomonas aeruginosa,Klebsiella pneumoni- ae, Proteus mirabilisand Staphylococcus sp.
Minimal inhibitory concentrations
Heavy metals and organomercurials minimal inhibitory concentrations (MICs) values registered in liquid culture media are shown in Table 1. The tests ob- tained in solid culture were essentially the same. The MICs in solid medium of Klebsiella pneumoniae and Pseudomonas aeruginosa to HgCl2and AgNO3are 2400 µM, those of Proteus mirabilisto NiCl2are 600 and 300 µM, and those of Staphylococcussp. are 300 and 150 µM respectively. Klebsiella pneumoniae was the most resistant species to all of the metals tested. The results obtained indicate that these strains are potent multiresistant. In fact, each strain shows a resistance at different compounds tested with higher MICs compared to those reported on literature such as Pseudomonas aeruginosa isolated from natural waters which was inhibited at 94 µM Ag+, 235 µM Hg2+, Yersinia enterocolitica 138A14 was inhibited at 480 µM HgCl2 (Blaghen et al., 1983), and Pseudomonas aeruginosaPAO9501 was inhibited at 960 µM HgCl2(Spangler et al., 1973).
Volatilization of mercury in bioreactors
Mercury volatilization was studied in both fluidized bed bioreactor with activated vermiculite and fixed bed bioreactor with cooper beech as support of fixation of bacteria. Figures 3 and 4 show a progressive decrease of the polluting load and this for the different effectuated experiences due to mercury detoxification. In fact mercury can bind with cell surface proteins, highly specific transport of Hg2+
into the cell in the protein-bound form. The bound mercury is delivered to the
TABLE 1 – Minimal inhibitory concentration (µM) of some heavy metals and organo mercurials in liquid culture media
Metal Klesiella Pseudomonas Proteus Staphylococcus
compounds* pneumoniae aeruginosa mirabilis sp.
Mercury 2400 2400 600 300
Silver 2400 2400 300 150
Nickel 600 75 150 75
Cadmium 600 150 150 75
CMB 300 150 150 75
HMB 300 150 150 75
Merthiolate 300 150 150 75
* see Materials and Methods.
cytosolic mercuric reductase in which the substrate-binding site is at the C ter- minus. Here, the Hg(II) is reduced to Hg(0) by electron transfer from FADH2in the active site. The active site of mercuric reductase is similar to that of glu- tathione reductase and lipoamide dehydrogenase, with redox active cysteines.
FIG. 3 – Evolution of mercury concentration according to time in the mercurial solution using fixed bed bioreactor. A: mercurial solution at 125 µM Hg2+; B: mercurial solution at 250 µM Hg2+; C: mercurial solution at 500 µM Hg2+. ◆, Pseudo- monas aeruginosa; ■, Klebsiella pneumoniae; ▲, Yersinia enterocolitica;
✱, abiotic control.
Time (h)
µM Hg2+
Time (h)
µM Hg2+
Time (h)
µM Hg2+
A
B
C
Hg0is volatilized out of the system due to its high vapor pressure (Distefano et al., 1990; Ghosh et al., 1996b).
The evolution of mercury volatilization in fluidized and fixed bed bioreactor according to the time and bacterial strains is represented in the Figs. 5 and 6.
FIG. 4 – Evolution of mercury concentration according to time in the mercurial solution using fluidized bed bioreactor. A: mercurial solution at 125 µM Hg2+; B: mer- curial solution at 250 µM Hg2+; C: mercurial solution at 500 µM Hg2+.
◆, Pseudomonas aeruginosa; ■, Klebsiella pneumoniae; ▲, Yersinia entero- colitica; ✱, abiotic control.
Time (h)
µM Hg2+
Time (h)
µM Hg2+
Time (h)
µM Hg2+
A
B
C
Pseudomonas aeruginosaand Klebsiella pneumoniaecaused a remarkable mercury concentration decrease with mercury removal efficiencies of 96% and 89.5%, respectively (Figs. 3 and 4), whatever tested concentrations and biore- actor used. This is due to their high mercury resistance in opposition toYersinia FIG. 5 – Evolution of mercury concentration according to time in the oxidizing solution using fixed bed bioreactor. A: mercurial solution at 125 µM Hg2+; B: mercurial solution at 250 µM Hg2+; C: mercurial solution at 500 µM Hg2+. ◆, Pseudo- monas aeruginosa; ■, Klebsiella pneumoniae; ▲, Yersinia enterocolitica;
✱, abiotic control.
Time (h)
µM Hg2+
Time (h)
µM Hg2+
Time (h)
µM Hg2+
A
B
C
enterocoliticathat presents a low mercury resistance. Yersinia enterocolitica stops to volatilize mercury at 500 µM Hg2+in mercurial solution (Figs. 3 and 4).
Volatilization rates, which are about 70% for Pseudomonas aeruginosaand 60% for Klebsiella pneumoniaein both of bioreactors (Figs. 5 and 6), confirm FIG. 6 – Evolution of mercury concentration according to time in the oxidizing solution using fluidized bed bioreactor. A: mercurial solution at 125 µM Hg2+; B: mercu- rial solution at 250 µM Hg2+; C: mercurial solution at 500 µM Hg2+.
◆, Pseudomonas aeruginosa; ■, Klebsiella pneumoniae; ▲, Yersinia entero- colitica; ✱, abiotic control.
Time (h)
µM Hg2+
Time (h)
µM Hg2+
Time (h)
µM Hg2+
A
B
C
their competence. The difference recorded between the quantities of mercury eliminated from the mercurial solution and that volatilized and recovered in the oxidizing solution was probably due to retention of the metal volatilized mercu- ry by the bacteria in the bioreactor (Von Canstein et al., 1999) and to a passive mercury fixation on the support of immobilization. In fact, in fixed bed bioreactor, the control experience using non-activated support shows that copper beech fixes 30% of total mercury concentration, which correspond to its mercury sat- uration percentage (Fig. 3), whereas it was only about 6% for the vermiculite used in the fluidized bed bioreactor (Fig. 4).
Reduction rates of each bacterium obtained using fluidized bed are widely higher than those obtained using fixed bed bioreactor (Table 2). For example, Pseudomonas aeruginosa reduction rates are about 0.60, 1.48 and 2.74 µmoles/h/1010 cells in fixed bed bioreactor and 1.60, 2.08 and 3.72 TABLE 2 – Reduction rates related to bioreactor used, expressed as µmoles/h/1010
cells
Bacteria tested Bioreactor Concentrations of Hg2+in inflow (µM) used
125 250 500
Pseudomonas Fixed bed 0.605 1.480 2.740
aeruginosa
Fluidized bed 1.600 2.080 3.720
Klebsiella Fixed bed 0.590 1.508 2.720
pneumoniae
Fluidized bed 1.408 2.080 3.600
Yersinia Fixed bed 0.130 0.320 N.D.
enterocolitica
Fluidized bed 0.350 0.460 N.D.
N.D.: Not determined.
TABLE 3 – Volatilization rates related to bioreactor used, expressed as µmoles/h/1010 cells
Bacteria tested Bioreactor Concentrations of Hg2+in inflow (µM) used
125 250 500
Pseudomonas Fixed bed 0.58 1.28 2.05
aeruginosa
Fluidized bed 1.06 1.60 2.72
Klebsiella Fixed bed 0.50 1.20 1.96
pneumoniae
Fluidized bed 0.96 1.30 2.40
Yersinia Fixed bed 0.12 0.22 0
enterocolitica
Fluidized bed 0.23 0.35 0
µmoles/h/1010cells in fluidized bed bioreactor at 125, 250 and 500 µM Hg2+, respectively. This may be due to the fact that vermiculite fixes the double of bacteria number than copper beech used in fixed bed bioreactor (Table 3) and also a great contact with bacteria and Hg2+in fluidized bed bioreactor (Taoufik, 1998).
The same result was found comparing volatilization rates in different biore- actors for the same bacteria Pseudomonas aeruginosa: 0.58, 1.28 and 2.05 µmoles/h/1010 cells in fixed bed bioreactor and 1.06, 1.60 and 2.72 µmoles/h/1010cells in fluidized bed bioreactor (Table 3).
As shown in Table 4, immobilized cells continuously volatilize mercury after 10 consecutive cycles of 24 h without any loss of bacterial activity and with re- duction rates almost constant.
CONCLUSION
The results obtained show that this technology allows specific transformations exclusively reproductive and high yield, during several days, without notable loss activity. Furthermore, improvement in reactors performances should be ob- tained by optimizing reactors design and operation (example the carrier materi- al used to immobilize the microbial biofilm). Moreover, the performances of mixed community biofilms should be examined.
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).
TABLE 4 – Purifying performances obtained using Klebsiella pneumoniae in cont nuous fluidized bed bioreactor (mercurial solution is at 500 µM Hg2+)
Cycle Hg removal Hg recovery Total Measured Hg
number efficiency (%) reduction rate reduction rate (%) (µmoles/1010cells) (µmoles/1010cells)
1 91 70 3.52 2.80
2 91 69 3.68 2.88
3 90 70 3.60 2.72
4 92 69 3.45 2.67
5 92 68 3.52 2.81
6 91 70 3.56 2.85
7 89 70 3.65 2.90
8 92 70 3.56 2.86
9 92 70 3.58 2.91
10 91 70 3.60 2.90
REFERENCES
Begley T.P., Walts A.E., Walsh C.T. (1986). Bacterial organomercurial lyase: overpro- duction, isolation, and characterization. Biochemistry, 25: 7186-7192.
Blaghen M., Lett M.C., Vidon D.J.M. (1983). Mercuric reductase activity in mercury- re- sistant strain of Yersinia enterocolitica. FEMS Microbiol. Lett., 19: 93-96.
Bryan G.W., Lanston W.J. (1992). Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries. A review.
Environ. Pollut., 76: 89-131.
Distefano M.D., Moore M.J., Walsh C.T. (1990). Active site of mercuric reductase re- sides at the interface and requires cys135 and cys140 from one submit and cys558 and cys559 from the adjacent submit: evidence from in vivoand in vitro heterodimer formation. Biochemistry, 29: 2703-2713.
Engasser J.M. (1988). Réacteurs à enzymes et cellules immobilisées. In: Biotechnolo- gie. Tec et Doc, pp. 468-486.
Essa A.M.M., Macaskie L.E., Brown N.L. (2002). Mechanisms of mercury bioremedia- tion. Biochem. Soc. Trans., 30: 672-674.
Ghosh S., Sadhukhan P.C., Ghosh D.K., Chaudhuri J., Mandal A. (1996a). Volatiliza- tion of mercury by resting mercury-resistant bacterial cells. Bull. Environ. Contam.
Toxicol. 56: 259-64.
Ghosh S., Sadhukan P.C., Chaudhuri J., Ghosh D.K., Madal A. (1996b). Volatilization of mercury by immobilized mercury-resistant bacterial cells. J. Appl. Bacteriol., 81:
104-108.
Silver S., Misra T.K. (1988). Plasmid mediated heavy metal resistances. Ann. Rev. Mi- crobiol., 42: 717-743.
Silver S., Walderhaug M. (1992). Gene relation of plasmid and chromosome deter- mined inorganic ion transport in bacteria. Microbiol. Rev., 56: 195-228.
Spangler W.J., Spigarelli J.L., Rose J.M., Filippin R.S., Miller H.H. (1973). Degradation of methyl mercury by bacteria isolated from environment samples. Appl. Microbi- ol., 25: 448-493.
Sumino T., Kon M., Mori N., Nakajima K. (1985). Development of wastewater treatment technique by immobilized microorganisms. J. Water Waste, 27: 52-57.
Sumino T., Nakamura H., Mori N., Kawaguchi Y., Tada M. (1992). Immobilization of ni- trifying bacteria in porous pellets of urethane gel for removal of ammonium nitro- gen from wastewater. Appl. Microbiol. Biotechnol., 36: 556-560.
Taoufik J. (1998). Epuration biologique des eaux polluées par le mercure et biodégra- dation des hydrocarbures aromatiques en bioréacteurs à lits fixe et fluidisé. Thèse de 3éme cycle, Faculté des Sciences, Université Hassan II - Aïn Chock, Casablanca.
Vidon D.J., Lett M.C., Delmas C.L. (1981). Mercury resistance in Yersinia enterocoliti- ca. Ann. Microbiol. (Paris), 132: 225-230.
Von Canstein H., Li. Y., Timmis K.N., Deckwer W. D., Wagner Döbler I. (1999). Re- moval of mercury from chloralkali electrolysis wastewater by a mercury resistant Pseudomonas pudidastrain. Appl. Environ. Microbiol., 65: 5279-5284.
Walsh C.T., Distefano M.D., Moore M..J., Shewchuk L.M., Verdine G.L. (1988). Molec- ular basis of bacterial resistance to organomercurial and inorganic mercuric salts.
FASEB J., 2: 124-130.
Zeroual Y., Moutaouakkil A., Dzairi F.Z., Talbi M., Chung P.U., Lee K. Blaghen M.
(2003). Purification and characterization of cytosolic mercuric reductase from Klebsiella pneumoniae. Ann. Microbiol., 53: 149-160.
Zilloux E.J., Porcella D.B., Benott J.M. (1993). Mercury cycling and effects in freshwa- ter wetland ecosystems. Environ. Toxicol. Chem., 12: 2245-2264.
364 F.Z. DZAIRI et al.
