search
for
 About Bioline  All Journals  Testimonials  Membership  News


International Journal of Environment Science and Technology
Center for Environment and Energy Research and Studies (CEERS)
ISSN: 1735-1472 EISSN: 1735-2630
Vol. 1, Num. 3, 2004, pp. 199-204

International Journal of Enviornmental Science and Technology, Vol. 1, No. 3, Autumn 2004, pp.199-204

Biological removal of cadmium by Alcaligenes eutrophus CH34

1*A. H. Mahvi and 2L. Diels

1Department of Environmental Health Engineering, School of Public Health, Center for Environmental Research,
Tehran University of Medical Sciences, Tehran, Iran
2Felemish Institute of Technological Research, V.I.T.O., MOL, Belgium

*Corresponding Author, E-mail: ahmahvi@yahoo.com

Code Number: st04024

Abstract

Some bacteria like the heavy metal resistant Alcaligenes eutrophos CH34 strains are able to promote biomineralization, being the biologically induced crystallization of heavy metals. In the presence of heavy metals, this strain may create an alkaline environment in the periplasmic space and outer cell environment appropriate induction of heavy metals resistance mechanisms. In such an environment metal hydroxides are formed together with metal bicarbonates resulting from the carbonates production by the cell. Also metals bind to out cell membrane proteins and the metal hydroxides and bicarbonates precipitate around these nucleation foci inducing further metal crystallization. A pilot-plant was set up in which Alcaligenes eutrophus CH34 were inoculated and reproduced in a composite membrane, based on polysulfone. The membrane is casted on a polyester support. The biological membrane was in continuous contact with nutrients from inside and the other side was in contact with wastewater flow containing 120-mg/l cadmium. Nutrients are used for growth and reproduction of bacteria and for development of bacteria resistance agents against heavy metals. At the effluent side immobilized bacteria induce metal precipitation and metal crystals. A column, which was in continuous contact with treated effluent, was continuously filled with glass bends to which the metal crystals bind and grow. The efficiency for Cd removal was over 99 percent. Cd removal could be recovered from the recuperation column by acid treatment without damaging the bacteria.

Key words: Alcaligenes eutrophos CH34, cadmium removal, biomineralization, polysulfone membrane, crystallization, industrial wastewater

Introduction

Recuperation of heavy metals can be realized by different techniques in function of their concentration. High concentration (>500 ppm) can be removed by electrolysis and low concentration (<500 ppm) can be removed by biosorption or ion exchange columns. At concentrations between 500 and 5 ppm precipitation with lime is possible generating high volumes of sludge with low metal/sludge ratios (Diels, et al., 1994). Biomineralization is a process in which bacteria actively induce the precipitation foci on their cell surface (Diels, 1990 and Michel et al., 1986). Bacteria, which are resistant to heavy metals and to oxyanions, were the subjects of numerous studies, which relay on various disciplines from microbial ecology to molecular biology. Alcaligenes were found in sediments or soils with a high content of heavy metals (zinc, cobalt, copper and cadmium in Belgium and Zaire). Samples from such places may contain up to 5-10% of these metals and still provide viable bacterial contents.

Most of Alcaligenes eutrophus strains share the following characteristics with: growth at the expense of a variety of organic substrates with the exclusion of sugars, oxidative metabolism, facultative chemolithotrophy, and presence of megaplasmids carrying heavy metals resistance genes (Collard, et al., 1993). The aerobic gramnegative bacterium Alcaligenes eutrophus CH34 contains two large plasmids that confer resistance to several heavy metals (Cooksey, 1993). PMOL28 is resistance to Co2+, Ni2+, CrO42-, Hg2+ and Ti+ and pMOL30 resistance to Cd2+, Co2+, Cu2+, Zn2+, Hg2+ and Ti+ (Diels and Mergeay, 1990). In the plasmid pMOL30, the CZC gene cluster ensures resistance to Cd2+, Co2+ and Zn2+ (Nies, et al., 1989). This gene cluster includes the operon (structural genes), upstream of which a regulatory region is located. When compared with the sequences of corresponding putative proteins, the strongly hydrophilic czcA emerges as a transmembrnae protein, which ensures a cation/protein anitiporter efflux. In association with czcB and czcC, czcA allows the efflux of heavy metal ions. On pMOL28, the structural genes cnrCBA correspond to the structural czc CBS genes. Figure 1 shows the similarity of cnrCBA and czcCBA gene products (Collard, et al., 1994). Figure 2 shows the efflux cation system and how heavy metals enter and exit from cells.

Figure 3 shows the colonization of bacteria in function of time and in Figure 4 bacterial induced formation Cd2+ crystals on membrane was presented (Diels, et al., 1994). The glass bead columns are utilized tp trap and remove the bacterial formed Cd(HCO3)2 and CdCO3 crystals. The growth of Cd(HCO3)2 and CdCO3 on the glass beads was presented in Figure 5.

Materials and Methods

A pilot-plant reactor for biological removing of Cd2+ was set up. A. eutrophus CH34 is immobilized on a polysulfone membrane. A reaction chamber was composed of two stainless steel cylinders mounted on each other and had two inlets and two outlets. A polysulfone membrane covered with the polyester active membrane at the inner surface separated the cylinders.The pore diameters were 1 µm on one side and the other side had a pore diameter of 5 µm. The membrane had a thickness of 150 µm and the thickness of the former pore was 100 µm. After colonization of bacteria on whole side of the polysulfone membrane, the pilot-plant reactor was operated for wastewater treatment. 1 mM CdCl2 was added to produce an effluent with a concentration of about 120 ppm of Cd. The following nutrient mediums were used in this study:

 
β-glycophosphate
294 mg/l
NH4NO3   1000 mg/l
MgSO4.7H2O   200 mg/l
FeSO4.7H2O   10 mg/l
Acetate   8000 mg/l
CaCl2.2H2O   10 mg/l

The effluent enters the reactor; has a contact with polysulfone membrane; and exit the reactor from outlet side. Through the opposite side a nutrient stream to keep the bacteria metabolizing flows into the reactor and without any contact with effluent leaves it. Solutions were pumped at a flow-rate of 15 ml/min for the raw wastewater containing Cd and 30 ml/min for the nutrient. Since each chamber had a known volume, the effluent was discharged at a same flow rate. After leaving the reactor, the effluent to be treated, passes over a column, bearing glass beads, before it recalculates to the effluent flask.

The precipitates of CdCO3 and Cd(OH)2 were trapped on the glass beads. The pilot was set up and run for eight weeks. It took about 2-3 weeks for pilot plant to be stabilized. The samples were taken twice a week. Similarity in results will represent the stabilized conditions.

Figure 6 provides the schematic presentation of the pilot-plant reactor. It was used to treat effluents containing Cd2+ in a continuous way.

The main purpose of this study is to remove cadmium from industrial wastewater by eutrophus CH34 bacteria on polysulfone membrane.

This study has been done in Felemish Institute of Technological Research, V.I.T.O., MOL, Belguim in 1999.

Results

The pilot was operated for a week without addition of wastewater in order to colonize A. eutrophus CH34 on the polysulfone membrane. At first glance, it was not obvious how the efflux cations (i.e. transport and maintenance of cations outside the cytoplasma) as a main mechanism of resistance would lead to a process of bioremediation. In fact, it was observed in cultures of Alcaligenes eutrophus CH34 grown in the presence of Cd2+, which the Cd2+ concentration in the supernatant decreased drastically in late log phase. The Cd2+ concentration in the culture was reduced from 200 ppm to 2 ppm and the Zn2+ concentration from 600 ppm to 10 ppm. This effect was always accompanied by a pH increase (up to 9) and an important precipitation of carbonates and hydroxides (Diels et al., 1993). The precipitation seemed to be initiated around bacterial cells, which under the scanning microscope looked to be coated with crystals, and continued into the culture of the antiporter efflux system, where the bacterial cell accumulated hydrogen ions in exchange for the extrusion of cations (Collard et al., 1994). To avoid any shock, raw wastewater gradually flowed into reactor. After 3 days and being assured that steady–state was provided, samples were taken in function of time and metal concentration measured. Since pH of the solution increased to a level of about 9, it was assured that the crystal of carbonate, bicarbonate and hydroxide of cadmium would occur in alkaline condition (Figure 7). The turbidity tests that were carried out on the wastewater and nutrient solution indicated bacterial growth in the system. Biological removal of Cd(HCO3)2, CdCO3 and Cd(OH)2 on the glass beads that is resulted in the color and shape change of them. Cd2+ content was measured by the atomic adsorption indicated a significant decrease of Cd2+ concentration (0.1 mg/l) in the effluent (Figures 8, 9,10). Cd2+ removal efficiency was calculated about 99 percent for this reactor. Figures 9 and 11 show removal efficiency of Cd2+ in the reactor.

The results of effluent pH, percentage of Cd2+ removal, the concentration of Cd2+ in the effluent or in the nutrient side in Figures 7, 8, 9, 10,11 show that the system was not stabilized at first and after 2-3 weeks it had been stabilized.

Discussion and Conclusion

A through physiological study of this biochemical reactions resulting in bioprecipitation of Cd2+ salt crystals on the beads and the reduction of Cd2+ concentration from the influent wastewater, with maintaining the pH regulation during the expression of heavy metal resistance mechanisms, helps the improvement of bioremediation processes that are the basic functions of these bacteria for metal fixation.

Different potential fields of applicability’s can be emphasized for heavy metal resistively of Alcaligenes eutrophus CH34. The development of bioreactors in which bacteria can be immobilized, could be in the pores of the membrane.

Upon this bacterial growth, the decontamination of heavy metals especially Cd2+ from influent wastewater can be achieved. Chromate, copper, and cobalt. Alcaligenes eutrophus CH34 has the capabilities in being resistance to many metals by mutations as reported for several systems such as mercury, arsenic and antimony, Also, the decontamination of mixed sources of pollution having different concentrations of heavy metals and organic xenobiotics is possible with high efficiencies. Obtaining about 99 percent reduction in Cd2+ concentration of polluted wastewater in this research is vary interesting and therefore can be applied for similar industrial wastewater.

The bioremediation of polluted soils is smother application of Alcaligenes eutrophus CH34.The development of these areas in fullscale commercial projects and applications needs a great multipurpose approach from molecular genetics and physiology biotechnology and biochemical engineering.

Acknowledgements

The Student Scholarships Office of Ministry of Health with cooperation of the Felemish Institute of Technological Research V.I.T.O., Mol, and Belguim generously helped this research during the sabbatical leave period of this first author.

References

  • Collard, J. M., A. Provoost, S. Taghavi, and M. Mergeay, A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system, J. of bacteriology. 779-784, 1993
  • Collard, J. M., P. Corbisier, L. Diels, Q. Dong, C. Jeanthon, M. Mergeay, S. Taghavi, D. van der Lelie, A. Wilmotte and S. Wuertz, Plasmid for heavy metal resistance in Alcaligenes eutrophus CH34: mechanisms and applications, FEMS Microbio. Reviews, 405-414, 1994
  • Cooksey, D. A., Copper uptake and resistance system in bacteria. Mol. Microbiol. 7, 1-5, 1993
  • Diels, L., Accumulation and precipitation of Cd and Zn ions by Alcaligenes eutrophus strains. Biohydrometallurgy Editors J. Salley, R. G. L. McCready, P. Z. Wichlacz, 369-377, 1990
  • Diels, L., and M. Mergeay, DNA probe mediated detection of resistance bacteria from soils with highly polluted by heavy metals. Appl. Environ. Microbiol. 56, 1485-1491, 1990
  • Diels, L., S. Van Roy, S. Taghavi, W. Doyen, R. Leysen, and M. Mergeay, The Use of Alcaligenes eutrophus immobilized in a tubular membrane reactor for heavy metal recuperation. Biohydrometallurgy Meeting, Jackson Hole, 2225, 1993
  • Diels, L., S. Van Roy, M. Mergeay, W. Doyen, S. Taghavi, R. Leysen, Immobilization of bacteria in composite membranes and development of tubular membrane reactors for heavy recuperation. Un megaplasmide de resistance aux metaux lourds, d’alcaligenes eutrophus, 275-293, 1996.
  • Michel L. J., L. E. Macaskie and A. C. R. Dean, Cadmium accumulation by immobilized cell of a citrobacter sp., using various phosphate donors. Biotechnol. Bioeng. XXVII, 1358-1365,1986
  • Nies, D. H., A. Nies, L. Chu and S. Silver, Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus. Proc, Nat., Acad. Sci. USA, 86, 7351-7355, 1989

© 2004 Center for Environment and Energy Research and Studies (CEERS)


The following images related to this document are available:

Photo images

[st04024f9.jpg] [st04024f4.jpg] [st04024f10.jpg] [st04024f7.jpg] [st04024f11.jpg] [st04024f5.jpg] [st04024f6.jpg] [st04024f8.jpg] [st04024f2.jpg] [st04024f1.jpg] [st04024f3.jpg]
Home Faq Resources Email Bioline
© Bioline International, 1989 - 2024, Site last up-dated on 01-Sep-2022.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Google Cloud Platform, GCP, Brazil