Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 103-105, 2005

AcceptedRole of Microorganisms in the Redistribution of Heavy Metals between Soil Phases:
Model Study
Perelomov Leonid,*, a Kandeler Ellen,b and Perelomova Irinaa
aTula State University, Boldin Street, 128, Tula 300600, Russia
University of Hohenheim, Emil Wolff – Str. 27, 70599 Stuttgart, Germanyb

Received: November 15, 2004; In Final Form: April 21, 2005
The effect of soil microorganisms on the redistribution of Zn and Pb compounds between solution and goethite
precipitated as a coating on a sand matrix was studied. Mixed colonies of microorganisms extracted from the soilof an urban park were used to prepare cell and microbial debris suspensions. Chemical states of metal ions retainedon the goethite were determined as either exchangeable (extractable by 1.0 M KNO3) and nonexchangeable(extractable by 0.3 M NH2OH-HCl in 1 M HNO3). Pure goethite without microorganisms sorbed about 99% of
the added Pb and 99.4% of the Zn although 96% of the sorbed Pb and 78% of the sorbed Zn were in the nonexchangeable state. Pb concentrations decreased on the surface of the iron mineral due to living microorganisms.
Sorption of Pb was inversely correlated with the concentration of organic carbon (Corg) in the solution. Microorganisms
clearly influenced the Zn sorption by goethite at concentrations of Corg > 450 mg L-1. The amount of retained Zn
was decreased primarily due to the decrease in its exchangeable fraction. Microbial debris prepared by autoclaving
reduced the Pb sorption in accordance with the results for living cells. Living microorganisms accumulated moreZn than did microbial debris.

1. Introduction
There is a continual influx of radionuclides and heavy metals
into the biosphere from both natural and anthropogenic sources.
Due to their potential mobility in ecosystems and their toxicity
to higher life forms, these elements have been prioritized as
major inorganic contaminants. Microorganisms play an important role in the biogeochemical cycling of radionuclides and heavymetals. Microbial processes such as chelations by metabolites,oxidation-reduction, changes of solution pH, biosorption by functional groups on the cell surface, bioaccumulation, formation of
stable compounds, biodegradation of organic complexes, andbiomethylation may affect the behavior of these toxic elementsin the environment.1 Some of these processes effectively immobilize them, whereas others cause their release or transformation to different species that may be more bioavailable and/ortoxic to organisms. Fundamental information on microbialmetal interactions and their implications for the environment ispresented in References 2 – 4 and others. However, the role ofmicrobes in the redistribution of stable and radioactive traceelements between various phases of soils has largely been
neglected. The object of this study was to determine the effectof soil microorganisms on the redistribution of Zn and Pb
compounds between the solution and the goethite in the modelexperiment.
Soil is a very complicated stochastic natural system. Numerous
ecological factors influence the mineral, organic and biologicalcomponents of such systems, and studying the interactions ofthe components at any specific moment is relatively difficult.
At the present time, this justifies the use of model soil systems
with a limited number of soil components for studying theseinteractions.
2. Material and Methods
Goethite-coated sand was prepared using a modification of*Corresponding author. E-mail: perelomov@rambler.ru. FAX:
+007-0872-331305.

the procedure of Kinniburgh et al.5 according to Stahl and James.6
Prior to oxide preparation, the quartz sand (Carl Roth GmbH&Co,Germany) was acid washed in 1.0 M HNO3 for 24 h and rinsed
in deionized water. Goethite (α-FeOOH) was precipitated on
500 g of sand by adding 87.5 mL of 0.17 M Fe(NO3)3 and 90
mL of 0.52 M NaOH in an evaporating dish. The mixture wasplaced in a drying oven at 105 ˚C for 72 h. The mixture wasstirred periodically to prevent crusting of the salts on the surface.
After this procedure, goethite-coated sand was washed indeionized water to remove the residual iron mineral.
Mixed colonies of microorganisms extracted from the top
horizons of soil from a public park were used to prepare thecell and microbial debris suspension. The properties of the soil
from which microorganisms were cultivated are shown in
Table 1.
TABLE 1: Properties of Urban Park Soil Used for theExtraction of Soil Microorganisms
pHCaCl2Corg,(%)

6.8

5.3

Biomass,
Pb total,µg Ninhydrin
mg kg−1−1
Ng
34.1

313

Zn total,
mg kg−1

Fe total,
mg kg−1

483

45000

The solid medium prepared to cultivate the microorganisms
comprised (in g L−1): tryptone, 5; yeast extract, 2.5; dextrose,
1; and agar, 15. Mixed colonies from urban park soil weregrown at 37 ˚C for 24 h. After growth, 15 mL of cold deionized water was added to a Petri dish with microorganisms inorder to wash colonies from the medium and to prepare thesuspension. Part of the suspension was autoclaved to evaluate
the effect of microbial debris on heavy metal sorption bygoethite. Concentrations of biomass and microbial debris in
the suspensions were determined as concentrations of organic
C using a DIMATOC analyzer.
Five mL of cell suspension of soil microorganisms withconcentrations of living cells of 0, 280, and 450 mg L−1 inorganic carbon, and 5 mL of suspension consisting of microbial debris after autoclaving of 810 mg L−1 in Corg, were added© 2005 The Japan Society of Nuclear and Radiochemical SciencesPublished on Web 07/31/2005

Perelomov

into tubes containing:
1) 1 g of the goethite-coated sand and 15 mL solution ofPb(NO3)2;
2) 1 g of the goethite-coated sand and 15 mL solution ofZn(NO3)2;
3) 1 g of the goethite-coated sand and 15 mL deionized water.
The experimental design is shown in Table 2. The concentrations of Zn and Pb in the solution were each 0.1 mM (6.54 mg L−1
and 20.7 mg L−1, respectively, or 130.8 and 414 mg kg −1 ofcoated sand). Tubes with microorganisms and corresponding
controls (no inoculum) were incubated unshaken at 22 ˚C for 20days. Sorption experiments were carried out in triplicate.

Pb, mg kg-1

104 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005

TABLE 2: Experimental Design
GOETHITE-COATED SAND
ZnPb

C solution = 6.54 mg L−1

C solution = 20.7 mg L−1

Without METALSC sand = 130.8 mg kg−1

C sand = 414 mg kg−1

Cell
Suspension
suspension from microbial
debrisCell
Suspension
suspension from microbial
debrisCell
Suspension
suspension from microbial
debrisOrganic C, mg l-1

Concentration of organic C, mg L−1450810

280450810

280
450810

To determine the loss of metals from solution, Pb and Znconcentrations in the equilibrium solutions were measured.
After the equilibrium, the solid phase was provided for thedesorption experiments after decanting, filtering the metal treatments solution and weighing the tubes to account for occluded
solution. The chemical states of the metals (exchangeable or nonexchangeable) were determined by adding 10 mL of 1.0 M KNO3and agitating for 1 h at 150 cycles min−1. Nonexchangeable Znand Pb were recovered by dissolving the oxide surface after
decanting the KNO3 solution. Goethite was dissolved using 40mL of 0.3 M NH2OH-HCl in 1 M HNO3. This extraction wasperformed in a hot water bath at 40 ˚C for 2 h.6
Microbial biomass was determined by the fumigationextraction method according to Amato and Ladd.7 All heavy
metal analyses were performed by atomic absorption spectrometry.

Figure 1. Influence of microorganisms (280 and 450 mg L −1 oforganic C) and microbial debris (810 mg L−1 of organic C)on theredistribution of Pb compounds between solution and goethite after 20days of incubation (Average values and Standard deviations).
Pb ex: the concentration of exchangeable-state Pb; Pb nonex: theconcentration of nonexchangeable-state Pb; Pb sol: the concentrationof Pb in the solution.

Zn, mg kg-1

280

3. Results and Discussion
The goethite-coated sand without microorganisms or microbial debris sorbed about 99% of the added Pb and 99.4% of theZn (Figure 1) indicating that pure goethite possesses a highzinc and lead sorption capacity. Iron oxides significantly
affect heavy metal reactions in soils because they occur as
coatings, concretions and as discrete particles of colloidal size;
they also have a strong affinity for metal ions.8 The sorption
ability of iron oxides is determined by the degree of crystallinity, the surface area, reaction time and pH.9 The pH ofthe solution changes the speciation of the metals in solution as
well as the nature of the adsorbent. Our study was carried out
at a weakly alkaline pH of the solution, which induced negatively charged goethite surfaces. Stahl and James6 showed that
goethite coatings increase sorption of Zn by fine quartz sand,principally in nonexchangeable form, at pH > 6. Our experiment also demonstrates that the nonexchangeable chemical
state plays a dominant role in the adsorption of both Zn and Pb
on the surface of the iron mineral in all treatments (Figures 1and 2). The amount of Pb retained in the nonexchangeablestate on the surface of the pure goethite was approximately
96.3% (321.4 mg kg−1 sand). The nonexchangeable fraction ofZn was less, averaging 78.4% (95.5 mg kg−1) of the sum absorbedby goethite.
The heavy metal concentrations in our experimental solution

Organic C, mg l-1

Figure 2. Influence of microorganisms (280 and 450 mg L −1 oforganic C) and microbial debris (810 mg L−1 of organic C) on theredistribution of Zn compounds between solution and goethite after 20days of incubation (Average values and Standard deviations).
Zn ex: the concentration of exchangeable-state Zn; Zn nonex: theconcentration of nonexchangeable-state Zn; Zn sol: the concentrationof Zn in the solution.

were no doubt below toxic levels. Mergeay et al.10 reported
minimal inhibitory concentrations for E. coli: Zn –1.0 mM, Pb– 5.0 mM.
Microorganisms had a significant effect on Pb and Zn sorption by goethite coatings from solution. Pb concentrations
increased in the sorption solutions and decreased on thesurface of the iron mineral due to living microorganisms(Figures 1 and 2). The absolute concentrations of the nonexchangeable-state metals were reduced to a greater extent thanthose of the exchangeable state, whereas the relative content of

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005 105

Role of Microorganisims in the Redistribution

both fractions dropped by a factor of approximately two. The
sorption of Pb by goethite is inversely correlated with theconcentration of organic carbon in the solution (Figure 1).
Microorganisms reduced the sorption of Zn only slightly
compared with Pb. They clearly influenced the Zn sorption bygoethite at concentrations of Corg ≥ 450 mg L−1. The amount ofretained metal is decreased primarily due to a decrease in its
concentration in the exchangeable state (Figure 2).
Most probably the main reasons for the retention of metals
by microorganisms are biosorption and bioaccumulation.
Biosorption of toxic metals and radionuclides is based on theuse of ligands or functional groups situated on the outersurface of the cell. The process requires neither an active
membrane transport mechanism nor metabolic energy in orderto function and is controlled in a non-directed physiochemical
reaction.11 Metals interact with reactive amphoteric groups(carboxyl and phosphate) within the constituent polymers ofbacterial cell walls and external sheaths and capsules. Somewall and capsular polymers react with dissolved ions as if they
were an open ion exchange resin, whereas others exhibit more
selectivity with a greater partitioning ability. 1 2 At pHs
between 5 and 8, these polymers are ionized and naturallyanionic.13 Under our experimental conditions the ionized cell
walls or cell debris accumulated high amounts of heavy metals
and were pushed away from goethite coatings with negative
charges, which reduced Pb and Zn sorption by goethite.
Both living and dead biomasses of microorganisms exhibit
biosorption.14 Non-viable or dead biomass can be obtained
through pretreatment of biomass. Physical pretreatment
methods such as heating, autoclaving, freeze-drying andboiling, and chemical pretreatments such as using acidic, alkaline and organic chemicals showed enhancement or reduction
in metal bioadsorption, depending on the strains and treatment
procedures used. 15, 16 In our experiment microbial debris
prepared by autoclaving reduced Pb sorption by goethite inaccordance with the results for living cells of microorganisms.
An opposite reaction was detected for Zn. Living microorganisms accumulated more Zn than did microbial debris. Thereduced bioadsorption capacity of the autoclaved suspension
for Zn versus live cells may be explained by the loss of intracellular uptake.
The principal driving force for metal ion sorption by biotic
and abiotic soil components is their net negative surface
charge. So, along with soil and microbial community composition the intensive properties of soil systems such as pH and
Eh have great importance for the fate of radioactive and stabletrace elements. On the other hand these properties may dependon the physiological activity of soil microorganisms. For asatisfactory understanding and prediction of the behavior oftoxic elements in the environment, further investigation
involving the modeling of soil systems is required.
Acknowledgements. The authors would like to acknowledge the funding and support of the German Academic
Exchange Service (DAAD) and the grant “Universities ofRussia”.
References(1) A. J. Francis, Biotechnology for Waste Management and Site
Restoration, NATO ASI Series, 2/34, eds. C. Ronneau andO. Bitchaeva, Kluwer Academic Publisher, DordrechtBoston-London (1997), p. 247.
(2) M. D. Mullen, D. C Wolf, F. G. Ferris, T. J. Beveridge, C.
A. Lemming, and G. W Bailey, Appl. Environ. Microbiol.
55, 3143 (1989).
(3) N. V. Ashly and D. J. W. Roach, J. Chem. Tech. Biotechnol.
49, 381 (1990).
(4) A. J. Francis, Experientia. 46, 840 (1990).
(5) D. G. Kinniburgh, J. K. Syers, and M. L. Jackson, Soil Sci.
Soc. Amer. Proc. 39, 464 (1975).
(6) R. S. Stahl and B. R. James, Soil Sci. Soc. Amer. J. 55,1287 (1991).
(7) M. Amato and J. N. Ladd, Soil Biol. Biochem. 20, 107 (1988).
(8) K. M. Spark, B. B. Johnson, and J. D. Wells, Europ. J. Soil.
Sci. 46, 621 (1995).
(9) L. M. Shuman, Soil Sci. Soc. Amer. J. 41, 703 (1977).
(10) M. Mergeay, D. Nies, H. G. Schlegel, J. Gerits, P. Charles,and F. van Gijsegem, J. Bacteriol. 162, 328 (1985).
(11) G. M. Gadd, Biotechnology – A Comprehensive Treatise,in 8 Volumes, Volume 6B, eds. H-J. Rehn and G. Reed,
VCH Publishers, Weinheim (1988), p. 401.
(12) T. J. Beveridge and R. G. E. Murray, J. Bacteriol. 127, 1502
(1976).
(13) T. J. Beveridge, Proc. Workshop on Biotechnology for theMining, Metal-Refining, and Fossil Fuel Processing Industries, Symposium N 16 “Biotechnology and Bioengineering”, eds. H. L Ehrlich and D. C. Holmes, J. Wiley
Interscience, New York (1986), p. 127.
(14) D. Brady, A. Stoll, and J. R. Duncan, Environ. Technol. 15,
429 (1994).
(15) A. Kapoor and T. Viraraghavan, Bioresour. Technol. 53,
195 (1995).
(16) Y. Guangyu and T. Viraraghavan, Water SA 26, 119 (2000).