Use of heavy metal resistant bacteria in a bioreactor concept (bacteria metal sludge reactor) to remove bioavailable heavy metals from polluted soils.

D. van der Lelie^*, P. Corbisier, S. Taghavi, M. Mergeay, M. De Smet, L. Hooyberghs, L. Kinnaer, L. Diels (Vlaamse Instelling voor Technologisch Onderzoek-VITO, Environmental Technology, B-2400 Mol, Belgium, Tel: +32-14-335166, Fax: +32-14-580523, e-mail: vdlelied@vito.be); N. Spelmans, J. Vangronsveld (Limburgs Universitair Centrum-LUC, Center for Environmental Sciences, B-3590 Diepenbeek, Belgium); G. Brox (Tekno Associates, Salt Lake City, USA)

ABSTRACT

A patented system (Diels et al., 1992), called Bio Metal Sludge Reactor (BMSR) consists of a continuous stirred tank reactor that is fed with contaminated soil to which water and nutrients are added. During the start-up period Ralstonia metallidurans CH34 was added to the slurry, which allowed the contaminating metals to be transferred from the soil particles to the bacterial cell walls. The bacterium has special properties, which makes that the settling of the bacteria goes only very slowly compared to the soil organic and clay particles. This allows the separation between the soil and the metal loaded bacteria in a settling device. After treatment, the Cd concentration is reduced from 5 mg/kg dry soil to concentrations below 1 mg Cd/kg soil. The measurement of bioavailability of the heavy metals gives a reduction of a factor 10.

Introduction

Historical emissions of old non-ferrous factories in the neighbourhood of the VITO in the Kempen lead to large geographical areas (>100 km² with Cd concentrations higher than 3 mg/kg soil) of contaminated sites. The only possible remediation methods are based on concentration and subsequent removal. Besides physico-chemical techniques, biotechnology offers some interesting possibilities. The solubilization of metals from soil, sludge or solid waste can be done via autotrophic or heterotrophic leaching, the use of metallophores or by chemical leaching followed by microbial treatments. Metals displaced in this way into the water phase or metals already available in wastewater can be desolubilized via biological induced adsorption, precipitation and transformation or complexation processes. In this case study, the heavy metal resistance, bioprecipitation capacity and improved soil flocculation characteristics of Ralstonia metallidurans CH34 lead to the development of a bioremediation method for heavy metal contaminated soils.

Methods

Bacterial strains

Heavy metal resistant bacteria were obtained by selection on Tris-minimal medium (Schlegel et al., 1961) with one of the following added metals: 0.8 mM Cd or Cu, 2.0 mM Zn, Ni or Co. The Alcaligenes eutrophus strain CH34, now called Ralstonia metallidurans, was used as starter culture for the BMSR reactor. This strain is considered as a model system for bacterial interactions with heavy metals in the environment (Taghavi et al., 1997). An interesting characteristic of strain CH34 is a removal of Cd or Zn ions from the solution during the late log phase and the stationary phase (Diels, 1990). This accumulation and precipitation is correlated with the concentration and kind of carbon source, with the progressive alkalinization of the medium and appears to be associated with the outer cell membrane. In this way a crystallisation process is induced on the cells and this will lead to a very high metal to biomass ratio (between 0.5 and 5.0).

Soil

Slightly contaminated sandy garden soils from the Kempen region in the north of Belgium were used. The Cd concentrations were in the range between 5 and 20 mg/kg. The soil was sieved to remove coarse materials. As it was observed that as well the fine as the coarse fraction contained high metal concentrations the soil was passed in an attrition cell before BMST treatment.

2.3. Soil slurry reactor

The characteristics and the operation of the BMSR soil slurry reactor were recently described by Diels et al (1999). The BMSR concept is presented in Fig. 1.

Fig. 1. General overview of the BMSR concept.

Results

Heavy metals bioextraction

The very coarse fraction (larger than 2 mm) of the soil contained very high concentrations of metals, especially of Cd. This fraction was removed as waste material. The fractions smaller than 2 mm were pumped into the 450BLA slurry reactor. The input concentration and output concentrations are presented in table 1. The concentration in the reactor (MKSR) increased due to the attrition and the increase in availability from 3.5 mg Cd/l to 5.0 mg Cd/l. The output metal concentration was measured after the settling of the soil and the removal of the water, containing the bacteria loaded with heavy metals. After a few repetitions (MKS06) of the treatment the metals concentration went down to 63 mg Zn/kg, 0.5 mg Cd/kg, 47 mg Pb/kg and 4 mg Cu/kg (Table 1). The Cd concentration was reduced from 5.0 mg Cd/kg to 0.5 mg Cd/kg dry soil, which is a 10 times reduction and below the flemish soil remediation standard of 1.0 mg Cd/kg dry soil.

Table 1. Metals concentration in the input and output of the BMSR reactor

Sample	Zn	Cd	Pb	Cu
MKSLF	471	8.0	384	66
MKSI	278	3.5	240	29
MKSR	344	5.0	215	32
MKSO2	194	2.8	167	21
MKSO4	128	1.5	103	12
MKSO5	100	1.0	67	7
MKSO6	63	0.5	47	4

Metal concentrations are expressed in mg metal/kg dry soil. MKSLF: fraction > 2 mm; MKSI: input soil (< 2 mm); MKSR: soil in the reactor; MKSO: output soil.

Water and biosludge

After settling of the soil the water with the metal loaded biosludge was removed and pumped to a decantation tank. Table 2 presents the heavy metal content before and after flocculation for the process water of MKS06. The flocculation could reduce the metal concentration with a factor 10. The heavy metals concentration in the biosludge is presented in table 3.

Table. 2. Heavy metals concentrations in the process water before and after flocculation.

Heavy metal	Before flocculation	After flocculation
Zn	3.7	0.3
Cd	0.06	0.00
Pb	3.5	0.2
Cu	0.4	0.03

Heavy metals concentrations are presented in mg metal /l process water.

Table 3. Heavy metals content in the biosludge

Sludge	Zn	Cd	Pb	Cu
MKB5	4590	80	2840	421
MKB7	4390	78	3150	427
MKB8	4560	86	3560	438

Heavy metals concentrations are presented in mg metal/kg dry sludge.

Heavy metals bioavailability

Heavy metals bioavailability was measured with the BIOMET-biosensor (Corbisier et al., 1996; 1999). The availability is expressed as a signal to noise ratio. An S/N below 2 is recognised as no bioavailability of heavy metals. The total soil had a S/N of 36.1. The fraction > 2 mm had a S/N of 50.1 and the fraction < 2mm (fraction added to the reactor) had a S/N of 25.4. The treated soil had a S/N between 0.2 and 2.9 which means a reduction of at least 10 times compared to the input value and a reduction to nearly no presence of bioavailable Cd or Zn. The indication of bioavailability by the BIOMET sensor was also supported by plant and earthworm tests (results not shown). In some cases shortly after the start of the treatment process an increase in the bioavailable metal fraction is observed. This is due to the shearing forces and attrition that makes the bound metals more available. Afterwards the metals are transferred to the bacteria and in that way removed from the soil.

Conclusions

The BMSR approach shows a soft soil treatment technology in a way that the soil treated with bacteria at neutral pH can be reused. The treatment of heavy metals contaminated soil by Ralstonia metallidurans CH34 allows the contaminating metals to be transferred from the soil particles to the bacterial cell walls. The bacterium has special properties, which makes that the settling of the bacteria goes only very slowly compared to the soil organic and clay particles. This allows the separation between the soil and the metal loaded bacteria in a settling device. Once the metal loaded bacteria are separated from the soil the bacteria can easily be removed from the water suspension by either flotation or a flocculation process. A highly contaminated biomass is obtained from the treatment process. The biomass contains between 8 and 25 g Zn/kg, 3-5 g Pb/kg and 160-250 mg Cd/kg. This biomass can be incinerated in a pyrometallurgical treatment. A highly metal loaded ash will be obtained. The metal oxides can be leached for reuse or the ash can be dumped in a landfill.

The presented method can be used for the removal of metals from sandy soils. Removal of metals from clay soils is not successful or worthwhile. Concentrations of Cd up to 70 mg Cd/kg soil can be removed to below 3 mg Cd/kg. Simultaneously the metals Zn, Cu and Pb are removed and mostly brought to below normal standards. The process water contained only very low heavy metals concentrations (< 0.1 mg Cd/l) and could be recycled in the system.

Acknowledgements

This project was supported by the European Fund for Regional Development (EFRO) and by the Public Authorities of Flanders for Waste (OVAM).

References

Corbisier, P., Thiry, E., Diels, L. (1996) Bacterial biosensors for the toxicity assessment of solid wastes. Environmental Toxicology and Water Quality 11, 171-177.

Corbisier, P., D. van der Lelie, B. Borremans, A. Provoost, V. de Lorenzo, N. L. Brown, J. R. Lloyd, J. L. Hobman, E. Csöregi, G. Johansson, and B. Mattiasson (1999). Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Analytica Chem. Acta. 387: 235-244.

Diels, L. (1990) Accumulation and precipitation of Cd and Zn ions by Alcaligenes eutrophus strains. in Biohydrometallurgy, (Salley J., McCready R.G.L., Wichlacz P.Z., editors), (CANMET publisher), 369-377.

Diels, L., Carpels, M., Geuzens, P., Mergeay, M., Rymen T. (1992) Method and device for cleaning soil polluted by at least one heavy metal. European Patent 92203049.9.

Diels, L. , De Smet, M., Hooyberhgs, L., Corbisier, P. (1999) Heavy metal bioremediation of soil. In Molecular biotechnology, Vol.12: 149-158

Schlegel, H.G., Kaltwasser, H., Gottschalk, G. (1961) Ein Sumbersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstum physiologische Untersuchungen. Arch. Mikrobiol. 38, 205-222.

Taghavi S., Mergeay M., Nies D. and van der Lelie D. (1997). Alcaligenes eutrophus as a model system for bacterial interactions with heavy metals in the environment., Res.Microbiol., 148, 536-551.