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Biosorption of toxic metals from aqueous solutions by bacteria strains isolated from metal-polluted soils

A.I. Zouboulis

^∗

, M.X. Loukidou, K.A. Matis

Division of Chemical Technology, School of Chemistry, Aristotle University of Thessaloniki, P.O. Box 116, Thessaloniki GR 541 24, Greece Received 18 November 2002; received in revised form 24 April 2003; accepted 19 May 2003

Abstract

The use of biological materials for effective removal and recovery of heavy metals from contaminated wastewaters has emerged as a potential alternative method to conventional treatment techniques. The aim of this paper was the laboratory study of biosorption of toxic metals from aqueous solution by the application of microorganisms (Bacillus laterosporus or Bacillus licheniformis), isolated from polluted (metal-laden) soil. Microorganisms have a high surface area-to-volume ratio, because of their small size and therefore, they can provide a large contact interface, which would interact with metals from the surrounding environment. Microbial metal accumulation has received much attention during recent years, due to the potential use of microorganisms for treatment of metal-polluted water or wastewater streams. Two toxic metals were selected as typical examples: a cation (cadmium) and an oxyanion (hexavalent chromium, and promising results were obtained, under optimized conditions.

Keywords: Biosorption; Cadmium; Chromate; Bacterial strains; Metal-polluted soils

1. Introduction

The increase of industrial activities has intensified environmental pollution problems and the deterioration of several ecosystems with the accumulation of many pollutants, such as toxic metals. Heavy metal pollution usually derives from electroplating, plastics manufacturing, fertilizers, pigments, mining and metallurgical processes. Growing attention is being given to health hazards presented by the ex- istence of heavy metals in the environment; their accumulation in living tissues throughout the food chain, poses a serious health problem. The imposition of stricter regula- tions increases the demand for innovate treatment technologies to remove metals from wastewaters and to attain to- day’s toxicity-driven concentration limits. When selecting the metals of interest in order to examine their removal or recovery options by the application of appropriate technologies, the following considerations are mainly taking under account: (a) environmental risk which could be based on a number of factors (technological uses, value) (b) the reserve

∗Corresponding author. Fax:+30-31099-7759.

E-mail address: [email protected] (A.I. Zouboulis).

depletion rate, which is used as an indicator of a probable future increase in the market price of metals[1]. However, the combination of these factors could change the priority sequence among the metals consideration. The cases of cadmium and chromium are considered as toxic metals of high priority, due to the aforementioned considerations.

Cadmium is widely used in rechargeable nickel–cadmium batteries, pigments, stabilizers, coatings, alloys and specific compounds for electronics, such as cadmium telluride (CdTe). The major categories of products, where cadmium may be present as an impurity, are non-ferrous metals (zinc, lead and copper), iron and steel, fossil fuels (coal, oil, gas, peat or wood), cement, and phosphate fertilizers [2]. The relevant EU Directive, as well as the US EPA, have set the maximum contaminant level (MCL) for Cd(II) cations in domestic water supplies to be 5␮g l⁻¹[3].

The wastewaters of dyes and pigments production, film and photography, galvanometry and metal cleaning, plat- ing and electroplating, leather production and mining oper- ations, may contain undesirable amounts of chromium(VI) anions, according to the respective water standards[4]. Due to severe toxicity of Cr(VI), the EU Directive and US EPA have set the maximum contaminant concentration level for Cr(VI) in domestic water supplies to be 50␮g l⁻¹[3].

doi:10.1016/S0032-9592(03)00200-0

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Table 1

Conventional metal removal technologies from wastewaters

Method Disadvantages Advantages

Chemical precipitation (a) difficult separation simple

(b) disposal of resulting toxic sludge relatively cheap (c) not very effective

Electrochemical treatment (a) applicable for high metal concentrations (b) sensitive to specific conditions, such as the presence of certain interfering compounds

metal recovery

Reverse osmosis (a) application of high pressures pure effluent/permeate (available for recycling) (b) membrane scaling/fouling

(c) expensive

Ion exchange (a) sensitive to the presence of particles effective

(b) expensive resins possible metal recovery

Adsorption (a) not very effective for certain metals conventional sorbents

Conventional technologies for removing metals from wastewaters are listed inTable 1. However, the application of these treatment processes is sometimes restricted, due to technological or economical constrains. The search for novel technologies has recently been directed to the application of bioremediation, which constitutes an attractive alternative to common by-applied physico-chemical reme- diation methods[5]. Biosorption involves a combination of active and passive transport mechanisms, starting with the diffusion of metal ions to the surface of microbial biomass.

Metal accumulative bioprocesses are generally divided into two broad categories:

a) biosorptive (passive) uptake by using non-living biomass, and

b) bioaccumulation by applying living cells[6].

Metal ion uptake by biosorption may involve the con- tribution of diffusion, adsorption, chelation, complexation, coordination or micro-precipitation mechanisms, depending on the specific substrate (biomass) [7]. A relevant enter- prise dealing with the application of biosorption has two main aspects, according to Volesky[7], such as the introduction of a new family of products (biosorbents) and the services involved. There are two types of pilot plants that could be used in relevant applications: a biomass process- ing pilot plant or a biosorption one. During the early 1990s, AlgaSORB^TM (non-living biomass, immobilized in silica gel polymer) and AMT-Bioclaim^TM(hard granular biomass derived from B. subtilis), were developed and commercial- ized[6]. The application of flotation for the subsequent separation of metal-laden biomass, when applied in suspension, was also suggested[8].

This aspect of pollutant immobilization on polluted soil particles was also examined, instead of conventional clean up, which appears to be a process less technically and eco-

nomically developed. This method is suited to agricultural soils, contaminated by toxic metals; it was proposed to in- oculate soils with viable microorganisms, which can accumulate considerable concentration of metals [9]. A major interest has been also shown for the application of biotopes, dominated by the confrontation between bacteria and heavy metals, such as mining sites or waste sites of non-ferrous industries[10].

The biosorption of hexavalent chromium was recently reported by the application of dead biomass of Aeromonas caviae, isolated from raw water wells [11]. On the other hand, the recovery of cadmium using Gram-positive bacteria such as actinomycetes have been previously investigated in a relevant E.U. project; it was found that some isolates from metal-contaminated soil did not show advantages in terms of binding capacity[12]. Nevertheless, certain strains exhibited much higher affinity for Cd, than the performance of commercial Streptomyces clavuligerus biomass. There- fore, it was considered that supplementary research in this field would be useful. The objective of the present study was to isolate and identify several strains with particular tolerance toward heavy metals from polluted soils of mining or industrial activities. This research was part of a large na- tional collaborative research programme, focused on three inter-influenced, subsequently applied scientific investiga- tions, based upon: microbiological, biochemical engineering and chemical technology aspects. Selected experimental results from the chemical technology group are presented.

2. Materials and methods

Polluted soil samples were initially collected from two appropriate areas: the mixed sulphides mining district of Stratoni-Chalkidiki (North Greece) and the industrial area of Sindos, nearby Thessaloniki. The isolation and identifica-

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tion of different consortia of microorganisms was then conducted at the Laboratory of Microbiology and Food Hygiene (Department of Agriculture, AUTh). Following preliminary screening tests for the removal of Cd(II) or Cr(VI) ions from synthetic aqueous solutions, selected isolated consortia of microorganisms were grown, under optimum conditions; the latter was carried out at the Laboratory of Organic Chem- istry (Chemical Engineering Department, AUTh).

All examined strains were grown in Luria Bertani broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl), tris-minimal medium at 28^◦C and pH 7, using a rotating shaker. The production of bacteria was monitored spectrophotometrically, by measuring the absorbance at 600 nm.

The biomass was separated by centrifugation at 4500 rpm for 10 min and washed several times, using a solution of NaCl (0.9%). After thermal treatment (autoclaving, 121^◦C), the resulting biomass (sterilized) was stored in the form of slurry at 4^◦C and used for the biosorption/bioaccumulation experiments at the Laboratory of General and Inorganic Chemical Technology (Department of Chemistry, AUTh).

Biomass was employed as a suspension (of 50 ml volume) in lab experiments, in order to investigate and to optimize the potential use of biosorption for the removal of toxic metals from synthetic wastewaters. Batch biosorption experiments were carried out with 1 g l⁻¹biomass, at ambient temperature (25±2^◦C), using a rotary shaker (at 140 rpm).

An aqueous solution of metals of known concentration (prepared from K2Cr2O7 or Cd(NO3)2·4H2O salt, respectively) was added to a biomass suspension, which was dispersed using a hand homogeniser (Jencons, with 45 ␮m clearance) in the appropriate aqueous volume, to produce the required concentration. pH was adjusted by the addition of HNO₃ or NaOH dilute solutions, whereas ccontact time was 120 min. The residual concentration of Cd(II) in solution was analyzed after the separation of used biosorbent by centrifugation, by atomic absorption spectrophotom- etry (AAS, Perkin–Elmer, model 2360), whereas chromium was chemically analyzed in solution using UV–visible spec- trophotometry (HITACHI UV-200), applying a colorimetric spectrophotometer method at 540 nm and using diphenyl- carbazide[13].

The surface tension measurements were carried out with an interfacial tensiometer (Krüss, type K-8), applying the ring method. Zeta potential values were measured with a Zeta-Meter (Rank Brothers, Mark II). Contact angle measurements to evaluate the hydrophobicity of this system, were conducted using a specific apparatus (Krüss, type G1).

3. Results and discussion

Soil is a dynamic, multi-component system, whose properties are continually modified by microbial, chemical, hy- drological and geological processes. The interaction between microorganism and metal ions, which are present in soil, is considered to be the result of metal competition by all the

other components in the system[14]. Some metals are es- sential to microorganisms and therefore required by them, whereas others are toxic, even in small quantities.

3.1. Isolation of bacterial strains from polluted soils and biomass production

Life in a polluted environment challenges the microorganisms in many ways, which is reflected in the fact of a greater demand for energy in order to cope with the toxicity of pollutants. The ability to grow even at high metal concentrations is found in many microorganisms and may be the result of intrinsic or induced mechanisms, as well as of other environmental factors (pH, speciation, redox etc.), which can reduce toxicity. Tolerance may be defined as the ability to cope with metal toxicity by means of intrinsic properties of the microorganism, while resistance is the ability of microorganisms to survive in higher concentrations of toxic metals by detoxification mechanisms, activated in direct response to the presence of metals[14].

The aim of the preliminary part of this work was to se- lect and identify the appropriate strains, which demonstrate greater resistance towards heavy metal toxicity. It was observed that Gram-positive were dominant accounting for 70% of isolated microbial populations. Yeasts and fungi were not isolated in significant numbers. Fungal cells have a relatively larger size with lower density, as well as lower mechanical strength and rigidity than bacterial cells [15].

Therefore, heavy metals even in small concentrations can induce morphological changes and more easily destroy fungal cells. Sorption of metals to the surface of cells is likely to play a critical role in all microbe–metal interactions[14].

The distribution and diversity of microbes inhabiting contaminated soil sites and of genes that code for phenotypes, facilitating metal–microbe interactions, are critical elements for metal bioremediation. The relevant microorganisms are ubiquitous in the environment and their frequency is often increased in contaminated soils. Both natural and engi- neered microbes have been demonstrated to show increased potential as bioremedial tools [16]. The main objective of the second part of this project (biochemical engineering) was the evaluation of screening tests for the selection of metal-resistant microorganisms, as well as the biomass detailed production in sufficient quantities for subsequent biosorption experiments. Bacillus laterosporus and Bacillus licheniformis, which were used in this study, were among the dominant strains of isolated and identified bacteria, showing good preliminary metal removal properties. Biosorption, the passive sequestration of metals by interactions with live or dead biological materials, is used as a practical and rather widely used approach for the bioremediation of soils, contaminated with metals or radionuclides[16].

Nature has created certain defence mechanisms against the contamination of the environment. The presence of microorganisms, such as bacteria, fungi and algae, which have developed resistance mechanisms for toxic metals, being

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also capable for degradation of many man-made xenobiotic organic compounds, opens up the possibility for use of these microorganisms in biological treatment processes, applied to effluents or sites contaminated with a wide range of chemi- cals[6].

The growth pattern of selected bacteria can be characterized by a lag phase, which occurs at the beginning of inoculation, where very little growth occurs as the bacteria are becoming ‘acclimatized’ to the new environmental conditions. This is followed by a rapid growth rate, where the cells are begin to grow and finally, a stationary phase, when the growth rate equals the death rate. The growth of Gram-positive bacteria reach the stationary phase at approximately 24 h, often longer than Gram-negatives, which may need only 12 h.

3.2. Biosorption studies

It is relatively simple and easy to obtain laboratory equilibrium sorption data for a certain biomass. A small amount of each examined biomass was brought into contact with aqueous solution, containing the respective metal. However, the specific conditions of the sorption system (particularly the pH value) have to be carefully controlled at preset values over the entire period of contact, until the sorption equilibrium is reached. During preliminary screening tests, Gram-positive bacteria demonstrated better biosorption removal capacity for Cd(II) in the range of (30–99%), than for Cr(VI) around (20–40%). Each of the examined metal ions, i.e. cationic Cd(II) or anionic Cr(VI) was separately biosorbed by B. licheniformis or B. laterosporus, i.e. by the dominant isolated strains from the polluted soil samples. To obtain the metal adsorption isotherm, biomass cells were ex- posed to various initial concentrations of toxic metal ions.

An adsorption isotherm is characterized by specific constants, the values of which express the surface properties and affinity of sorbent towards the studied metal and can be also used to compare the biosorptive capacity of different biomasses for different toxic metals. The Langmuir type isotherm was adopted and found suitable, similarly with other relevant cases of literature[2]. This model is based on the assumption that maximum adsorption occurs, when a saturated monolayer of solute molecules is present on the adsorbent surface, the energy of adsorption is constant and that there is no migration of adsorbate molecules in the surface plane.

3.2.1. Removal of Cd(II) by biosorption

The isotherms regarding Cd(II) adsorption at constant pH are presented inFigs. 1 and 2. Metal ion adsorption on both non-specific and specific sorbents is pH dependent, as the pH affects the availability of metal ions in solution (speciation), as well as the metal binding sites onto cell surface.

The optimum pH for Cd(II) adsorption was found during preliminary experiments to be 7, i.e. below the pH value of 9, at which cadmium precipitates as insoluble hydrox-

Fig. 1. Comparison of biosorption capacity for cadmium ions using non-living or living cells of B. licheniformis; the Langmuir isotherm was applied: qe=(qmaxbCe)/(1+bCe), where qmax and b are constants and qethe specific metal uptake at equilibrium (mg g⁻¹).

Fig. 2. Comparison of biosorption capacity for cadmium using non-living or living cells of B. laterosporus; Langmuir isotherm at pH 7.

ide onto the cell surfaces. Zeta-potential values of Bacillus biomasses were determined at various pH values of deion- ized water, as shown in Fig. 3 and it was found that they were approximately the same. As the pH was lowered, the

Fig. 3. Zeta potential measurements of non-living cells of B. laterosporus and of B. licheniformis.

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Table 2

Selected literature survey; comparison of different biosorbents, regarding Cd(II) or Cr(VI) removal capacity

Biosorbent qmax (mg l⁻¹) Reference

Cr(VI) Saccharomyces cerevisiae 3 [21]

Zooglea ramigera 3 [21]

Rhizopus arrhizus 4.5 [21]

Rhizopus arrhizus 8.8 [22]

Chlorella vulgaris 3.5 [21]

B. licheniformis 62 this paper

B. laterosporus 72.6 this paper

Cd(II) Rhizopus arrhizus 26.8 [23]

Gram negative bacteria 13.5 [24]

Arthrobacter viscosus 1.4 [17]

Zooglea ramigera 26 [25]

B. licheniformis 142.7 this paper

B. laterosporus 159.5 this paper

overall surface charge of cell surface will become positive, whereas at higher pH values the overall surface charge will become negative, resulting in an increase of cationic Cd(II) biosorption.

The best biosorption capacity was observed for non-living cells of both Bacillus strains. The maximum removal capacity (qmax) of cadmium, using non-living cells of B. licheni- formis, was 142.7 mg g⁻¹, while the respective qmaxvalue, using non-living cells of B. laterosporus, was 159.5 mg g⁻¹. These values appear to be significantly higher in comparison with relevant uptake values of cadmium(II), when applying other biosorbents (literature data,Table 2).

The equilibrium uptake of cadmium(II) ions onto B.

licheniformis biomass was not significantly influenced by temperature (Fig. 4).Table 3displays the results of fitting the Langmuir model to experimental data. The temperature of the adsorption medium could be important for energy dependent mechanisms in metal biosorption. Energy-independent

Fig. 4. Effect of temperature on the biosorption of Cd(II) ions by of B.

licheniformis; conditions: contact time: 120 min, volume: 50 ml, shaking speed: 140 rpm, pH value: 7.

Table 3

Influence of temperature on Langmuir model regression constants, using non-living cell of B. licheniformis

Temperature Cd(II) Cr(VI)

qmax b r² qmax b r²

25^◦C 142.73 0.03 0.99 62.02 0.02 0.99

37^◦C 140.41 0.04 0.98 63.98 0.03 0.99

50^◦C 138.45 0.04 0.98 70.98 0.03 0.99

mechanisms are less likely to be affected by temperature, since the processes responsible for biosorption in this case seems to be largely physico-chemical (electrostatic forces) in nature[17].

3.2.2. Removal of Cr(VI) by biosorption

The isotherm adsorption of B. licheniformis and B. lat- erosporus regarding the sorption of Cr(VI) anions are presented in Figs. 5 and 6, respectively, at the optimum pH value of 2.5, as it was determined during preliminary experiments, because as the pH was lowered to 2.5, adsorption of Cr(VI) was promoted. For each isotherm, the initial Cr(VI) concentration was varied, whereas the biomass concentration was kept constant (1 g l⁻¹). Cr(VI) appears as an oxyanion (CrO₄²⁻ or Cr₂O₇²⁻) in aqueous solution, hence it can not bind effectively to a negatively charged functional group[18]. However, as the pH was lowered to 2.5, the overall surface charge of biomass surface becomes positive (Fig. 3), promoting adsorption of anionic Cr(VI) species.

The maximum removal capacity (q_max) of Cr(VI), using dead cells of B. licheniformis, was 62 mg g⁻¹, whereas the respective qmax value, using dead cells of B. laterosporus, was 72.6 mg g⁻¹. InTable 2 the adsorption capacities of other biosorbents (literature data) are presented for comparison reasons. The values of chromium(VI) specific up-

Fig. 5. Comparison of biosorption capacity for chromates using non-living or living cells of B. licheniformis (Langmuir isotherm); the other experimental conditions were the same, as in the aforementioned experiments.

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Fig. 6. Comparison of biosorption capacity for chromates using non-living or living cells of B. laterosporus (Langmuir isotherm); the other experimental conditions were the same, as in the aforementioned experiments.

take, as they were determined in this work, were significantly higher in comparison with these reported in the literature.

Fig. 7 indicates that the rise of temperature may influence slightly the biosorption of Cr(VI) by B. licheniformis.

Table 3displays the results of fitting the Langmuir model to experimental data. The increase in metal uptake at increased temperatures can be attributed to either higher affinity of sites for this metal or to an increase of binding sites on the biomass, under the applied experimental conditions.

At higher temperatures the energy of the system seems to facilitates Cr(VI) attachment onto cell surface, but when the temperature is even higher, a decrease of metal sorption may be expected, due to damage of certain surface sites of cell, available for metal biosorption[19].

Fig. 7. Effect of temperature on the biosorption of Cr(VI) ions by of B. licheniformis at pH 2.5 (Langmuir isotherm); the other experimental conditions were the same, as in the aforementioned experiments.

3.3. Effect of pH and presence of surfactants on biomass hydrophobicity

Hydrophobicity in a solid/liquid/gas system is a complex phenomenon, resulting from different interactions. In order to apply dispersed-air flotation as a suitable separation method to harvest the metal-loaded suspended biomass, surface tension and contact angle measurements of this system were measured. Surface tension is a measure of wetting ability of a medium (usually aqueous) and it is largely affected by the addition of surface-active agents (flotation collectors, such as dodecylamine, DA). Also the addition of ethanol (a convenient frother) decreased the size of air bubbles and stabilized the froth layer. The values of surface tension were decreased with the addition of dodecylamine (as expected).

The smaller values were observed as the respecting pH values were increased, up to 12 (Fig. 8). However, the presence of surfactant was found to produce a positive effect for subsequent separation by flotation.

Fig. 9presents the results of contact angle measurements, as they were influenced by the respective pH values. Con- tact angle values were slightly increased with the addition of surfactant for the studied pH range. The natural hydropho-

Fig. 8. Surface tension measurements of non-living cells of B. laterosporus and B. licheniformis; [䊏] biomass, [䊉] in the presence of 3×10⁻⁴ M DA and 0.6% ethanol [䉱] in the presence of 3×10⁻⁴M DA and 0.6%

ethanol, 5 mg l⁻¹ metal solution.

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Fig. 9. Contact angle measurements of non-living cells of B. laterosporus and B. licheniformis; [䊏] biomass, [䊉] in the presence of 3×10⁻⁴ M DA and 0.6% ethanol [䉱] in the presence of 3×10⁻⁴ M DA and 0.6%

ethanol, 5 mg l⁻¹metal solution.

bicity of bacteria cells is mainly due to specific properties of the cell wall (i.e. the biosorption sites), which strongly de- pend upon the presence of various polysaccharides, proteins and lipids that form a biopolymer layer, although it can be increased by the presence of surface-active agents, influenc- ing positively the subsequent separation by the application of flotation.

3.4. General discussion

The uptake of metal ions by the Bacillus strains, applying batch system equilibrium experiments, occurs in two subsequent stages: an initial rapid stage (mainly due to passive uptake), followed by a slower process (due to active uptake).

The first stage can be attributed mainly to physical adsorption or ion exchange interactions with the biomass surface.

Biosorption utilizing the ability of non-living biomass to accumulate heavy metals from wastewaters is considered as a more competitive, effective and economically attractive treatment method than bioaccumulation, became maintain- ing a viable biomass during the metal removal process can be rather difficult. Additionally, when using living biomass, higher difficulty in biomass separation can be encountered,

mass loss after regeneration and insufficient understanding of process[1,20].

Biosorption offers a competitive waste treatment alternative, the basis of which need to be well understood, in order to prevent application failures. This innovate treatment technology based on biosorption, is gradually improved, although for its continued success it requires further research efforts. The combined environmental pressures and cost factors make the removal and recovery of heavy/toxic metals from industrial effluents an important priority, which rep- resents business opportunities coupled with scientific chal- lenge. It was found that the application of biosorptive processes could reduce capital costs by 20%, operating costs by 36% and total treatment costs by 28%, as compared with convenient ion exchange systems[1].

4. Conclusion

Metal accumulation by appropriate biological substrates can counteract metal mobilization into the environment.

Microorganisms provide a large contact area that can interact with metals in the surrounding environment. Biosorption has received great attention during the last years, due to the potential use of microorganisms for cleaning metal-polluted water or wastewater streams. The aim of this work was to show the ability of several microorganisms, isolated from metal-polluted soils to biosorb and remove toxic metals from aqueous solutions and to compare biosorption with bioaccumulation, which can be attributed to the result of intrinsic mechanisms.

Increase of temperature did not affect the removal of Cd(II) by B. licheniformis, whereas Cr(VI) removal was slightly affected by temperature changes. The maximum removal capacities of Cd(II) using the non-living cells of B.

licheniformis and B. laterosporus were 142.7 and 159.5 mg g⁻¹, respectively. Regarding the case of Cr(VI) removal using the same bacterial biomass, the relevant values were 62 and 72.6 mg g⁻¹. Hence, the studied biomass proved very effective for the removal of toxic metal anions or cations.

The potential of using different species of (native) microorganisms as biosorbents for the removal of Cd(II) cations or Cr(VI) anions, examined in this study as representative toxic metals, has been clearly indicated.

Acknowledgements

Thanks are due to Profs. E. Tzanetaki (Dept. Agric.) and M. Liakopoulou-Kyriakidou (Dept. Chem. Eng.) for their collaboration in this PENED research program of the Greek General Secretariat of Research and Technology, to Mr K. Jachristas and to Dr M. Palaiomylitou for experimental collaboration. Part of these results were presented during the 12th Intern. Biodeterioration and Biodegrada- tion Symp. (Prague, July 14–18, 2002), with partial funding

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by the EC Environment programme (acronym Metasep, no.

EVK1-CT-2000-00083).

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