**4. Assessment of the remediation of lead by microorganisms**

## **4.1. Microorganisms adapted for lead absorption**

[89-92]. Transgenic plants which could increase the volatility of heavy metals or decrease the toxicity of heavy metals may be the best candidates because the remediation process can be continuously carried out without removing the plants. Transgenic *B. juncea*, for example, expressing the cystathionine gamma-synthase gene of *Arabidopsis thaliana* L. could convert selenium to volatile dimethylselenium [93, 94], and a plant expressing the methylmercury lyase gene decreased the toxicity by reducing methylmercury to mercury [95]. However, vaporization is not acceptable for lead because methylated lead diffuses into the air and

Thus, the following two mechanisms have been proposed. One method is to enhance the number of compounds capable of combining heavy metals, such as metallothionein, gluta‐ thione, and phytochelatin. For example, the absorption efficiency of transgenic *B. juncea* expressing adenosine triphosphate sulfurylase, glutamyl-cysteine synthetase, and glutathione synthetase genes was 4.3 times higher than that in the wild plant [96]. Moreover, the accumu‐ lation in *Nicotiana glauca* expressing phytochelatin synthase was also enhanced [97]. The other mechanism is to obtain a high lead tolerance by enhancing the transport into the cell and vascular membranes. Higher tolerance and accumulation of Zn, Mn, and Cd were realized by the plants transformed with a zinc transporter (*ZAT* or *AtMTP1*), *ShMTP*, *CAX2*, *AtMHX* [89-91] or the *AtNramp, AtPDR8*, and *AtATM3* genes of ABC transporters [98, 99]. For lead accumulation, the following transgenic plants were studied: tobacco plants expressing the calmodulin-binding protein gene of *Nicotiana tabacum* (*NtCBP4*) [100] and *Arabidopsis* plants expressing the *ZntA* [101], which codes for the zinc transporter in *E. coli*, and an enhanced accumulation of lead, as well as other heavy metals, was observed. The yeast *YCF1* gene codes for a transporter of vacuolar storage of Cd/Pb. *A. thaliana* expressing the *YCF1* gene showed a high resistance to Cd and Pb and accumulated those heavy metals [102]. Transgenic poplar trees expressing the *YCF1* gene also developed a high resistance to Cd and Pb [103]. Moreover, a study conducted by Mizuno et al. showed that transgenic *A. thaliana* had longer roots (2.5 times longer) and a higher (3–14 times higher) accumulation of lead when the *FeMRP3* gene

The author assessed the efficiency of phytoextraction in contaminated soil by lead. The most advantageous point of phytoremediation is its profitability. By the author's rough estimate, the income generated by the phytoremediation process is approximately 340, 000 dollars/ha for cases where it is assumed that (1) pollution is present at 1 m in depth and 10 g/kg of lead is contained in the soil (density: 1.7), (2) 100 % of the lead is extracted from the soil, and (3) the price of lead is 2, 000 dollars/ton. However, the approximation of the necessary expenses is much higher according to some reports and are estimated to be as high as 300, 000–5, 000, 000 dollars/ha (lowest estimation: 2, 500–15, 000) [104]. The difference in the costs suggests that further efforts are necessary to decrease the expenses in order to improve the application of

The high necessary expenses of phytoextraction are due to the low yield per treatment period and the time-consuming posttreatment heavy metal recycling from the biomass. Even in

exhibits a high toxicity as previously described.

258 Advances in Bioremediation of Wastewater and Polluted Soil

of buckwheat was expressed in *A. thaliana*.

phytoextraction.

**3.6. Assessment of efficiency of phytoextraction**

Some microorganisms contain a high ability to adsorb and absorb lead [106-108], and the mechanism through which microorganisms achieve this can be classified into four mechanisms (Table 3). The first mechanism is the absorption of lead by secreting extracellular polymers. The typical extracellular polymer is polysaccharide, which rapidly combines lead at a high affinity. *Halomonus* sp. [109], *Staurastrum* sp. [110], *Bacillus firmus* [111], *Paenibacillus jamilae* [112], and *Pseudomonas* sp. are known as microorganisms that secrete polysaccharides. The polysaccharide secreted by *B. firmus* cells, for example, was capable of adsorbing 98.3 % of Pb at an optimum pH, and 2 g/L of polysaccharide produced by *P. jamilae*, an endospore-forming bacillus, specifically adsorbed 230 mg/g of lead.

The second mechanism is adsorption at the cell wall. *Bacillus* sp. [113], *Pseudomonas aerugino‐ sa* [114], *Synechococcus* sp. [115], *Saccharomyces cerevisiae* [116], and fungi (such as *Aspergillus flavus* [117] and *Corollospora lacera* [118]) were highly efficient in the adsorption of lead; the amounts of lead absorbed by *P. aeruginosa*, *S. cerevisiae*, and *C. lacera* were 123, 250, and 270.3 mg/g dry biomass, respectively.

The third mechanism is the binding of lead inside the cell through phytochelatins, metallo‐ thioneins, and siderophores. Phytochelatins are produced by some microorganisms, such as *Schizosaccharomyces* sp. Metallothioneins produced by *Bacillus*[119], *Streptomyce*s sp. [120], and *P. aeruginosa* [121] are capable of combining with Pb(II), although metallothioneins typically combine with copper or zinc ions. Moreover, the yellow-green fluorescent pyoverdine and pyochelin produced by *Pseudomonas putida* KNP9 [122] and *P. aeruginosa* PAO1 [123] were capable of combining with Pb(II).

The fourth mechanism is the precipitation of lead inside the cell. For example, *Staphylococcus aureus* [124], *Vibrio harveyi* [125], and *Enterobacter cloacae* [126] are capable of producing Pb3(PO4)2, Pb6(PO4)6, and Pb(PO4)3Cl, respectively, by binding with phosphate, and sulfurreducing bacteria produce PbS [127].

Furthermore, using genetic techniques, these abilities could be enhanced. For example, the amount of Cd accumulation was seven times higher when the phytochelatin synthesis gene of *Schizosaccharomyces pombe* was expressed in *P. putida* KT2440. The genes associated with metallothioneins, siderophores, and phytochelatins were precisely examined [128-131], and recombinants expressing these genes at a high level may be useful for enhancing the accumu‐ lation of lead.

#### **4.2. Novel bioremediation process of heavy metals using microorganisms**

As introduced in Section 4.1, some microorganisms showed a high ability for lead accumula‐ tion (> 300 mg/g), which was higher than the plant hyperaccumulators. However, few microorganisms have been utilized for the bioremediation of soil polluted by lead. The reason is that the collection of such microorganisms from soil followed by adsorption is extremely difficult. If the microorganisms can be readily collected from the soil, then bioremediation with microorganisms becomes an effective process. Thus, the author developed a novel bioreme‐ diation method which combines the immobilized technique with landfarming, referred to as the landfarming with immobilized microorganisms (LIM) method.

The LIM method consists of four steps, shown in Figure 4. In the first step, the beads (approx‐ imately 0.35–0.4 cm in diameter) immobilized with microbial cells which demonstrate a high ability of absorption to lead are prepared and mixed with contaminated soil while plowing the field by the landfarming process. The soil is oxygenated by the operation, and the immo‐ bilized microbial cells contained in the beads are activated by the increased oxygen supply. In the second step, the plowed soil containing the cell beads is incubated for a defined period. The lead is absorbed (or adsorbed) by the microbial cells during this period. In the third step, the soil containing the beads is collected, and the beads are separated from the soil with sieves of adequate mesh sizes (0.25 and 0.50 cm). Thus, the beads can be easily collected. In the fourth step, lead absorbed (or adsorbed) in the cells is extracted with a small amount of nitric acid. The separated soil by the sieves is recycled by returning it to its point of origin, and the beads flowed by extraction are reused in the next remediation.

An assessment of the Causes of Lead Pollution and the Efficiency of Bioremediation by Plants and Microorganisms http://dx.doi.org/10.5772/60802 261


**Table 3.** Various mechanisms to adsorb or absorb lead by microorganisms

Step 1: The field is plowed and the soil is mixed with beads of the immobilized cells. Step 2: The plowed field is incu‐ bated for a defined period to absorb lead. Step 3: The beads are separated using sieves. Step 4: Lead is extracted from the beads, and the resultant beads and sifted soil are recycled.

**Figure 4.** The LIM method.

The third mechanism is the binding of lead inside the cell through phytochelatins, metallo‐ thioneins, and siderophores. Phytochelatins are produced by some microorganisms, such as *Schizosaccharomyces* sp. Metallothioneins produced by *Bacillus*[119], *Streptomyce*s sp. [120], and *P. aeruginosa* [121] are capable of combining with Pb(II), although metallothioneins typically combine with copper or zinc ions. Moreover, the yellow-green fluorescent pyoverdine and pyochelin produced by *Pseudomonas putida* KNP9 [122] and *P. aeruginosa* PAO1 [123] were

The fourth mechanism is the precipitation of lead inside the cell. For example, *Staphylococcus aureus* [124], *Vibrio harveyi* [125], and *Enterobacter cloacae* [126] are capable of producing Pb3(PO4)2, Pb6(PO4)6, and Pb(PO4)3Cl, respectively, by binding with phosphate, and sulfur-

Furthermore, using genetic techniques, these abilities could be enhanced. For example, the amount of Cd accumulation was seven times higher when the phytochelatin synthesis gene of *Schizosaccharomyces pombe* was expressed in *P. putida* KT2440. The genes associated with metallothioneins, siderophores, and phytochelatins were precisely examined [128-131], and recombinants expressing these genes at a high level may be useful for enhancing the accumu‐

As introduced in Section 4.1, some microorganisms showed a high ability for lead accumula‐ tion (> 300 mg/g), which was higher than the plant hyperaccumulators. However, few microorganisms have been utilized for the bioremediation of soil polluted by lead. The reason is that the collection of such microorganisms from soil followed by adsorption is extremely difficult. If the microorganisms can be readily collected from the soil, then bioremediation with microorganisms becomes an effective process. Thus, the author developed a novel bioreme‐ diation method which combines the immobilized technique with landfarming, referred to as

The LIM method consists of four steps, shown in Figure 4. In the first step, the beads (approx‐ imately 0.35–0.4 cm in diameter) immobilized with microbial cells which demonstrate a high ability of absorption to lead are prepared and mixed with contaminated soil while plowing the field by the landfarming process. The soil is oxygenated by the operation, and the immo‐ bilized microbial cells contained in the beads are activated by the increased oxygen supply. In the second step, the plowed soil containing the cell beads is incubated for a defined period. The lead is absorbed (or adsorbed) by the microbial cells during this period. In the third step, the soil containing the beads is collected, and the beads are separated from the soil with sieves of adequate mesh sizes (0.25 and 0.50 cm). Thus, the beads can be easily collected. In the fourth step, lead absorbed (or adsorbed) in the cells is extracted with a small amount of nitric acid. The separated soil by the sieves is recycled by returning it to its point of origin, and the beads

**4.2. Novel bioremediation process of heavy metals using microorganisms**

the landfarming with immobilized microorganisms (LIM) method.

flowed by extraction are reused in the next remediation.

capable of combining with Pb(II).

260 Advances in Bioremediation of Wastewater and Polluted Soil

reducing bacteria produce PbS [127].

lation of lead.

A. Soil containing the beads. Fifty beads were mixed with 50 g of soil (Hyoko, Japan). a. Beads made of alginate gel (0.38 mm in diameter). B. The soil and beads were separated by sieves with mesh sizes between 2.5 mm and 5 mm. B+ and B-. The impassable soil through the 5 mm mesh sieve and the passed soil through the 2.5 mm mesh sieve are shown. C. The washed soil and beads in water. D. Extraction of lead from the beads by nitric acid.

**Figure 5.** Schematic illustration of the LIM method using alginate gel beads.

The beads made of alginate gel are most suitable for the LIM method because (1) they can be easily and inexpensively produced at a uniform size, (2) they can immobilize microbial cells at a high density (approximately 100–1, 000 mg dry cells/cm3 ), and (3) they have an appropriate hardness. If it is assumed that the immobilized cell can accumulate lead at 300 mg/g, one bead should be able to absorb 3–30 mg of lead. Additionally, if cells secreting polysaccharides are utilized, each bead may be applied several times for remediation because polysaccharides are not leaked from the beads. Therefore, alginate gel beads can be utilized as a superior absorbent of lead.

Figure 5 shows the separation experiment of the beads and soil from the soil and beads mixture (Fig. 4, Step 3). The experiment was performed to examine the separation efficiency of the beads; the absorption by the immobilized cells was not conducted. Fifty beads (0.38 mm in diameter) were mixed with 50 g of soil (Fig. 5A) and separated with 2.5 mm and 5 mm mesh sieves. All beads were collected between 2.5 mm and 5 mm mesh sieves (Fig. 5B) and the soil was eliminated from the 2.5 mm mesh sieve by rinsing with water (Fig. 5C). Heavy metals were extracted by a small amount of nitric acid (Fig. 5D). Following extraction, the beads may be reused in the next remediation because the beads are not broken by the operation and can be easily separated with small stones (Fig. 5D).

The advantage of the LIM method is that the processing time is short and the beads may be readily collected and reused for the extraction operation. Therefore, the LIM method has a high potential for remediating the soil contaminated by lead. This method may become an impor‐ tant process for remediation of soil in the future, although the proper procedure and efficiency of the LIM method must be further investigated.
