immobility in fly ash induced by bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dumps

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Bioresource Technology 99 (2008) 1305–1310

Evaluation of metal mobility/immobility in fly ash induced by bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dumps Sadhna Tiwari, Babita Kumari, S.N. Singh

*

Environmental Science Division, National Botanical Research Institute, Lucknow-226001, India Received 24 October 2006; received in revised form 6 February 2007; accepted 6 February 2007 Available online 26 March 2007

Abstract In this investigation, 11 bacterial strains were isolated from the rhizospheric zone of Typha latifolia. All the strains were aerobic, showed positive result with indole production and were able to grow in MacConkey agar. However, four strains were gram positive and others gram negative. These strains were inoculated separately in the fly ash with additional source of carbon to test their ability to increase the bioavailability or immobilization of toxic metals like Cu, Zn, Pb, Cd and Mn. It was observed that most of the bacterial strains either enhanced the mobility of Zn, Fe and Mn or immobilized Cu and Cd. However, there were a few exceptions. For example, in contrast to other bacterial strains, NBRFT6 enhanced immobility of Zn and Fe and NBRFT2 of Mn. On the other hand, in place of immobility induced by most of the bacterial strains, NBRFT8 and NBRFT9 enhanced bioavailability of Cu. However, in case of Cd, all the strains without any exception immobilized this metal. The results also indicated that the mobility/immobility of trace metals from the exchangeable fractions was the specific function of bacterial strains depending upon the several edaphic and environmental factors. Based on the extractability of metals from fly ash, a consortium of high performer bacterial strains will be further used to enhance the phytoextraction of metals from fly ash by metal accumulating plants. On the other hand, bacterial strains responsible for immobilization of metals may be used for arresting their leaching to water bodies.  2007 Elsevier Ltd. All rights reserved. Keywords: Heavy metals; Bioavailability; Immobilization; Bacteria; DTPA

1. Introduction Despite of other energy sources, coal still continues to be a major source of energy in India. More than 70% of energy is today generated by coal-based thermal power plants. Since Indian coal contains around 40% ash, these power plants generate enormous amounts of fly ash which is dumped in the near by areas. As per available estimate, the production of coal ash in India, including both fly ash and bottom ash, is likely to touch 140 million tons per year by 2020. Disposal of huge amount of fly ash not only requires more than 30,000 hectares of agricultural *

Corresponding author. Tel.: +91 522 2205831 35; fax: +91 522 2205839/2205836. E-mail address: drsn_s@rediffmail.com (S.N. Singh). 0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.010

and forest lands, but also becomes a potential source of metal contamination in surface and ground water, threatening human health (Kalra et al., 1998). Fly ash contains a number of toxic trace elements like Si, Al, Fe, Cd, Mn, B, As, Hg which gradually leach out to contaminate the water reservoirs. Removal of metals from fly ash by conventional methods is not only uphill and cost-intensive task, but it is also eco-unfriendly. In view of these constraints, bioremediation of metals from fly ash is thought to be an alternate technology which is self-sustainable, eco-friendly and cost effective. In this technology, microbes may be employed, as they are known to play various functions in metal transformations. Generally metal transformations may be divided into two broad categories (i) redox conversions of inorganic forms and (ii) conversions from inorganic to organic forms through

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methylation and demethylation process. They utilize metals as terminal electron acceptors in anaerobic respiration (Niggemyer et al., 2001; Quilntana et al., 2001), while they derive energy from metal oxidation for their growth in aerobic respiration (Tebo et al., 1997; Santini et al., 2000). In addition, the microbes possess reduction mechanisms, which are not coupled to respiration, but impart metal resistance. Aerobic and anaerobic reduction of Cr(VI) to Cr(III) (Quilntana et al., 2001), Se(VI) to elemental Se (Lloyd et al., 2001), U(VI) to U(IV) (Francis, 1998; Chang et al., 2001) and Hg(II) to Hg(0) (Brim et al., 2000) are widely known detoxification mechanisms in the bacteria. In redox conversion and methylation reaction, acidophilic iron and sulfur oxidizing bacteria are reported to leach very high concentration of As, Cd, Cu, Ni and Zn from contaminated soils (Seidel et al., 2000; Groudev et al., 2001; Lo¨ser et al., 2001). Reversely, metals can be precipitated as insoluble sulfides indirectly by the metabolic activity of sulfate reducing bacteria (White et al., 1997; Lloyd et al., 2001). Although a lot of works have been done on the use of fly ash as a soil amender to boost up crop production due to presence of some essential element in fly ash (Jala and Goyal, 2006), only a few reports are available on the microbe-assisted remediation of metals from wastes to check surface and ground water metal contamination. Xu and Ting (2004) used Aspergillus niger – a fungus for bioleaching of metals from incinerator ash of municipal solid waste. Roy et al. (2006) reported immobilization of heavy metals by sorption and in situ bioprecipitation processes. However, no report is available on microbial remediation of metals from fly ash. Hence, this study was planned to measure the ability of fly ash tolerant bacteria in solublization and immobilization of metals in order to develop a microbe-assisted phytoremediation technology (Roy et al., 2006; Jala and Goyal, 2006). 2. Methods Fly ash samples were collected from the stands of Typha latifolia growing naturally on the fly ash disposal sites of NTPC coal-based Thermal Power Plant, located at Unchahar, Raibraelli, UP, India.

method and using nutrient agar (NA composition (1 L): 10 g peptone, 10 g beef extract, 5 g sodium chloride and 12 g agar) plates. Plates were incubated at 37 C for 24 h and the bacterial colonies were counted to find out the CFU. This value was calculated as 2.27 · 108 bacteria/g of fly ash. The isolated colonies were picked up by a sterilized loop and then used to make a series of parallel nonoverlapping streaks on the surface of the solidified agar plates After 24 h of incubation, the isolated pure colonies developed on the agar plates. Isolated bacterial strains from fly ash were named for convenience before identification as: NBRFT1, NBRFT2, NBRFT3, NBRFT4, NBRFT5, NBRFT6, NBRFT7, NBRFT8, NBRFT9, NBRFT10, NBRFT11. 2.3. Experimental setup All the bacterial strains were inoculated separately in nutrient broth (NB composition (1 L): 5 g peptic digest of animal tissue, 1.5 g yeast extract, 1.5 g beef extract and 5 g sodium chloride) in glass conical flask and then incubated at 37 C in an incubation shaker (180 rpm) for 24 h. Then after, 500 ml inoculum (CFU: 3.2 · 1012– 9.65 · 1013 bacteria/ml, pH 7.4) was added to 1 kg air-dried fly ash placed in earthen pots (triplet for each bacterial strain) to study their growth. However, nutrient broth without bacteria was added to control pots to simulate the conditions. Fly ash in pots was kept moist by adding 300 ml double distilled water (DDW) in each pot on alternate days and homogenized by using sterile spatula to facilitate the growth of bacteria. Bacterial growth was examined through serial dilution at different time intervals to know the time required for the optimum growth of bacteria. CFU values of all bacterial strains showed their growth at 7, 15 and 30 days of inoculation in fly ash as reflected in Table 1. However, the CFU value ranged from 1.33 · 108 to 39.3 · 108 bacteria/g at Zero days incubation. For the extraction of metals from fly ash, a synthetic chelate like diethylene triamine penta acetic acid (DTPA) was used as per standard procedure (Lindsay and Norvell, 1978). 2.4. Metal extraction

2.1. Physico-chemical analysis of fly ash Electrical conductivity and pH of fly ash was determined by Orion electrical conductivity meter and Orion pH meter. Metal concentration of fly ash was determined using Pan atomic absorption spectrophotometer (G B C Avanta A A S) after the digestion of fly ash samples following the method EPA 3050B. 2.2. Bacterial isolation from fly ash Bacterial isolation was carried out from the fly ash of rhizospheric zone of T. latifolia following the serial dilution

Since DTPA has the potential to strongly chelate Fe, Cu, Mn and Zn from the exchangeable fraction, it is normally used as a metal extractant from the alkaline soils and fly ash (Lindsay and Norvell, 1978; Gupta and Sinha, 2006). The amount of metals extracted by the DTPA gives an idea of the pool size of available metals to plants. Therefore, after 7, 15, and 30 days of inoculation, a synthetic chelate DTPA was used for the extraction of metals from the fly ash incubated with all the bacterial strains separately. The pH of the fly ash with addition of DTPA was measured to be 7.35. DTPA extractable fraction was obtained by mechanically shaking of 10 g fly ash for 2 h

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with 40 ml of 0.5 M DTPA, 0.01 M CaCl2 and 0.1 M TEA buffered at pH 7.3 (Lindsay and Norvell, 1978) and then filtered through 44 Whatman. Using P an atomic absorption spectrophotometer (G B C Avanta A A S), metal content was determined in the DTPA extractions. 3. Results 3.1. Fly ash composition Fly ash, a residue of coal consumption is primarily made up of oxides of Al and Si, but also enriched with several other essential (Zn, Fe, Mn, B, and Mo) and non essential metals (Ni, Cr, Pb, Al, Si). Its particle size ranges between 10 and 150 lm, pH 7.5 and the electrical conductivity is very high (389 lS/cm). With no organic carbon, it can not support bacterial growth. Hence, nutrient broth was added as an additional source of organic carbon to enhance bacterial augmentation. 3.2. Growth of bacteria CFU values of bacterial strains inoculated in the fly ash indicated that the bacteria multiplied very fast using the additional source of carbon in the form of NB during first 7 days of incubation and then additional carbon was perhaps depleted. Hence the multiplication of bacterial cells was arrested as reflected by the CFU values beyond 7 days of incubation (Table 1). 3.3. Biochemical characterization The biochemical characteristics of the bacterial strains isolated from the fly ash dumps indicated that out of 11 strains, four were found gram positive and remaining strains were gram negative. All of them were aerobic bacteria. As none of the bacterial strain could grow in Pseudomonas agar, this ruled out the possibility of presence of Pseudomonas species in the bacterial strains isolated from the fly ash.

Table 1 CFU/g of different fly ash tolerant bacterial strains Bacterial strains

CFU value 7 days

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

15 days 10

13.3 · 10 39.9 · 108 19.9 · 1010 29.3 · 1010 19.9 · 1010 36.5 · 1010 26.6 · 1010 19.9 · 1010 19.9 · 1010 23.3 · 1010 19.9 · 1010 32.2 · 108

30 days 10

93.1 · 10 86.5 · 1010 53.2 · 1010 55.9 · 1010 53.2 · 1010 46.5 · 1012 43.3 · 1010 53.2 · 1010 53.2 · 1010 39.9 · 1010 53.2 · 1010 39.6 · 109

46.6 · 1010 33.3 · 1010 13.3 · 1010 19.9 · 1010 13.3 · 1010 29.9 · 1012 19.9 · 1010 13.3 · 1010 13.3 · 1010 13.3 · 1010 13.3 · 1010 19.3 · 109

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3.4. Metal mobility/immobility When different bacteria strains isolated from the fly ash were re-inoculated in the fly ash in high inoculum separately, they presented variable trends of solublization and immobilization with incubation period. The data on the metal mobility/immobility by the different bacterial strains have been shown in Tables 2–6. As far as the Cu immobility is concerned, there was no significant change in the metal immobility with the incubation of any of the bacterial strains for 7 days with respect to control. However, when the incubation period was increased to 15 and 30 days, it was observed that most of the bacterial strains except NBRFT8 and NBRFT9, enhanced immobilization of Cu in the fly ash. The extent of Cu immobilization varied from 6% to 37% in 15 days incubation and from 10% to 90% in 30 days incubation period. However, bacterial strain NBRFT8 enhanced Cu mobility by 49% in 15 days and by 94% in 30 days incubation in contrast to other bacterial strains. Similarly, bacterial strain NBRFT9 induced Cu mobility by 47% in 15 days and 43% in 30 days incubation period (Table 2). On the other hand, Table 3 indicates the mobility of Zn in the fly ash induced by the different bacterial strains. The solubility of Zn varied remarkably depending upon the bacterial strains incubated in fly ash for metal extractability. It was observed that most of the bacterial strains could enhance Zn metal bioavailability significantly after 15 days and 30 days of incubation period except NBRFT8, which indicated Zn immobilization to some extent. Many of the bacteria showed Zn immobilization at 7 days incubation period and then switched over to mobilization. The metal Table 2 Mobility/immobility of Cu (lg/g dw) in fly ash influenced by different fly ash tolerant bacterial strainsa Bacterial strain

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

Metal concentration (lg/g dw) incubation period

Metal bioavailability (%) incubation period

7 days

15 days

30 days

7 days

15 days

30 days

0.811 0.823 0.835 0.851 0.803 0.743 0.787 0.935 0.991 0.767 0.843 0.865

0.344 0.369 0.386 0.396 0.298 0.331 0.384 0.710 0.704 0.405 0.444 0.476

0.084 0.037 0.236 0.244 0.056 0.272 0.292 0.796 0.588 0.368 0.200 0.410

6.2 4.8 3.5 1.6 7.2 14.1 9.0 8.1 14.6 11.3 2.5

27.7 22.5 18.9 16.8 37.4 30.5 19.3 49.2 47.9 14.9 6.7

79.5 90.9 42.4 40.5 86.3 33.6 28.7 94.1 43.4 10.2 51.2

Source

df

SS

MS

F

ANOVA_Table for Cu Between days Between strains Error Total

2 11 22 35

1.88 0.59 0.19 2.67

0.94 0.05 0.01

105.50 6.06

a

Average of three replicates.

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Table 3 Mobility/immobility of Zn (lg/g dw) in fly ash influenced by different fly ash tolerant bacterial strainsa

Table 5 Mobility/immobility of Mn (lg/g dw) in fly ash influenced by different fly ash tolerant bacterial strainsa

Bacterial strain

Metal concentration (lg/g dw) incubation period

Metal bioavailability (%) incubation period

Bacteria strain

Metal concentration (lg/g dw) incubation period

Metal bioavailability (%) incubation period

7 days

15 days

30 days

7 days

15 days

7 days

15 days

30 days

7 days

15 days

30 days

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

1.550 4.820 1.615 1.320 1.817 1.310 1.558 1.705 1.695 1.960 1.390 1.750

2.412 4.666 1.980 2.492 2.860 3.608 2.137 1.628 2.236 2.892 1.928 1.772

3.441 3.265 2.755 3.337 3.480 3.023 2.535 1.607 2.335 2.640 2.035 1.820

11.4 175.4 7.7 24.5 3.8 25.1 10.9 2.5 3.1 12.0 20.5

36.1 163.3 11.7 40.6 61.4 103.6 20.6 8.1 26.2 63.2 8.8

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

0.600 0.335 0.560 0.620 0.939 0.640 0.750 0.800 0.640 0.830 0.630 0.475

0.324 0.156 0.387 0.388 0.506 0.434 0.477 0.504 0.424 0.572 0.502 0.292

0.156 0.035 0.265 0.195 0.248 0.250 0.220 0.253 0.260 0.250 0.318 0.145

26.3 29.4 17.8 30.5 97.6 34.7 57.9 68.4 34.7 74.7 32.6

10.9 46.5 32.5 32.8 73.3 48.6 63.3 72.6 45.2 95.9 71.9

7.6 75.8 82.7 34.5 71.0 72.4 52.1 74.5 79.3 72.4 119.3

Source

df

ANOVA_Table for Zn Between day Between strains Error Total a

2 11 22 35

SS

MS

4.56 15.58 7.12 27.26

2.28 1.41 0.32

30 days 89.1 79.4 51.3 83.3 91.2 66.1 39.3 11.7 28.3 45.1 11.8

F 7.05 4.38

Table 4 Mobility/immobility of Fe (lg/g dw) in fly ash influenced by different fly ash tolerant bacterial strainsa

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

Metal concentration (lg/g dw) incubation period

Metal bioavailability (%) incubation period

7 days

15 days

30 days

7 days

15 days

30 days

6.2900 6.045 7.795 7.900 7.235 3.690 5.790 6.635 7.795 6.970 5.120 4.365

9.820 9.868 10.248 9.532 8.668 4.336 9.092 8.328 10.868 10.884 9.848 5.740

12.00 9.80 11.91 12.30 10.10 4.20 11.10 8.80 9.10 11.72 10.46 6.40

44.1 38.5 78.6 80.9 65.7 15.4 32.6 52.0 78.5 59.6 17.2

71.1 71.9 78.5 66.1 51.0 24.4 58.4 45.1 89.3 89.6 71.5

87.5 53.1 86.1 92.2 57.8 34.4 73.4 37.5 42.2 83.1 63.4

Source

df

SS

MS

F

ANOVA_Table for Fe Between day Between strains Error Total

2 11 22 35

80.50 108.21 22.30 211.02

40.25 9.84 1.01

39.71 9.71

a

df

SS

MS

F

ANOVA_Table for Mn Between days Between strains Error Total

2 11 22 35

1.13 0.40 0.09 1.63

0.56 0.04 0.004

131.76 8.49

a

Average of three replicates.

Bacterial strain

Source

Average of three replicates.

mobility after 15 days incubation period varied between 8% and 163% and after 30 days between 11% and 91% depending upon the specific bacterial strain inoculated in fly ash. Unlike other bacterial strains, NBRFT2 showed maximum Zn solubility (175%) initially after 7 days and then declined to 79% at 30 days incubation period.

Average of three replicates.

Table 6 Mobility/immobility of Cd (lg/g dw) in fly ash influenced by different fly ash tolerant bacterial strainsa Bacterial strain

NBRFT1 NBRFT2 NBRFT3 NBRFT4 NBRFT5 NBRFT6 NBRFT7 NBRFT8 NBRFT9 NBRFT10 NBRFT11 Control

Metal concentration (lg/g dw) incubation period

Metal bioavailability (%) incubation period

7 days

15 days

30 days

7 days

15 days

30 days

0.065 0.066 0.078 0.062 0.045 0.075 0.070 0.065 0.081 0.080 0.086 0.090

0.088 0.036 0.060 0.028 0.016 0.095 0.089 0.032 0.008 0.028 0.044 0.132

0.089 0.025 0.049 0.013 0.017 0.065 0.065 0.085 0.021 0.025 0.033 0.137

27.8 26.6 13.3 31.1 50.0 16.6 22.2 27.7 10.0 11.1 4.4

33.3 72.7 54.5 78.8 87.8 28.0 32.5 75.7 93.9 78.7 66.6

35.0 81.7 64.2 90.5 87.6 52.5 52.5 37.9 84.6 81.7 75.9

Source

df

SS

MS

F

ANOVA_Table for Cd Between days Between strains Error Total

2 11 22 35

0.003 0.022 0.011 0.035

0.001 0.002 0.0005

2.73 4.17

a

Average of three replicates.

Fe extractability from the fly ash with the incubation of bacterial strains has been shown in Table 4. It was noted that all the bacterial strains induced more Fe bioavailability than other heavy metals. However, NBRFT6 immobilized Fe in the fly ash. Four bacterial strains NBRFT1, NBRFT3, NBRFT4 and NBRFT7 showed an increasing trend of metal bioavailability with the increasing incuba-

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tion period, but NBRFT2, NBRFT9 and NBRFT11 indicated maximum Fe extractability at 15 days incubation period and then there was a decline. Rest two bacterial strains showed maximum Fe bioavailability at 7 days incubation period and then after there was gradual decline with the increasing incubation period. As compared to control, Fe mobility was enhanced between 17% and 81% at 7 days, between 45% and 89% at 15 days and between 37% and 92% at 30 days incubation period. Thus, these strains showed variable trends of metal mobility in the fly ash with the incubation period. As far as Mn mobility is concerned, most of the bacterial strains enhanced its bioavailability during their incubation in the fly ash. It is evident from Table 5 that barring NBRFT2, all the bacterial strains induced Mn mobility between 18% and 97% at 7 days, between 10% and 95% at 15 days and between 7% and 82% at 30 days incubation period. However, NBRFT2 showed immobilization between a minimum of 29% at 7 days incubation period and a maximum of 75% at 30 days incubation period. Among 10 bacterial strains, NBRFT3, NBRFT4, NBRFT6, NBRFT8, NBRFT9 and NBRFT11 enhanced Mn mobility with incubation period, while NBRFT1 and NBRFT5 showed reverse trend with incubation period. The remaining strains NBRFT7 and NBRFT10 indicated maximum metal extractability at 15 days incubation period, followed by a decline at 30 days incubation period. Like other metals, Cd mobility was also influenced by the incubation of different bacterial strains. In this case, all the bacterial strains without any exception enhanced immobilization of Cd in the fly ash, restricting its leaching to water streams. As evident from Table 6, these strains immobilized Cd between a minimum of 4.4% to maximum of 50% at 7 days, between 28% and 94% at 15 days and between 35% and 90% at 30 days incubation period. Except two strains NBRFT8 and NBRFT9, which showed maximum Cd immobilization at 15 days incubation, all the strains exhibited an increasing immobilization with the incubation period.

4. Discussion Metal contamination of rivers, ponds and lakes due to disposal of metal-loaded industrial and sewage wastes is a serious environmental concern today. To overcome this problem, bioremediation which involves microbes, plants and animals is considered as an alternative technology which is promising, cost-effective and eco-friendly in present scenario. No doubt microbes, being ubiquitous in nature, are being used for a long time to degrade pesticides, sludge and other xenobiotic compounds like PCB, PAH, TNT etc. but their application in bioremediation of metals from industrial wastes is a new dimension. Being integral component of biogeochemical cycle, they can be used to either solubilize the toxic metals, thereby increasing their bioavailability or immobilize them to check their migration

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to water reservoirs (Wainwright and Gadd, 1997; White et al., 1997; Gadd et al., 2001). When the bacterial strains, isolated from T. latifolia growing naturally on fly ash dumps, were augmented in the fly ash and metals were extracted by DTPA after different incubation periods, it was observed that most of the bacterial strains induced the bioavailability of Zn, Fe and Mn in fly ash, but immobilized Cu and Cd on other hand. However, NBRFT8 and NBRFT9 induced mobility of Cu in contrast to other strains. Similarly, NBRFT8 caused immobilization of Zn, while other 10 strains enhanced bioavailability of Zn. As against other strains, NBRFT6 induced Fe immobilization. This shows that metal mobility/immobility is the specific function of the bacterial strains in both aerobic and anaerobic conditions. No doubt, solublization and immobilization of metals are also governed by several edaphic and environmental factors. Besides, the extent of metal solubilization and immobilization also varied significantly among the bacterial strains and with incubation periods. Among 11 bacterial strains, better performers for more than 80% metal mobility were NBRFT1, NBRFT2, NBRFT4 and NBRFT5 for Zn, NBRFT2, NBRFT3, NBRFT4, NBRFT9 and NBRFT10 for Fe, NBRFT3, NBRFT5 and NBRFT11 for Mn and for metal immobility NBRFT2, NBRFT5 and NBRFT8 for Cu and NBRFT2, NBRFT5, NBRFT9, NBRFT10 and NBRFT11 for Cd. In our studies, most of the fly ash tolerant bacteria induced the bioavailablity of Fe and Mn which may be linked to biologically mediated reduction processes. Soil bacteria have been shown to exude organic compounds which stimulate bioavailability and thereby facilitate root absorption of various metal ions, Fe (Bural et al., 2000) and Mn (Barber and Lee, 1974). Caccavo et al. (1994) isolated Geobacter sulfurrenducens from hydrocarbon contaminated ditch which was the first bacterium described to couple the oxidation of hydrogen (or acetate) to Fe(III) reduction. Similarly, Lovley (1995) attributed reduction of Mn(IV) to Mn(II) to biological process, affecting its mobilization. Besides, mobilization and immobilization of metals are also governed by various physico-chemical characteristics, more particularly pH of the contaminated sites. However, in fly ash which has shown pH around neutrality, metal mobility/immobility can be attributed to bacterial actions. Increased organic matter also enhances both soluble and exchangeable metal levels in soil (Yoo and James, 2002). Zn and Cd occur in soil primarily in soluble precipitates (PO24 , CO23 , and hydroxyl-oxide) and hence unavailable to plants, but they are made available to plants by the microbial action. Fly ash has also high affinity to adsorb these elements. In our studies, most of the bacterial strains enhanced the bioavailability of Zn, Mn and Fe and immobilized Cd and Cu. While metal mobilization can be achieved by autotrophic and heterotrophic leaching, chelation by microbial metabolites and siderophores and methylation which can result in volatilization, immobilization is

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attributed to sorption to cell components, intracellular sequestration or precipitation as insoluble organic and inorganic compounds e.g. oxalates (Sayer and Gadd, 1997; Gharieb et al., 1998), sulphides (White and Gadd, 1996a,b) or phosphates (Young and Macaskie, 1995). Thus bacteria involve various metabolic process to enhance the solubilization and immobilization of heavy metals in the soils or industrial wastes, as metals serve as electron donors or acceptors in anaerobic and aerobic respirations. 5. Conclusion Since the extraction of metal contaminants from industrial wastes by physical and chemical methods is very cumbersome and cost-intensive, bacteria may be exploited to either enhance the bioavailability of toxic metals to be removed in the phytoextraction process or immobilize them to check their leachability to water reservoirs. Acknowledgements Authors are thankful to Director, National Botanical Research Institute, Lucknow for providing laboratory facilities and to CSIR for providing funds to networked project (COR008). References Barber, D.A., Lee, R.B., 1974. The effect of microorganisms on the absorption of manganese by plants. New Phytol. 73, 97–106. Brim, H., McFarlan, S.C., Fredrickson, J.K., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J., 2000. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments. Nat. Biotechnol. 18, 85–90. Bural, G.I., Dixon, D.G., Glick, B.R., 2000. Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. Can. J. Microbiol. 46, 237–245. Caccavo, F.J., Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F., McInerney, M.J., 1994. Geobacter sulfurreducens sp. nov., a hydrogenand acetate-oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 60, 3752–3779. Chang, Y.-J., Peacock, A.D., Long, P.E., Stephen, J.R., McKinley, J.P., MacNaughton, S.J., Hussain, A.K.M.A., Saxton, A.M., White, D., 2001. Diversity and characterization of sulfate-reducing bacteria in groundwater at a uranium mill tailings site. Appl. Environ. Microbiol. 67, 3149–3160. Francis, A.J., 1998. Biotransformation of uranium and other actinides in radioactive wastes. J. Alloy. Compd. 271–273, 78–84. Gadd, G.M., Bridge, T.A.M., Gharieb, M.M., Sayer, J.A., White, C., 2001. Microbial processes for solublization of metals and metalloids and their potential for environmental bioremediation. Ind. Environ. Biotechnol., 55–80. Gharieb, M.M., Sayer, J.A., Gadd, G.M., 1998. Solubilization of naturalgypsum (CaSO4 2H2O) and the formation of calcium oxalate by Aspergillus nigerand Serpula himantioides. Mycol. Res. 102, 825– 830. Groudev, S.N., Spasova, I.I., Georgiev, P.S., 2001. In situ bioremediation of soils contaminated with radioactive elements and toxic heavy metals. Int. J. Miner. Process. 62, 301–308.

Gupta, A.K., Sinha, S., 2006. Role of Brassica Juncea (L.) Czern. (var. Vaibhav) in the phytoextraction of Ni from soil amended with fly ash: selection of extractant for metal. J. Hazard Mater. 136 (2), 371–378. Jala, S., Goyal, D., 2006. Fly ash as a soil ameliorant for improving crop production – a review. Bioresource Technol. 97, 1136–1147. Kalra, N., Jain, M.C., Joshi, H.C., Choudhary, R., Harit, R.C., Vasta, V.K., Sharma, S.K., Kumar, V., 1998. Fly ash as a soil conditioner and fertilizer. Bioresource Technol. 64, 163–167. Lindsay, Norvell, 1978. Development of DTPA soil test for Zn, Mn and Cu. Soil Sci. Am. J. 42, 421–428. Lloyd, J.R., Mabbett, A.N., Williams, D.R., Macaskie, L.E., 2001. Metal reduction by sulphate-reducing bacteria: physiological diversity and metal specificity. Hydrometallurgy 59, 327–337. Lo¨ser, C., Seidel, H., Hoffmann, P., Zehnsdorf, A., 2001. Remediation of heavy metal-contaminated sediments by solid-bed bioleaching. Environ. Geol. 40, 643–650. Lovley, D.R., 1995. Bioremediation of organic and metal contaminants with dissimilatory metal reduction. J. Indus. Microbiol. 14, 85–93. Niggemyer, A., Spring, S., Stackebrandt, E., Rosenzweig, R.F., 2001. Isolation and characterization of a novel As(V)-reducing bacterium: implications for arsenic mobilization and the genus Desulfitobacterium. Appl. Environ. Microbiol. 67, 5568–5580. Quilntana, M., Curutchet, G., Donati, E., 2001. Factors affecting chromium(VI) reduction by Thiobacillus ferrooxidans. Biochem. Eng. J. 9, 11–15. Roy, S.V., Vanbroekhoven, K., Dejonghe, W., Diels, L., 2006. Immobilization of heavy metals in the saturated zone by sorption and in situ bioprecipitation processes. Hydrometallurgy 83, 195–203. Santini, J.M., Sly, L.I., Schnagl, R.D., Macy, J.M., 2000. A new chemolitoautotrophic arsenite-oxidizing bacterium isolated from a gold-mine: phylogenetic, physiological, and preliminary biochemical studies. Appl. Environ. Microbiol. 66, 92–97. Sayer, J.A., Gadd, G.M., 1997. Solubilization and transformation of insoluble inorganic metal compounds to insoluble metal oxalates by Aspergillus niger. Mycol. Res. 101, 653–661. Seidel, H., Ondruschka, J., Morgenstern, P., Wennrich, R., Hoffmann, P., 2000. Bioleaching of heavymetal contaminated sediments by indigenous Thiobacillus spp.: metal solubilization and sulfur oxidation in the presence of surfactants. Appl. Microbiol. Biotechnol. 54, 854–857. Tebo, B.M., Ghiorse, W.C., van Waasbergen, L.G., Siering, P.L., Caspi, R., 1997. Bacterially-mediated mineral formation: insights into manganese(II) oxidation from molecular genetic and biochemical studies. Rev. Mineral. 35, 225–266. Wainwright, M., Gadd, G.M., 1997. Industrial Pollutants. In: Wicklow, D.T., Soderstrom, B. (Eds.), The Mycota, Volume V: Environmental and Microbial Relationships. Springer-Verlag, Berlin, pp. 85–97. White, C., Gadd, G.M., 1996a. Mixed sulphate-reducing bacterial cultures for bioprecipitation of toxic metals: factorial and response-surface analysis of the effects of dilution rate, sulphate and substrate concentration. Microbiology 142, 2197–2205. White, C., Gadd, G.M., 1996b. A comparison of carbon/energy and complex nitrogen sources for bacterial sulphate-eduction: potential applications to bioprecipitation of toxic metals as sulphides. J. Ind. Microbiol. 17, 116–123. White, C., Sayer, J.A., Gadd, G.M., 1997. Microbial solubilization and immobilization of toxic metals: key biogeochemical processes for treatment of contamination. FEMS Microbiol. Rev. 20, 503–516. Xu, T.J., Ting, Y.P., 2004. Optimization on bioleaching of incinerator fly ash by Aspergillus niger use of central composite design. Enzyme Microbial. Technol. 35, 444–454. Yoo, M.S., James, B.R., 2002. Zinc extractability as a function of pH in organic waste-contaminated soils. Soil Sci. 167, 246–259. Young, P., Macaskie, L.E., 1995. Removal of tetravalent actinide thorium from solution by a biocatalytic system. J. Chem. Technol. Biotechnol. 64, 87–95.