Title Page

Abstract

Contents

I. INTRODUCTION 11

II. LITERATURE REVIEW 13

1. ARSENIC ACCUMULATION IN FOOD PLANTS 13

1.1. Arsenic toxicity 13

1.2. Uptake of arsenic in plants 13

1.3. Arsenic accumulation in crop and vegetable plants in contaminated areas 15

2. BIOFERTILIZERS IN SOIL REMEDIATION 16

2.1. Conventional methods for soil remediation 16

2.2. Biofertilizers in soil remediation 18

III. MATERIALS AND METHODS 25

1. Site description and sample collection 25

2. Isolation of arsenic-resistant bacteria 27

3. Evaluation of arsenic resistance 28

4. Arsenic accumulation by bacterial cells 28

5. Identification of selected bacterial strains 28

6. Physiological characterization 29

7. Evaluation of other toxicants tolerance 29

8. Adsorption capacity of carrier 30

9. Biofertilizer formation 30

10. Plant cultivation and analysis 31

11. Data quality control and statistical analysis 33

IV. RESULTS AND DISCUSSION 35

1. Isolation of arsenite-resistant bacteria 35

2. Evaluation of arsenite resistance 37

3. Arsenic accumulation by growing cells 39

4. Identification of selected bacterial strains 40

5. Physiological characterization 41

6. Evaluation of other toxicants tolerance 43

7. Adsorption capacity of carrier 45

8. Biofertilizer formation 46

9. Plant cultivation and analysis 47

V. CONCLUSIONS 55

VI. FUTURE STUDY 56

VII. REFERENCES 57

Table II.1. Total arsenic concentrations (tAs) in several crop plants sampled from contaminated sites 16

Table III.1. Total arsenic concentrations in soil samples 27

Table III.2. Metal solutions used to study competitive adsorption behavior 31

Table III.3. Certified values (mg/kg D.W) and recovery rates of heavy metals of various Standard Reference Materials (SRM) 34

Table IV.1. Minimum inhibition concentration (MIC) for arsenite of bacterial isolates 38

Table IV.2. Physiological characteristics of the strain DE12 and Bacillus megaterium in other studies 42

Table IV.3. Minimum inhibition concentration (MICs) for arsenate and heavy metals of the strain DE12 44

Figure II.1. Uptake routes of arsenic in terrestrial, emergent and submerged plants 14

Figure II.2. Schematic representation of the different processes evolved by prokaryotes to cope with arsenic 20

Figure II.3. (a) SEM image showing the cross-section of rice husk at high magnification (b) Schematic diagram demonstrating the hierarchical microstructure in (a) 23

Figure II.4. SEM image of rice straw cross section 24

Figure II.5. SEM image of coconut fiber adsorbent 24

Figure III.1. Location of soil sampling sites from Dongjin mine area 25

Figure III.2. Location of soil sampling sites from Duckum mine area 26

Figure III.3. Schematic diagram demonstrating different cultivating treatments 32

Figure IV.1. Colony morphology of isolated strains 36

Figure IV.2. Morphology of bacterial cell of isolated strains 37

Figure IV.3. Arsenic accumulating abilities of isolated strains 39

Figure IV.4. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain DE12 and related bacteria 40

Figure IV.5. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain DE26 and related bacteria 41

Figure IV.6. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship between strain DE52 and related bacteria 41

Figure IV.7. Results of physiological characteristics of the strain DE12 by biochemical test 43

Figure IV.8. MIC test for Pb of the strain DE12 using agar dilute method 44

Figure IV.9. MIC test for Cr of the strain DE12 using broth dilute method 44

Figure IV.10. Adsorption capacity of studied agricultural plant wastes 46

Figure IV.11. Biofertilizer in incubation and packaging process 47

Figure IV.12. Lettuce samples after cultivation period with different treatments 48

Figure IV.13. Water spinach samples after cultivation period with different treatments 49

Figure IV.14. Sweet basil samples after cultivation period with different treatments 49

Figure IV.15. Total arsenic concentration in edible parts of studied plants 51

Figure IV.16. Total arsenic concentration in root of studied plants 51

Figure IV.17. Lead concentration in edible parts of studied plants 53

Figure IV.18. Lead concentration in roots of studied plants 53