BIOREMEDIATION OF ACID MINE DRAINAGE CONTAMINATED SOIL BY
PHRAGMITES AUSTRALIS AND RHIZOSPHERE BACTERIA
A Dissertation
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Lin Guo August, 2014 BIOREMEDIATION OF ACID MINE DRAINAGE CONTAMINATED SOIL BY
PHRAGMITES AUSTRALIS AND RHIZOSPHERE BACTERIA
Lin Guo
Dissertation
Approved: Accepted: ______Advisor Department Chair Dr. Teresa J. Cutright Dr. Wieslaw K. Binienda
______Committee Member Dean of the College Dr. Stephen Duirk Dr. George K. Haritos
______Committee Member Dean of the Graduate School Dr. Lan Zhang Dr. George R. Newkome
______Committee Member Date Dr. John M. Senko
______Committee Member Dr. Chelsea Monty ii ABSTRACT
Acid mine drainage (AMD) with low pH and high levels of heavy metals affects many regions. Experiments were conducted to investigate the bioremediation potential of AMD contaminated soils via Phragmites australis, rhizosphere acidophilic heterotrophs and/or Fe(II) oxidizing bacteria (Fe(II)OB), and citric acid
(CA).
Field characterizations indicated that Fe plaque amounts on reeds were related to the abundance of Fe(II)OB in soil. The metal concentrations in roots of reeds also depicted a strong correlation with the soil concentrations. The Fe concentrations in soil were 413.63±7.75 mg/g which were much higher than Mn (0.04±0.00 mg/g) and
Al (1.39±0.03 mg/g), while the Fe amounts in roots (13.43±5.98 mg/g) were also higher than Mn (0.09±0.03 mg/g) and Al (0.08±0.01 mg/g). Histological staining found that most of Fe and Al were stored in exodermis and endodermis of roots.
Laboratory experiments indicated that Fe(II)OB enhanced the formation of Fe plaque. The root Fe plaque (108.08±12.05 mg/g) of reeds cultured in spiked soil inoculated with Fe(II)OB were higher than that without adding Fe(II)OB (88.47±5.26 mg/g). CA inhibited the formation of Fe plaque. However, acidophilic heterotrophs consumed CA and enhanced the growth of Fe(II)OB. Metal plaque may decrease the accumulation of Fe and Mn into reeds while had no important influence on Al uptake.
iii CA enhanced Fe and Al entering stele of roots and increased metals uptake in reeds. Compared with non-contaminated reeds, wild reeds initially grown in AMD sites accumulated more metals due to the adaptability to the hostile environments. For instance, wild reeds cultured in soil added with 33.616 g/kg CA accumulated
0.32±0.01 mg/g Mn, 96.99±5.75 mg/g Fe and 3.17±0.51 Al in roots, while purchased reeds uptake 0.20±0.00 mg/g Mn, 79.21±5.95 mg/g Fe and 0.74±0.02 Al mg/g.
CA, rhizosphere bacteria and reeds had interconnected impacts on remediation of
AMD sites. CA significantly enhanced phytoremediation efficiency. Rhizosphere microorganisms also influenced metal bioavailability and metal uptake in reeds. Wild reeds in spiked soil or solution amended with 33.616 g/kg CA and without bacteria uptake the most metals. Further investigations are required to study the effect of CA and rhizosphere bacteria on phytoremediation of real AMD contaminated field.
iv ACKNOWLEDGEMENTS
First of all, I would like to express my sincere appreciation to my advisor Dr.
Teresa, J. Cutright for providing me the opportunity to work on this project. Her encouragement, guidance, patience, help and support motivate me to move forward.
I would like to thank all my committee members Dr. John M. Senko, Dr. Stephen
Duirk, Dr. Lan Zhang and Dr. Chelsea Monty. Your comments and suggestions improved my experiments and dissertation. Also, I deeply appreciate the help of Dr.
Donald W. Ott and Dr. Ron Salisbury who inspired me and helped me to do histological experiments.
I would like to thank Mr. Thomas J. Quick who taught me to use ICP and made it available for my convenience. I would like to thank Dr. Randy Mitchell for providing me place to grow reeds in greenhouse. I would like to thank Dr. Richard L. Einsporn for helping me with statistical analysis of my data.
I would like to appreciate the support of my group partner Kevin Freese, especially Ziya Erdem who always encouraged me and helped me collect samples. I am also grateful to the help of Chris Menge and Justin Brantner.
Finally, I would like to thank my family and friends for loving me, encouraging me and supporting me through my life.
v TABLE OF CONTENTS Page LIST OF TABLES…………………………………………………………………….xi
LIST OF FIGURES………………………………………………………………….xiv
CHAPTER
I. INTRODUCTION……………………………………………………..…….……...1
1.1 Introduction………………………………………………………………...... 1
1.2 The formation of AMD……………………………………………………….1
1.3 The impact of AMD………………………………………………………….3
1.4 The treatment of AMD…………………………………………………….....4
1.5 Research objectives and approaches………………………………………. ...6
II. LITERATURE REVIEW…………………………………………………...... 10
2.1 Introduction………………………………………………………………....10
2.2 Microorganisms in AMD…………………………………………………....10
2.3 What is phytoremediation…………………………………………………. .12
2.4 Hyperaccumulator…………………………………………………………. .14
2.5 Chelate-assisted phytoremediation………………………………………….15
2.6 Rhizosphere microorganims in phytoremediation………………………….19
2.7 Impacts of chelators on rhizosphere microorganisms………………….…...22
2.8 Histological research in phytoremediation………………………………….23
vi III. MATERIALS AND METHODS…………………………...…………………….25
3.1 Reagent sources……………………………………………………………..25
3.2 Sampling site………………………………………………………………..26
3.3 Spiked soil…………………………………………………………………..28
3.4 Plant source and preparation………………………………………………..29
3.4.1 Purchased non-contaminated reeds……………………………...……29
3.4.2 Wild reeds………………………………………………………...…...30 3.5 Hydroponic experiements cultured with reeds propogated from wild rhizomes………………………………………………...31
3.6 Hydroponic experiements cultured with reeds collected from site E…………………………………………………………….33 3.7 Rhizosphere bacteria: isolation and enrichment…………………………….34
3.8 Rhizobacteira inoculation…………………………………………………...35
3.9 Plates counts for acidophilic heterotrophs and Fe(II)OB…………………...36
3.10 Soils incubation experiments……………………………………………...37
3.11 Establish Fe(II) calibration curve………………………………………….38
3.12 CA biodegradation experiment……………………………………………39
3.13 Measure pH, dissolved oxygen (DO) and conductivity………………...…40
3.14 Analysis of mobile metals in soils…………………………………………40
3.15 Soil digestion: analysis of total metals in soils……………………………41
3.16 DCB extraction and plant digestion……………………………………….41
3.17 ICP-MS method…………………………………………………………....42
3.18 Histological experiments…………………………………………………..43
vii 3.19 Statistical analysis…………………………………………………………44
IV. RESULTS AND DISCUSSIONS………………………………………………...45
4.1 Analysis of samples collected from field………………………………...... 45
4.1.1 Enumeration of acidophilic heterotrophs and Fe(II)OB…...... 45 4.1.2 Soil incubation experiments to assess Fe(II) oxidation kinetic rates………………………………………………………………………....47 4.1.3 Soil digestion and soil pH in field ……………………………..…...... 53
4.1.4 DCB extraction of reeds collected from field………………………...56
4.1.5 Plant digestion of reeds collected from field……………...…….…….62
4.1.6 Histological experiments of reeds collected from field……..……..….68
4.1.6.1 Cross sections of fresh root and rhizome before staining……...68
4.1.6.2 Fe staining for reeds collected from field…………………..…..69
4.1.6.3 Al staining for reeds collected from field…………………...... 75
4.1.7 Summary of field experiments………………………..…….……..….81
4.2 CA biodegradation experiment……………………...………………………82
4.3 Reeds cultured in spiked soil………………………………………………..84
4.3.1 Acidophilic heterotrophs and Fe(II)OB in spiked soil………………..84
4.3.2 pH of soil……………………………………………..……………….90 4.3.3 DCB extraction of reeds cultured in spiked soil for 4, 8 and 12 weeks………………………………………….…………………………….93
4.3.4 Plant digestion of reeds cultured in spiked soil for 4, 8 and 12 weeks……………………………………………………………………....107 4.3.5 Metals concentrations in spiked soil…………………..……...... 127 4.3.6 Histological experiments for Fe in reeds cultured in greenhouse…...140
viii 4.3.6.1 Fe staining for purchased uncontaminated reeds grown in clean soil…...... 141
4.3.6.2 Fe staining for wild reeds cultured in clean and spiked soil…...... 142
4.3.7 Histological experiments for Al in reeds cultured in greenhouse…...... 150
4.3.7.1 Al staining for purchased uncontaminated reeds grown in clean soil…...... 150
4.3.7.2 Al staining for wild reeds cultured in clean and spiked soil…...... 151
4.3.8 Summary of spiked soil experiments treated with purchased and wild reeds……………………….…………………………………..…..…159 4.4 Hydroponic experiments for reeds propogated from wild rhizomes……....160 4.4.1 Rhizosphere bacteria in solution cultured with reeds propagated from wild rhizomes………………………………………………….….....161
4.4.2 pH change of solution cultured with reeds propagated from wild rhizomes…………………………………………...……………...... 167
4.4.3 DCB extraction of reeds propagated from wild rhizomes cultured in solution……………………………………………………...... 171
4.4.4 Digestion of reeds propagated from wild rhizomes cultured in solution…………………………………………………………..…...... 185
4.4.5 Mass balance for metals in solution………………………………....207
4.4.6 Summary of hydroponic experiments of reeds propagated from wild rhizomes………………………...... 211
4.4.7 Comparison between HM hydroponic experiments and spiked soil experiments…………………………..……………...... 213
4.5 Hydroponic experiments for wild reeds collected from site E …………....214
4.5.1 Rhizosphere bacteria in solution cultured with reeds collected from site E…………………………………………………….……………...... 214
ix 4.5.2 pH, conductivity and DO change of solution cultured with reeds collected from site E……………………………………..………..…..…...218
4.5.3 DCB extraction of reeds collected from site E cultured in solution………………………………………………….………..…....…..222
4.5.4 Digestion of reeds collected from site E cultured in solution…………………………..…………………………….……..…....229
4.5.5 Histological experiments of reeds collected from site E cultured in solution……………………………………………………...…………...... 241
4.5.5.1 Fe staining for reeds collected from site E cultured in solution…………………………………………………………...... 241
4.5.5.2 Al staining for reeds collected from site E cultured in solution…………………………………………………………...... 246
4.5.6 Mass balance for metals in solution cultured with reed collected from site E……………………………..…...... 249
4.5.7 Summary of hydroponic experiments cultured with reeds collected from site E…………………………...... 251
4.5.8 Comparison between MM hydroponic experements cultured with reeds propagated from wild rhizomes and with wild reeds collected from site E……………………………………………………………….……....253
V. CONCLUSIONS AND REOMMEDATIONS…………………………...….…..255
5.1 Conclustions………………………………………………………….……255
5.2 Recommedations…………………………………………………………..259
REFERENCES……………………………………………………………………...261
x LIST OF TABLES Table Page
2.1 Research about the effects of CA on metal uptake of plants………………18
3.1 Compositions of solutions in hydroponic experiments...... 32
3.1 Amounts of CA added in hydroponic experiments………………………... 32
4.1 Rhizosphere bacteria in rhizosphere soil collected from different sites...... 45
4.2 pH and concentration of Fe(II) of sterile (abiotic) samples………………. 48
4.3 pH and concentration of Fe(II) of non-sterile (biotic) samples...... 48
4.4 pH and total metals in rhizosphere soils collected in March, 2012……….. 53
4.5 Volume of 1M NaOH to neutralize C6H8O7·H2O solution...... 83
4.6 Numbers of rhizosphere bacteria in soils cultured with reeds (a) for 4 weeks...... 84 (b) for 8 weeks…………………………………………………………….. 85 (c) for 12 weeks…………………………………………...... 86
4.7 pH of spiked soil before and after adding CA...... 90
4.8 pH of spiked soil under different treatments……………………………… 90
4.9 Background values of metal plaque on reeds cultured in spiked soil……...94
4.10 Background values of metal accumulations in reeds cultured in spiked soil………………………………………………………………………...107
4.11 Shoot concentration and translocation factor of purchased reeds cultured in different treatment conditions (a) for 4 weeks………………………... 120 (b) for 8 weeks…………………………………………………………… 121 (c) for 12 weeks………………………………………………………….. 122
xi 4.12 Shoot concentration and translocation factor of wild reeds cultured in different treatment conditions for 4 and 12 weeks………………………..123
4.13 (a) Metals in spiked soil cultured with purchased reeds on 8/29/2012……………………………………………………………... 128 (b) Metals in spiked soil cultured with wild reeds on 8/29/2012………………………………………………………………....128
4.14 (a) Metals in spiked soil cultured with purchased reeds on 8/31/2012………………………………………………………....…... 129 (b) Metals in spiked soil cultured with wild reeds on 8/31/2012………………………………………...………………………. 129
4.15 Metals in soil (mg/kg) cultured with purchased reeds (a) for 4 weeks (b) for 8 weeks………………………………………………………….... 131 (c) for 12 weeks………………………………………………………….. 132
4.16 Metals in soil (mg/kg) cultured with wild reeds (a) for 4 weeks…………132 (b) for 8 weeks (c) for 12 weeks…………………………………………. 133
4.17 Mass balance for (a) Mn in spiked soil cultured with purchased reeds for 12 weeks (b) Fe in spiked soil cultured with purchased reeds for 12 weeks (c) Al in spiked soil cultured with purchased reeds for 12 weeks………...138
4.18 Mass balance for (a) Mn in spiked soil cultured with wild reeds for 12 weeks (b) Fe in spiked soil cultured with wild reeds for 12 weeks (c) Al in spiked soil cultured with wild reeds for 12 weeks……………………. 139
4.19 Numbers of rhizosphere bacteria in (a) LM solution cultured with reeds for 4, 8 and 12 weeks…………………………………………………….. 162 (b) MM solution cultured with reeds for 4, 8 and 12 weeks……………...163 (c) HM solution cultured with reeds for 4, 8 and 12 weeks……………....164
4.20 (a) pH of LM solution under different treatment conditions (b) pH of MM solution under different treatment conditions……………………….168 (c) pH of HM solution under different treatment conditions……………..169
4.21 Background values of metal plaque on reeds propagated from wild rhizome…………………………………………………………………... 171
4.22 Concentrations of free inorganic and organically complexed (a) Fe in LM solution (b) Mn in LM solution (c) Al in LM solution……………… 183
xii 4.23 Concentrations of free inorganic and organically complexed (a) Fe in MM solution (b) Mn in MM solution (c) Al in MM solution…………….184
4.24 Concentrations of free inorganic and organically complexed (a) Fe in HM solution……………………………………………………………… 184 (b) Mn in HM solution (c) Al in HM solution…………………………… 185
4.25 Background values of metal accumulations in reeds propagated from wild rhizomes……………………………………………………………. 185
4.26 Shoot concentration and translocation factor of metals in reeds cultured in (a) LM solution for 4, 8 and 12 weeks ………………………………...203 (b) MM solution for 4, 8 and 12 weeks………………………………….. 204 (c) HM solution for 4, 8 and 12 weeks………………………………...... 205
4.27 Mass balance for metals in (a) LM solution cultured with reeds for 12 weeks ………………………………………………………………….208 (b) MM solution cultured with reeds for 12 weeks……………………… 209 (c) HM solution cultured with reeds for 12 weeks………………………. 210
4.28 Numbers of rhizosphere bacteria in solution cultured with reeds collected from site E for 4, 8 and 12 weeks………………………………………... 216
4.29 (a) pH of solution under different treatment conditions (b) Conductivity (ms/cm) of solution under different treatment conditions (c) DO (mg/L) of solution under different treatment conditions………………………… 220
4.30 Background values of metal plaque on reeds collected from site E cultured in solution…………………………………………………………………222
4.31 Background values of metal accumulations in reeds collected from site E cultured in solution……………………...……………………………….. 229
4.32 Shoot concentration and translocation factor of metals in reeds collected from site E cultured in solution for 4, 8 and 12 weeks…………………... 239
4.33 Mass balance for metals in solution cultured with reeds collected from site E for 12 weeks………………………………………………………. 250
xiii LIST OF FIGURES Table Page
3.1 Aerial image of sampling site……………………………………………… 27
3.2 Rhizosphere soils collected from the field………………………………… 27
3.3 Pictures of Phragmites australis (a) propagated from purchased uncontaminated rhizomes (b) collected from the wild……………………. 31
3.4 Fe(II) calibration curve…………………………………………………….. 39
3.5 CA and NaOH calibration curve…………………………………………... 40
4.1 (a) Fe2+ concentration and pH of soil collected from sampling point A on March 16, 2012 (b) Fe2+ concentration and pH of soil collected from sampling point B on March 16, 2012……………………………………... 49 (c) Fe2+ concentration and pH of soil collected from sampling point C on March 16, 2012 (d) Fe2+ concentration and pH of soil collected from sampling point D on March 16, 2012 (e) Fe2+ concentration and pH of soil collected from sampling point E on March 16, 2012………………… 50
4.2 Mobile metals in rhizosphere soils collected on March 16, 2012…………. 54
4.3 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on March 16, 2012……………………………………………………………. 57
4.4 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on January 9, 2013……………………………………………………………. 58
4.5 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on May 20, 2013……………………………………………………………… 59
4.6 (a) Mn (b) Fe and (c) Al in reeds collected on March 16, 2012…………… 63
4.7 (a) Mn (b) Fe and (c) Al in reeds collected on January 9, 2013…………… 64
4.8 (a) Mn (b) Fe and (c) in reeds collected on May 20, 2013…………………65
4.9 Cross sections of rhizome prior to staining (a) rhizomes at 1 cm scale (b) 10 µm cross section of depiction (c) vascular bundle of rhizome……... 69
xiv 4.10 Cross sections of non-stained Phragmites australis root (a) root at 1 cm scale (b) 10 µm cross section of root (c) subsection of the root (d) part of root……………………………………………………………………... 69
4.11 Fe stained cross section of root of reed collected from site A in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 70
4.12 Fe stained cross section of root of reed collected from site B in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 70
4.13 Fe stained cross section of root of reed collected from site C in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 70
4.14 Fe stained cross section of root of reed collected from site D in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 71
4.15 Fe stained cross section of root of reed collected from site E in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis……71
4.16 Fe stained cross section of root of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013………………………... 71
4.17 Fe stained cross section of rhizome of reeds collected from site A in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection that to clearly show the sequestration of iron…………………. 73
4.18 Fe stained cross section of rhizome of reeds collected from site B in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 73
4.19 Fe stained cross section of rhizome of reeds collected from site C in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 73
4.20 Fe stained cross section of rhizome of reeds collected from site D in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 74
4.21 Fe stained cross section of rhizome of reeds collected from site E in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 74
4.22 Fe stained cross section of rhizome of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013………………... 74
4.23 Al stained cross section of root of reed collected from site A in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 76
xv 4.24 Al stained cross section of root of reed collected from site B in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 76
4.25 Al stained cross section of root of reed collected from site C in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…....76
4.26 Al stained cross section of root of reed collected from site D in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 76
4.27 Al stained cross section of root of reed collected from site E in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis…... 77
4.28 Al stained cross section of rhizome of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013………………... 77
4.29 Al stained cross section of rhizome of reeds collected from site A in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 78
4.30 Al stained cross section of rhizome of reeds collected from site B in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 78
4.31 Al stained cross section of rhizome of reeds collected from site C in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 79
4.32 Al stained cross section of rhizome of reeds collected from site D in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection ……………………………………………...…………………. 79
4.33 Al stained cross section of rhizome of reeds collected from site E in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection…………………………………………………………………. 79
4.34 Al stained cross section of rhizome of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013………………... 80
4.35 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks………………………………….. .94
4.36 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks…………………………………... 95
4.37 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks…………………………………. 96
4.38 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks…………………………………... 98 xvi 4.39 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks…………………………………... 99
4.40 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks………………………………... 100
4.41 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks…………………………………. 103
4.42 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks…………………………………. 104
4.43 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks………………………………... 105
4.44 Mn concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks…………………………………. 108
4.45 Mn concentration (a) in the tissues of purchased reed and (b) in the leaves of wild reed cultured in spiked soil for 8 weeks………………….. 109
4.46 Mn concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks……………………………………... 110
4.47 Fe concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks…………………………………. 111
4.48 Fe concentration (a) in the tissues of purchased reed and (b) in the leaves of wild reed cultured in spiked soil for 8 weeks…………………………. 112
4.49 Fe concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks……………………………………... 113
4.50 Al concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks………………………………………. 114
4.51 Al concentration (a) in the tissues of purchased reed and (b) in the leaves of wild reed cultured in spiked soil for 8 weeks…………………………. 115
4.52 Al concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks………………………………... 116
4.53 The fate of metals in spiked soil experiments……………………………. 137
4.54 Fe stained cross section of root of uncontaminated reeds grown in clean soil, (a) magnified image of stele(b) magnified image of exodermis…….141
xvii 4.55 Fe stained cross section of rhizome of uncontaminated reeds grown in clean soil, (a) full view of the cross section with image (b) a magnified subsection………………………………………………..141
4.56 Fe stained cross section of root of wild reed grown in clean soil, (a) magnified image of stele (b) magnified image of exodermis………....142
4.57 Fe stained cross section of root of reed grown in spiked soil HCNB, (a) magnified image of stele (b) magnified image of exodermis………... 142
4.58 Fe stained cross section of root of reed grown in spiked soil HCWB, (a) magnified image of stele (b) magnified image of exodermis………... 143
4.59 Fe stained cross section of root of reed grown in spiked soil MCNB, (a) magnified image of stele (b) magnified image of exodermis………... 143
4.60 Fe stained cross section of root of reed grown in spiked soil MCWB, (a) magnified image of stele (b) magnified image of exodermis………... 143
4.61 Fe stained cross section of root of reed grown in spiked soil LCNB, (a) magnified image of stele (b) magnified image of exodermis………... 144
4.62 Fe stained cross section of root of reed grown in spiked soil LCWB, (a) magnified image of stele (b) magnified image of exodermis………... 144
4.63 Fe stained cross section of root of reed grown in spiked soil NCNB, (a) magnified image of stele (b) magnified image of exodermis…………144
4.64 Fe Stained cross section of root of reed grown in spiked soil NCWB, (a) magnified image of stele (b) magnified image of exodermis………... 145
4.65 Fe stained cross section of rhizome of reeds in clean soil, (a) full view of the cross section with image (b) a magnified subsection that to clearly show the sequestration of iron……………………………. 146
4.66 Fe stained cross sections of rhizomes of reeds in spiked soil HCNB, (a) full view of the cross section with image (b) a magnified subsection...147
4.67 Fe stained cross sections of rhizomes of reeds in spiked soil HCWB, (a) full view of the cross section with image (b) a magnified subsection...147
4.68 Fe stained cross sections of rhizomes of reeds in spiked soil MCNB, (a) full view of the cross section with image (b) a magnified subsection...147
4.69 Fe stained cross sections of rhizomes of reeds in spiked soil MCWB, (a) full view of the cross section with image (b) a magnified subsection...148
4.70 Fe stained cross sections of rhizomes of reeds in spiked soil LCNB, (a) full view of the cross section with image (b) a magnified subsection...148 xviii 4.71 Fe stained cross sections of rhizomes of reeds in spiked soil LCWB, (a) full view of the cross section with image (b) a magnified subsection...148
4.72 Fe stained cross sections of rhizomes of reeds in spiked soil NCNB, (a) full view of the cross section with image (b) a magnified subsection...149
4.73 Fe stained cross sections of rhizomes of reeds in spiked soil NCWB, (a) full view of the cross section with image (b) a magnified subsection...149
4.74 Al stained cross section of root of uncontaminated reed grown in clean soil, (a) magnified image of stele (b) magnified image of exodermis……151
4.75 Al stained cross section of rhizome of uncontaminated reed grown in clean soil, (a) full view of the cross section with image (b) a magnified subsection………………………………………………..151
4.76 Al stained cross section of root of wild reed grown in clean soil, (a) magnified image of stele (b) magnified image of exodermis………... 152
4.77 Al stained cross section of root of reed grown in spiked soil HCNB, (a) magnified image of stele (b) magnified image of exodermis………... 152
4.78 Al stained cross section of root of reed grown in spiked soil HCWB, (a) magnified image of stele (b) magnified image of exodermis………... 152
4.79 Al stained cross section of root of reed grown in spiked soil MCNB, (a) magnified image of stele (b) magnified image of exodermis………... 153
4.80 Al stained cross section of root of reed grown in spiked soil MCWB, (a) magnified image of stele (b) magnified image of exodermis………... 153
4.81 Al stained cross section of root of reed grown in spiked soil LCNB, (a) magnified image of stele (b) magnified image of exodermis………... 153
4.82 Al stained cross section of root of reed grown in spiked soil LCWB, (a) magnified image of stele (b) magnified image of exodermis ………...154
4.83 Al stained cross section of root of reed grown in spiked soil NCNB, (a) magnified image of stele (b) magnified image of exodermis 154
4.84 Al stained cross section of root of reed grown in spiked soil NCWB, (a) magnified image of stele (b) magnified image of exodermis………... 154
4.85 Al stained cross section of rhizome of wild reeds in clean soil, (a) full view of the cross section with image (b) a magnified subsection...155
4.86 Al stained cross sections of rhizomes of reeds in spiked soil HCNB, (a) full view of the cross section with image (b) a magnified subsection...156
xix 4.87 Al stained cross sections of rhizomes of reeds in spiked soil HCWB, (a) full view of the cross section with image (b) a magnified subsection...156
4.88 Al stained cross sections of rhizomes of reeds in spiked soil MCNB, (a) full view of the cross section with image (b) a magnified subsection...156
4.89 Al stained cross sections of rhizomes of reeds in spiked soil MCWB, (a) full view of the cross section with image (b) a magnified subsection...157
4.90 Al stained cross sections of rhizomes of reeds in spiked soil LCNB, (a) full view of the cross section with image(b) a magnified subsection... 157
4.91 Al stained cross sections of rhizomes of reeds in spiked soil LCWB, (a) full view of the cross section with image (b) a magnified subsection...157
4.92 Al stained cross sections of rhizomes of reeds in spiked soil NCNB, (a) full view of the cross section with image (b) a magnified subsection...158
4.93 Al stained cross sections of rhizomes of reeds in spiked soil NCWB, (a) full view of the cross section with image (b) a magnified subsection...158
4.94 Mn plaque on the roots and rhizomes of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks………………………………………………. 173
4.95 Mn plaque on the roots and rhizomes of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks………………………………………………. 174
4.96 Fe plaque on the roots and rhizomes of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks………………………………………………. 176
4.97 Fe plaque on the roots and rhizomes of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks………………………………………………. 177
4.98 Fe plaque on the roots and rhizomes of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks………………………………………………. 178
4.99 Mn concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...186
4.100 Mn concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...187
4.101 Mn concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...188
4.102 Fe concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...189
xx 4.103 Fe concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...190
4.104 Fe concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...191
4.105 Al concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...192
4.106 Al concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...193
4.107 Al concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks…………………………………………………...194
4.108 Mn plaque on the roots and rhizomes of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks…………………………. 223
4.109 Fe plaque on the r. oots and rhizomes of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks…………………………. 226
4.110 Mn concentration in the organs of reeds collected from site E cultured in solution for(a) 4 (b) 8 (c) 12 weeks…………………………………… 231
4.111 Fe concentration in the organs of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks………………………………….. .232
4.112 Al concentration in the organs of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks…………………………………... 233
4.113 Fe stained cross section of reed grown in control solution (a) root (b) Rhizome…………………………………………………………………...241
4.114 Fe stained cross section of roots of reeds grown in treatment solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e)NCFB (f) WCFB (g) NCAFB (h) WCAFB………………………………………………… 242
4.115 Fe stained cross section of rhizomes of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e)NCFB (f) WCFB (g) NCAFB(h) WCAFB…………………………………………………. 245
4.116 Al stained cross section of reeds grown in control solution (a) root (b) rhizome………………………………………………………………. 246
4.117 Al stained cross section of roots of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e) NCFB (f) WCFB (g) NCAFB (h) WCAFB………………………………………………… 247
xxi 4.118 Al stained cross section of rhizomes of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB(d) WCAB (e)NCFB (f) WCFB (g) NCAFB (h) WCAFB………………………………………………… 248
xxii CHAPTER I
INTRODUCTION
1.1 Introduction
Acid mine drainage (AMD) is a serious environmental issue which is mainly caused by both working and abandoned mine operations (Whitehead and Prior, 2005).
Many regions of the United States and other countries have been adversely affected by AMD (Batty and Younger, 2004; Bird, 1987; Herlihy et al., 1990).
The typical characteristics of AMD include low pH, low amounts of organic compounds, high levels of sulfate, high concentrations of metals such as Fe, Mn, Al and Cu, and high conductivity (Batty et al., 2000; Herlihy et al., 1990). AMD can cause serious pollution of surface water, groundwater and soil upon its emergence to the surrounding environment.
1.2 The formation of AMD
AMD is produced when sulfide minerals which were initially under anoxic conditions are exposed to oxygen and water during mine activity (Akcil and Kodals,
2006). AMD is usually associated with iron sulfide, which is the most common sulfide mineral (Akcil and Kodals, 2006). However, AMD can form in other sulfide minerals, such as covellite (CuS) and sphalerite (ZnS) (Skousen et al., 2000).
1 The formation of AMD is site specific, because many factors can affect its formation such as mineralogy particle size, presence of oxidizers (Akcil and Kodals,
2006). The main factors in the chemical and biological reactions for AMD generation include sulfide minerals, water or a humid atmosphere and an oxidant, particularly oxygen from the environment (Akcil and Kodals, 2006). The production of AMD can be illustrated by taking FeS2 as an example.
2- As shown in equation (1) (Akcil and Kodals, 2006), iron is dissolved and S2 is oxidized to produce hydrogen ions and sulfate when exposed to oxygen-rich waters.
2- 2+ + FeS2 (S) + 3.5 O2 + H2O → 2 SO4 + Fe + 2 H (1)
2+ 2- + The dissolved Fe , SO4 and H can cause a decrease in pH of the water. Under lower pH conditions, ferrous iron (Fe2+) can be further oxidized to ferric iron (Fe3+) according to equation (2) (Akcil and Kodals, 2006),
2+ + 3+ 2 Fe + 0.5 O2 + 2 H → 2 Fe + H2O (2)
Oxidation of the ferrous ion to ferric ion occurs more slowly at lower pH values, but some bacteria can catalyze this reaction (U.S. EPA, 1994). For instance, at pH between 3.5 and 4.5, iron oxidation can be accelerated by a variety of a filamentous bacterium, Metallogeniu. When pH is below 3.5, iron bacterium such as
Acidithiobacillus ferrooxidans is able to catalyze the iron oxidation (U.S. EPA, 1994).
3+ When Fe is brought into contact with pyrite FeS2, pyrite can be dissolved according to equation (3), thus producing more Fe2+ and acidity (Akcil and Kodals,
2006). The dissolution of pyrite by ferric iron (Fe3+) (equation (3)) combing with the oxidation of the ferrous ion (equation (2)) consist a cycle of dissolution of pyrite 2 (Younger et al., 2002). As shown in equation (4), Fe3+ can precipitates as iron hydroxide Fe(OH)3, a yellow, red or orange precipitate (Akcil and Kodals, 2006),
3+ 2- 2+ + 14Fe + FeS2 (s) + 8H2O → 2SO4 + 15Fe + 16H (3)
3+ + 2Fe + 6H2O → 2Fe(OH)3 (s) + 6H (4)
Fe(OH)3 coats plants, macroinvertebrates and sediments in the stream beds, damage habitats and pollute environments (Senko et al., 2008).
1.3 The impact of AMD
AMD can severely contaminate soils, affect water quality and pollute ecological environments because of the low pH and high concentrations of metals and other toxic elements (Peppas et al., 2000). About 20,000 to 50,000 mines were reported as producing acid on Forest Service lands; the AMD from these mines impacting 8,000 to 16,000 km of streams (U.S. EPA, 1994). Once it enters local water, AMD can acidify and decrease pH of water to as low as 2.0 to 4.5, which is harmful to most of aquatic life (Hill, 1974). The results of National Stream Survey (NSS) conducted by the U.S. Environmental Protection Agency showed that 4590±1670 km of streams were acidic because of AMD and another 5780±2090 km of streams were also affected by mine drainage without being acidic in the eastern United States in 1989
(Herlihy et al., 1990). AMD can cause metals, such as Fe, Al, Mn, Zn and Cd, to leach from mine wastes, enter waters and soils and then become available to organisms. Most of these metals are toxic which can affect human health and cause ecological damages. The formation of the red or orange colored precipitate Fe(OH)3 3 found in thousands of miles of streams affected by AMD may physically coat the surface of stream sediments and streambeds, damaging habitat, reducing availability of clean gravels used for spawning, and decreasing fish food items (Jennings et al.,
2008). The repots of Forest Service indicated that the environmental damages caused by metal load were greater than the acidity produced by AMD (U.S. EPA, 1994).
1.4 The treatment of AMD
The main aims of AMD treatment are to neutralize acidity and remove dissolved metals. Passive and active technologies can be utilized to treat AMD. Active AMD treatment systems consist of highly engineered water treatment facilities such as chemical dosing, and nanofiltration (Jennings et al., 2008; Mohapatra et al., 2011).
Alkaline material such as lime (CaO) is often added into AMD to raise alkalinity and pH. A passive treatment technology often operates without mechanized assistance, but use the physical/chemical, or biological processes that often occur naturally in the environment (Jennings et al., 2008; Mohapatra et al., 2011). Most of passive treatment technologies are based on wetland ecosystems. The major advantage of wetlands is their low cost to establish and maintain in comparison to traditional active treatment system. Wetlands have been widely used to treat AMD for several years because of its low costs and ecological benefits. According to the U.S. Bureau of Mines, over 400 wetlands had been established and successfully used in the United States to treat
AMD in 1991 (Mitsch and Wise, 1998). Wetlands are very complex ecosystems. The mechanisms involved in AMD treatment in wetlands are also complicated which 4 include ion change, metal uptake by plants, filtration of colloidal and suspended materials and microbial mediated transformation and so on (Jonson, 1995a).
Microorganisms and plants play an important interconnected role in wetlands for
AMD mitigation.
Vegetation is a significant composition of wetlands designed to treat AMD
(Batty and Younger, 2004). Phytoremediation is one of the main heavy metals removal mechanisms that occur in wetlands. Phytoremediation includes various plant-based technologies that use either naturally growing, or genetically engineered plants to restore contaminated environment (Ghosh and Singh, 2005). Wetland plants species that can survive under atrocious conditions (i.e. low pH and low levels of nutrients, high concentrations of metal concentrations), such as common reed
Phragmites australis and cattail Typha latifolia are commonly used in passive treatment systems (Stoltz and Greger, 2006; Batty and Younger, 2004).
Metal bioaccumulation by aquatic plants has been widely studied in wetlands treating AMD. A number of experimental studies have shown that plants, such as
Phragmites australis, Typha latifolia and Eriophorum angustifolium can be used to accumulate metals from AMD contaminated soil (Ye et al., 1997a; Stoltz et al., 2006;
Szymanowska et al., 1999). However, the strategy by which the plants can acclimate to and survive in the highly metalliferous environments is still unclear (Batty et al.,
2000). Furthermore, detains pertaining to metal uptaken and distribution in the plants and the factors that affect the metal accumulation is also limited (Weis and Weis,
2004). 5 1.5 Research objectives and approaches
The overall goal of this research was to study the bioremediation potential of an
AMD contaminated site via common reed Phragmites australis and rhizosphere bacteria. To achieve this goal, several field and laboratory experiments were conducted. Use of field samples (plants, rhizosphere soil and bacteria) under lab conditions could afford realistic conditions without disrupting the site. Laboratory assessments were used to get a better understating of interconnected complex factors prior to initiating any changes at a site. The specific research objectives included:
1. Field characterization
The main hypotheses in filed experiments were that rhizosphere bacteria and
plants in the field may have adapted to the AMD sites and may play a role in
affecting the bioremediation potential of the sites. The main tasks included:
(1) Study the physicochemical and biological conditions of the site to gain
critical insight of key parameters prior to initiating the lab experiments:
a: Determine the pH and the concentrations of heavy metals Fe, Al and Mn in
rhizosphere soil from different locations.
b: Quantify the populations of acidophilic heterotrophs and Fe(II) oxidizing
bacteria (Fe(II)OB) in rhizosphere soil at sampling locations and assess if there
are enough bacteria can be used for lab experiments.
c: Assess the role of indigenous Fe(II)OB in Fe(II) oxidation and determine the
kinetic rates of Fe(II) oxidation.
6 (2) Study the phytoremediation potential of the indigenous plants Phragmites australis:
a: Determine the amounts of metal plaques formed on the surface of roots and rhizomes of reeds collected from different locations at the AMD contaminated site and in different seasons;
b: Study the metal contents and distribution in plant tissues and translocation factors for different metals in reeds collected from different locations and different seasons.
2. Laboratory assessment
The main hypotheses of lab experiments were that the interactions between chelator citric acid (CA) and rhizosphere bacteria may affect metal mobility, metal plaque formation and metal uptake in different types of reeds (wild reeds collected from an AMD contaminated site; reeds propagated from purchased uncontaminated rhizomes or reed grown from wild rhizomes collected from the
AMD contaminated field). The main tasks included:
(1) Study the effect of CA and rhizosphere bacteria (acidophilic heterotrophs and
Fe(II)OB) on metal plaque formation on different types of reeds cultured in spiked soil or hydroponic solution for different time periods. Both soil and hydroponic systems were studied as Phragmites australis can grow in both terrestrial and hydroponic habitats. In addition, hydroponic experiments may afford additional insight as they have less mass transfer limitations that that associated with soil. 7 (2) Determine the influence of CA and rhizosphere bacteria on enhancing metal uptake in different reeds cultured in spiked soil or hydroponic solution for different time periods (4 weeks, 8 weeks or 12 weeks).
(3) Assess the change of pH, dissolved oxygen (DO), conductivity, population of rhizosphere bacteria, total and mobile metals in spiked soil or hydroponic solution for different time periods (4 weeks, 8 weeks or 12 weeks).
(4) Ascertain the specific plant organs that sequestered metals in fresh roots and rhizomes to assess whether there were histological alternations in roots and rhizomes grown under different conditions.
(5) Determine the best combinations of CA and rhizosphere bacteria to optimize the metal accumulation in reeds and find the potential method to enhance remediation of sites.
The specific approaches utilized to achieve above objectives included:
(1) Different types of reeds were cultured in spiked soil or hydroponic solutions treated with different levels of CA and rhizosphere bacteria for different growth periods (4 weeks, 8 weeks or 12 weeks);
(2) Every 4 weeks, reeds from each treatment were sacrificed to analyze the extent of metal plaque formed on the surface of fresh root and rhizome. Next, the amounts of heavy metals accumulated in different tissues of reeds were determined.
(3) At the same time reeds were harvested, rhizosphere soil or solution was collected to enumerate acidophilic heterotrophs and Fe(II)OB, as well as to 8 determine the pH, DO, conductivity, total and mobile metals contents in the rhizosphere soil or hydroponic solution.
(4) A mass balance of the metals were used to analyze the relationship among plants type, growth period, amounts of CA, pH, DO, conductivity and metals concentrations in soil or solution, growth of rhizosphere bacteria and metal accumulations in plants.
9 CHAPTER II
LITERATURE REVIEW
2.1 Introduction
AMD is a serous environmental pollution problem in many regions of the world.
Wetlands have been established to remove toxic metals from AMD contaminated water and soil for many years (Wildeman and Laudon, 1989; Dodds-Smith et al.,
1995; Scholes et al., 1998). Biological activity plays an important role in wetland for
AMD mitigation. For instance, many processes occur in wetlands, such as ammonification, denitrification and reduction of sulphur have been identified as microbially-driven (Johnson, 1995a). Macrophytes also play important roles in contaminants removal and marsh biogeochemistry through their active and passive circulation of elements (Weis and Weis, 2004).
2.2 Microorganisms in AMD
AMD was initially thought to have limited microbial activity, but in fact AMD microbial environments are diverse and variable (Leduc et al., 2002). The variation and complexity of microorganisms can be very dramatic even within a short distance in the same wetland (Johnson, 1995a). Most microorganisms which are active in
AMD are acidophic and acid-tolerant microorganisms (Johnson, 1995a). pH for the
10 growth of acidophic microorganism are between 2 and 4. The suitable pH for the growth of acid-tolerant microorganism is above the typical pH value in AMD site, but they still can be active in low pH site (Johnson, 1995a). Iron-oxidizing chemolithotrophs such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans in AMD have been widely studied (Baker and Banifield, 2003). The oxidation of ferrous iron to ferric in AMD is greatly accelerated by iron oxidizing bacteria. For instance, the oxidation rates at low pH can be increased even by five orders of magnitude by the iron oxidizing bacteria (Baker and Banifield, 2003).
Acidophilic, non-iron oxidizing microorganisms are also important groups in
AMD. Some of the species can metabolize organic materials that are toxic to autotrophic iron-oxidizing bacteria, while others can reduce ferric iron and be involved in iron cycling in AMD (Johnson, 1995a). Fungi and yeasts have also been identified from AMD, although the diversity is less than that of eukaryotic life (Tuttle et al., 1968; Wichlacz and Unz, 1981).
Quantification and understanding the roles of different microorganisms in AMD may be helpful to select suitable a method to both control and treat AMD. One approach to treat AMD is to add organic materials into AMD contaminated media or cause AMD to flow through reactors packed with organic matter and microorganisms
(Johnson, 1995a). The organic matter may inhibit the activity of autotrophic, iron-oxidizing organisms by destroying the cellular membrane (Marchand and
Silverstein, 2003). Organic matter may also increase the growth of acidophilic heterotrophs and decrease dissolved oxygen levels, then stimulate the reduction of 11 ferric iron (Johnson, 1995a). According to the research of Kolmert and Johnson
(2001), the use of immobilized sulfate-reducing bacteria were also capable of increasing the pH of AMD. Some metal-immobilizing acidophilic bacteria acting as biosorbents for the accumulation of metals can also be used to detoxify AMD
(Brierley, 1990).
2.3 What is phytoremediation
Phytoremediation is defined as the use of plants, either naturally growing, or genetically engineered plants to remove, destroy or sequester contaminants from various media such as soil, water and air and to restore contaminated environment
(Karami and Shamsuddin, 2010; Ghosh et al., 2005). As a clean, simple, cost effective, non-environmentally disruptive green technology, phytoremediation is widely accepted by the public (Hemen, 2011).
There are different categories of phytoremediation based on the mechanisms for cleaning the polluted medium. Phytoextraction is defined as plants absorb contaminants such as metals, from soil through the root systems and transfer them to harvestable shoots (Hemen, 2011). Phytovolatilization occurs when plants uptake certain metals from soil and then release them into the air by volatilization (Hemen,
2011). For instance, during phytovolatilization, mercuric ion is transformed into less toxic elemental mercury (Ghosh et al., 2005). In phytostabilization, the plant roots absorb the pollutants from the soil and prevent them from leaching by holding them in the rhizosphere (Lone et al., 2008). In phytofiltration, the plants roots or seedlings are 12 used to absorb/adsorb metals from water and aqueous waste stream (Hemen, 2011).
The use of plants and rhizosphere bacteria to degrade organic pollutants is phytodegradation (Garbisu et al., 2001). Some researchers have further separated phytoremediation into some other categories, such as phytoextraction, phytofiltration, rhizodegradation, phytotransformation, etc. (Alkorta et al., 2004). In any event, all these phytoremediation technologies are not exclusive and may be used simultaneously.
Abundant research has been conducted to study the uptake and accumulation of metals in various plants. More than 500 plant species from 101 families have been investigated and reported (Hemen, 2011), such as Pteris vittata (ladder brake),
Brassica juncea (Indian mustard) and Thlaspi caerrulescens (Alpine Pennygrass) and
Helianthus annuus (dwarf sunflowers). Some wetland plants have also been studied for their potential to phytoremediation, such as Typha latifolia (cattail) and
Phragmites australis (common reed). Phragmites australis is most abundant in North
America and is one of the most widespread plant species in the world (Reale et al.,
2012). It has been widely used in natural and constructed wetlands to remove and accumulate contaminants because of its many advantages: such as strong adaptability to different climatic conditions; ability to grow in hydroponic or terrestrial habitats; high production of biomass and high tolerance to some heavy metals and organic matters (Baldantoni et al., 2009).
As a relatively new technology, phytoremediation has its own limitations. First, suitable climatic conditions, appropriate soil properties and essential nutritional 13 materials, are needed to support the normal growth of plants (Karami et al., 2010).
Second, the long time required of this technique also limits its application (Karami et al., 2010). Other disadvantages include the low biomass of some plants, the limited tolerance of plants at high concentration of contaminants and the limitations when the contaminated soil layer is deep (Karami et al., 2010). Another concern pertains to the contaminated plant materials. The main methods include land filling, composting, incineration (Keller et al., 2005). However, these methods may enable the contaminants to migrate into other mediums of the environment, such as ground water.
For instance, landfilling may lead the risk of groundwater pollution (Karami et al.,
2010).
The use of phytoremediation is still in the development phase. Efforts have been made to overcome the above mentioned limitations. Recent research has focused on adding chelators to increase the bioavailability of heavy metals, thereby enhancing the uptake of metals by plants; injecting rhizosphere microorganisms to stimulate the growth of plants or to increase metals accumulation and application of fertilizers to increase the biomass of plants and then increase the metal accumulation.
2.4 Hyperaccumulator
The term hyperaccumulator was first applied for Ni uptake. The term was used for concentrations of more than 1000 mg/kg Ni in plant tissues during the study of
Homalium and Hybanthus from different sites throughout the world
(Gardea-Torresdey et al., 2005). However, the criterion was modified since some 14 heavy metals such as Cd and Cr are more toxic for plants than Ni (Gardea-Torresdey et al., 2005). At present, the definition of a hyperaccumulator is more extensive.
Hyperaccumulator species are the plants whose shoots contain>100 mg/kg Cd, 1000 mg/kg Pb, Al, Ni and Cu or>10000 mg/kg Zn, Fe and Mn (dry weight) when grown in metal-rich media (Marques et al., 2009). The translocation factor (ratio of shoot to root concentration) and bioaccumulation factor (the shoot to soil concentration ratio) are often taken into consideration to assess the ability of plants to uptake metals from media (Marques et al., 2009).
2.5 Chelate-assisted phytoremediation
Chelate-enhanced phytoextraction is when chelators are applied to contaminated soil, to enhance metal bioavailability and subsequent increase accumulation by plants
(Garbisu and Alkorta, 2001). There are different definitions of bioavailability (Semple et al., 2004). The terms “availability” and “bioavailability” can be utilized interchangeably (Kirkham, 2006). Availability means the rate and extent at which a chemical is released from a medium or can be obtained by living receptors such as plants roots by direct contact or uptake (Kirkham, 2006). In general, only a fraction of metals in soil is available for plants. Some metals occur primarily as soluble or exchangeable forms which are available for plants (Lasat, 2002). Some metals bind within the soil matrix that can limit the potential for phytoremediation (Lasat, 2002).
Chelating agents can be used to increase the bioavailability of heavy metals in soil by different mechanisms (Komárek et al., 2007). For instance, chelators can be used to 15 dissolve precipitated compounds or desorp of sorbed chemicals. Chelating agents can also prevent precipitation and sorption of the metals in soil through the formation of metal-chelate complexes with metals, thereby maintaining their availability for plant uptake (Marques et al., 2009). There are several reports about the interactions of metal ions and cheltors. (Lopez-quintela et al., 1984) indicated that strong complexes can be formed between metal ions (e.g. A13+ and Fe3+) and chelating ligands (e.g. polycarboxylic acids). ARP and Meyer (1985) studied the associated formation constants between metal ions (Fe3+ and A13+) and lower molecular weight organic acids (i.e. salicylic, phthalic, oxalic and citric acid). The exact mechanism of organic metal-ion chelation probably involves several reactive groups such as carboxyl, hydroxyl, carbonyl, amine, and quinone groups (Arp and Meyer, 1985). Many factors affect the complex reaction between chelators and metals, such as pH and stability constant. According to the research of Yuchi et al. (1986), citrate is more effective for cmplexing aluminum than ethylenediaminetetraacetic acid (EDTA) due to the lower conditional formation constants of the mixed ligand complexes. Aslo, the pH affected the formation of complex (Yuchi et al., 1986). Guardado et al. (2007) indicated that the stability constants and pH also affect the formation phosphate-metal-humic complexes.
Several chelating agents, such as EDTA, hydroxyethylethylene-diaminetriacetic acid (HEDTA), ss-ethylenediaminedissucinic acid (EDDS), nitrilotriacetic acid
(NTA), citric acid (CA) have been studied and applied to increase the bioavailability of heavy metals and enhance the metals accumulation in plants. Huang (1997) 16 reported that the application of EDTA to soil artificially spiked with 2500 mg/kg Pb could increase shoot lead concentration of Zea mays (corn) and Pisun sativum (pea) from about 500 mg/kg to more than 10,000 mg/kg. Zhao et al. (2011) showed that 10 mmol/kg EDTA increased the shoot Cu concentration in Lolium perenne L. by 3.2 fold and enhanced the Zinc and Pb concentrations in shoots by 1.2 and 2.1 fold.
Duquène et al. (2009) reported that 19, 34, and 37 folds increase were achieved in shoots of Indian mustard (Brassica juncea) for U, Pb, and Cu with 5 mmol/kg EDDS addition. Shen et al. (2002) indicated that 1.5 mmol/kg NTA increased Pb concentration in the shoot of cabbage by 8 fold. According to Quartacci et al. (2007), a 2 to 3 fold increase of Cd, Cu, Pb and Zn concentrations in shoots of Indian mustard has been observed after application of 5 mmol kg-1 NTA.
Nevertheless, some concerns have been proposed about the potential risk related to this technique. The high concentration of chelators may be toxic to plants by mobilizing too high of a metal concentration. Chlorosis, necrosis, and impairment of plant growth have been observed in plants growing in soils treated with EDTA, EDDS and NTA (Marques et al., 2009). Besides, the use of non-biodegradable chelating agents, such as EDTA, can create a new source of pollution by causing the leaching of metals into the ground water (Santos et al., 2006). Grčman et al., (2001) reported that addition of 10 mmol/kg EDTA soil resulted in the increased Pb, Zn and Cd concentrations in the shoot of Brasica rapa, however, 40% of the total applied EDTA was leached into the soil profile. Similar trends were also observed for EDDS
(Marques et al., 2009). 17 Alternatively, some other chelators, such as CA, can be easier to be biodegraded and have gotten greater social acceptance (Muhammad et al., 2009). It has also been widely reported that CA can increase the bioavailability of heavy metals in soils thus enhance the metals accumulation in plants. According to the results of Elkhatib et al.
(2001), 2 mmol/kg CA was more effective than EDTA and HEDTA in increasing Cd accumulation in sunflower. It increased the Cd shoot uptake in sunflower from 125 to
256 mg/kg. The results of the study of do Nascimento et al. (2006) also proved that the removal of Cd, Zn, Cu, and Ni by Indian mustard from multi-metal contaminated soils can be enhanced by CA. Mihalík et al. (2010) found that the accumulation of the total U content achieved 88 and 108 mg/kg in the above ground parts of sunflower and willow, respectively, when CA was used (Mihalík et al., 2010). A number of recent research conducted in relation to the effects of CA (CA) on the metal uptake of plants are shown in Table 2.1.
Table 2.1 Research about the effects of CA on metal uptake of plants Metals Plants CA Effects References (dose) 9.2 ppm Cd, or Crotalaria 3842 ppm Mn in the roots Alidoust et al., 156.3 ppm Zn, or juncea and shoots 2009 1480 ppm Mn increased about 2- and 4-fold Multi-metals: 7.3 marigold 1921 Maximum increase Sinhal et al., ppm Zn, 7.5 ppm ppm, in accumulation 2010 Cu, 3.7 ppm Pb 3842 ppm was with 3842 and 0.2 ppm Cd or 5763 ppm CA; ppm However, 5763 ppm CA reduced metal accmulation in plants
18 Table 2.1 (cont.) Research about the effects of CA on metal uptake of plants Metals Plants CA dose Effects References Multi-metals: Barnyard 1921 ppm CA increased shoot 40 ppm Cd, 100 grass concentrations of Cd, ppm Cu and Cu, Pb by 1.6, 5.0 600 ppm Pb and 22.8 fold respectively Juncus 960.5 ppm increased in Mn Najeeb et al., 27.5 ppm Mn effusus L. accumulation (from 2009 175.98 ppm to 195.26 ppm in shoot) Multi-metals: Sunflower 240.1 Metal concentrations Turan and 100 ppm B, 400 ppm, in shoots was greatly Angin, 2004 ppm Cd, 10 480.3 increased by ppm Mo, ppm, 1921ppm CA: 85 and 100 ppm 960.5 ppm ppm B, 520 ppm Cd, Pb or 921 320 ppm Mo and 15 ppm ppm Pb 50, 100, 150 or Brassica 3842 ppm enhanced shoot Cd Quartacci et 200 ppm Cd juncea L. concentration by 5% al., 2005
Multi-metals: Typha 480.2 Maximum increase Muhammad et 20 ppm Cd, 10 angustifolia ppm, was associated with al., 2009 ppm Cr, 50 960.5 ppm 1921 ppm CA: ppm Cu and 10 or 1921 increased shoot Cd, ppm Pb ppm Cu, and Cr concentrations from 2.5 ppm, 18 ppm, 6 ppm to 10 ppm, 22 ppm 13 ppm, respectively
2.6 Rhizosphere microorganisms in phytoremediation
The term “rhizosphere” referred to the narrow zone of soil adherent to the plant roots (Karami et al., 2010). Attributed to the root exudates which can be nutrient sources for soil microorganisms, microbial density and activity is much higher in the rhizosphere than that in the surrounding bulk soil (Pinton et al., 2007). The
19 interactions between rhizosphere soil microorganisms and plants can influence plants growth and metal uptake by plants. Rhizosphere microorganisms mainly include rhizobacteria and mycorrhizal fungi (Karami et al., 2010). Rhizosphere bacteria have been shown to both decease and increase metal uptake by plants, depending on the specific metal-plant combination. Bacterial immobilization of heavy metals may lead to the reduction of heavy metal uptake in plants, while bacterial activity that enhances the mobility of heavy metals may cause the increase of heavy metal accumulation by plants.
Some rhizosphere microorganisms can release organic acids from bacterial cells, such as citrate and oxalate, which can lower the pH of soil and then promote mobility and bioavailability of metals (Kamnev and van der Lelie, 2000). The organic acids can also cause the dissolution of metal phosphates, during which the heavy metal cations and the essential nutrient phosphate are released simultaneously (Gupta et al.,
2002). For instance, the Ni uptake by Alyssum murale can be increased by inoculation with phosphate solubilizing rhizosphere bacteria (Abou-Shanab et al., 2003). In addition, heavy metal adsorption by both bacterial cells and their extracellular metabolites can decrease the amount of mobile heavy metals in soil, thus decrease the bioavailability of the metal to plants and influence the metals uptake of plants
(Pishchik et al., 2009).
Some other rhizosphere microorganisms can change the solubility of heavy metals by redox transformations. For example, Lovley (1995) pointed out that biological process can reduce Mn(IV) to Mn(II), affecting its mobilization. It has also 20 been reported that the rhizosphere bacteria can transform Cr(VI) to Cr(III) under aerobic conditions (Gutiérrez et al., 2010)
The wetland rhizosphere is an interface between aerobic and anaerobic conditions, includes a diverse community of both aerobic and anaerobic microorganisms (Neubauer et al., 2007). According to the survey results of Weiss et al. (2003), Fe(II)OB has been found on the roots of 92% of wetland plants collected from 13 aquatic environments in USA and may consist up to 5% of the total microbial community in the rhizosphere of these aquatic habitats. Rhizosphere Fe(II)OB account for a large pool in the formation of iron oxyhydroxide coatings (i.e., Fe plaque) on the root surface of many wetland plants (Neubauer et al., 2007). In laboratory studies, Fe(II)OB accounts for 45-90% of Fe(II) oxidation (Weiss et al.,
2003). Neubauer et al. (2002) found a rhizosphere-isolated Fe(II)OB mediated about
62% of the total Fe(II) oxidation and accelerated total Fe(II) oxidation rates by up to
18%.
Iron plaques have been observed on the root surface of many wetland plants
(Batty et al., 2000; Ye et al., 1998; St-Cyr et al., 1988). However, the information about the function and role of iron plaque in metal uptake by plants is unclear and even conflicting. It was reported that the formation of iron plaque around the roots of wetland plants was an adaptation and protection mechanism of plants, since the iron plaque and may reduce the uptake of phytotoxic metals (Batty et al., 2000). For example, iron plaques can prevent the movement of other metals such as copper, zinc, nickel and cadmium into aboveground tissues (Batty et al., 2000; Greipsson, 1994; 21 Wang and Peverly, 1996). Although plaque can inhibit movement, the majority of research stated that the iron plaque can not completely impede the uptake of toxic metals. For instance, St-Cyr and Crowder (1987) reported that iron plaque did not prevent the uptake of Cu and Zn in Phragmites australis. It also did not reduce the accumulation of Zn and Pb in Typha latifolia (Ye et al., 1998). Conversely, other reports even indicated that iron plaque may enhance the uptake of metals by plants.
For example, Greipsson (1994) indicated that iron plaque enhanced the uptake of iron by rice and lessened the uptake of zinc.
2.7 Impacts of chelators on rhizosphere microorganisms
Chelate-assisted phytoremediation can artificially enhance heavy metal solubility in soil and thus increase heavy metal bioavailability. However, there is a concern about whether chelators have potential effects on the microbial activity in soil.
Chelators enhance metal mobilization in soil, and increase metal availability for soil microorganisms at the same time (Welp and Brümmer, 1997). Soil microorganisms uptake food and water from the soil solution directly or indirectly, so elevated metal concentrations in soil could lead to toxic effects for these organisms (Römkens et al.,
2002).
According to Grčman et al. (2003), EDTA can increase the stress index of microbial populations. In Zn-contaminated soils, the addition of EDDS, and EDTA caused a decrease in the root colonization of Solanum nigrum by microorganisms
(Marques et al., 2007). 22 Römkens et al. (2002) conducted greenhouse experiments to study the phytoremediation potential of 0.01 M glycoletherdiamine tetra acetic acid (EDGA) and 0.01 M (CA) and to evaluate its effects on microbial activity in soils contained 2 mg/kg Cd and 200 mg/kg Zn. The results showed that addition of EDGA and CA resulted in an increased of the bacterial biomass from 17 µg C/g soil to 50 µg C/g soil and 60 µg C/g soil, respectively.
Chen et al. (2006) studied the potential effects of chelator (glucose and CA) amendment on phytoextraction of copper and microbial community composition in
Elsholtzia splendens and Trifolium repens. The results indicated that chelator addition facilitated plant uptake of Cu without inducing a negative effect on microbial community.
Although most research has focused on finding efficient chelators to enhance phytoremediation, there is limited information about the potential effect of chelator amendment on the microbial activity and community composition in soil (Chen et al.,
2006). Further research is needed to study the effect of chelators on the soil microorganisms in phytoremediation of heavy metal contaminated soils.
2.8 Histological research in phytoremediation
In addition to the experiments to investigate the concentrations of contaminants accumulated in the organs of plants, some research have been conducted to study the anatomy and morphology of plants to better understand their mechanism to adapt to adverse environments. Beyer et al. (2009) found that cylindrospermopsin (CYN) 23 changed reed growth and anatomy through the alteration of microtubules organization and induced the formation of necrosis in root cortex. Baldantoni et al. (2009) determined the concentration of some macro and trace elements in roots and analyzed the histological differences between water root and sediments of common reed in a volcanic lake in Italy. They indicated that the two roots showed a different capacity to accumulate macro and trace elements and differed in the average size of the aerenchyma which may relate to oxygen levels (Baldantoni et al., 2009). The macroscopic and histological traits involved in the reed die-back syndrome were studied by Reale et al. (2012). However, no differences in the anatomical structures of rhizomes and roots were found among the analyzed healthy and die back Phragmites australis. Liu et al. (2012) studied the difference of adaptational characteristics such as leaf surface micro-morphology, anatomical structures, among three different reed ecotypes grew in different natural habitats. Their results showed dune reed and gobi salt reed had higher bundle-sheath cell areas and a lower xylem/phloem ratio than swamp reed, and different ecotypes had different mechanisms to their habitats.
However, there are limited studies on the effects of metals on the growth and histological organization within plants. Investigations into the histological alternations in plants caused by heavy metals can help us better understand the adaptation mechanisms of plants and provide us more information about phytoremediation.
24 CHAPTER III
MATERIALS AND METHODS
3.1 Reagent sources
The key chemicals used for FETSB (Senko et al., 2008) medium to culture Fe oxidizing bacteria and WAYE (Johnson, 1995b) medium for acidophilic heterotrophs included (NH4)2SO4, MgSO4.7H2O, agarose, trypticase soy broth, FeSO4·7H2O, glucose and H2SO4. All were purchased in biotechnical grade from Fisher Scientific.
The chemicals used in synthetic acidic mine drainage (SAMD) solution included FeSO4·7H2O, CaSO4·2H2O, MgSO4·7H2O, Na2SO4, Al2(SO4)3·16H2O, and
MnSO4·H2O. Chemicals not already on hand for use in culture medium were purchased from Fisher Scientific at technical grade form.
The chemicals used in soils incubation experiment included CaSO4·2H2O,
Na2SO4, Al2(SO4)3·16H2O, MnSO4·H2O, (NH4)2Fe(SO4)2·6H2O, ferrozine, NaOH,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and formaldehyde
(37%). All were purchased at biotechnical grade.
Na3C6H5O7·2H2O, NaHCO3, Na2S2O4 were the main chemicals for cold dithionite–citrate–bicarbonate (DCB) method to extract metal plaques from the root and rhizome surface. Concentrated HCl, H2O2 (30%) and HNO3 (70%) were used for total metal digestions in soil. CaCl2 were used to extract mobile metals from soil. KCl
25 was used for soil pH measurement. Perchloric acid (70%), HNO3 (70%) were utilized for plant digestion. All were purchased at technical grade from Fisher Scientific.
The main reagents used in hydroponic experiments for culturing common reeds included FeSO4·7H2O, CaSO4·2H2O, MgSO4·7H2O, Na2SO4, Al2(SO4)3·16H2O,
MnSO4·H2O and H2SO4. C6H8O7·H2O, NaOH and phenolphthalein indicator were used in the experiment for CA biodegradation. HCl, potassium ferrocyanide, phloxine
B, phosphotungstic acid, 95% ethanol, fast green FCF and glacial acetic acid were utilized in the histological experiments.
3.2 Sampling site
The sampling site is located in Beaver Township, North Lima, Ohio, USA. The site has been abandoned for more than 18 years due to the AMD from nearby mining activity. There is a small house at the site. The AMD has filled the basement of the house, where it exits from windows and flows across the field as showed in Figure 3.1.
On March 16th, 2012, Phragmites australis and rhizosphere soils were collected from five sampling points which were selected according to the emergence from the basement, pathway of AMD, and presence of plants. Sampling point A was not in the pathway of AMD and was selected as the control point. Sampling point B represented the source of the AMD as it emerged from the basement window of house. Sampling points C, D and E were in the pathway of the AMD. Rhizosphere soil collected from different sampling points on March, 2012 was showed in Figure 3.2. The soil of different sampling points presented different colors. For example, soil of point A was 26 black, soil of point B was red and soil of point C was brown. This may be relate to the degree of contamination and the amounts of metals in it.
Reeds from different points were collected again on January 9th and May 20th,
2013 to compare the metal accumulation and plaque formation with reeds collected in
March, 2012. Rhizosphere soils from site A were collected in August, 2012 to culture rhizosphere bacteria. Reeds were collected from site E in September, 2012 and August,
2013 to initiate spiked soil experiments and hydroponic experiments.
D B
C A
E North
Figure 3.1 Aerial image of sampling site. Image from Bing Maps accessed on 2012
Figure 3.2 Rhizosphere soils collected from the field. From left to right are the soils from sampling point A, B, C, D, and E.
27 3.3 Spiked soil
Soil collected from a clean residential garden center in northeastern Ohio was used as the source of spiked soils. At day 0, 0.5 kg of air-dried soils was weighed into individual small pans (21.9 cm×11.5 cm×5.9 cm). Soil as the control group was rehydrated with nutrient solution which contained 0.250 g N (NH4NO3), 0.060 g Mg
(MgSO4·7H2O), 0.109 g P (KH2PO4), and 0.207 g K (KH2PO4 and K2SO4) per kg soil
(Mench, 1994). Soil to be used as the treatment group was further artificially spiked with metal solution of which the pH was 3.5 and contained 42.94 g Fe (FeSO4.7H2O),
0.27 g Al (Al2(SO4)3·16H2O), 0.05 g Mn (MnSO4·H2O), 0.59 g Mg (MgSO4·7H2O),
1.24 g Ca (CaSO4.2H2O), 0.28 g Na (Na2SO4) per kg soil. The concentrations of metals in spiked soils were based on the characteristics of synthetic acid mine drainage (SAMD) (Senko et al., 2008) and the results of previous research conducted on the actual site (Sivaram, 2010; Bertel, 2011). The three small pans were placed into a large pan (33.9 cm×22.1 cm×3.4 cm) to collect any leacheate for subsequent analysis (Chen and Cutright, 2002). The soils were mixed thoroughly everyday and allowed to equilibrate for 4 weeks (28 days) in greenhouse. At day 26, part of the small pans were amended with 200 ml of solutions which contained different levels of
CA (2.101 g, 17.859 g or 33.616 g C6H8O7·H2O per kg soil), while the rest of the pans were added with the same amounts of DI water. The dosages of CA were selected based on previous research (Mihalík et al., 2010) and the concentration of metals in spiked soils. At day 28, Phragmites australis were transferred into the spiked soil and bacteria were inoculated into the rhizosphere of some reeds. This approach was used 28 for the non-contaminated (i.e. purchased, never exposed to previous contaminants) and wild reeds (collected from the contaminated site) (section 3.4).
There are nine different conditions per experiment: clean soil without adding CA and rhizosphere bacteria (clean soil); spiked soil without CA and rhizosphere bacteria
(NCNB); spiked soil inoculated with rhizosphere bacteria without CA (NCWB); spiked soil amended with low level of CA and without inoculating with rhizosphere bacteria (LCNB); spiked soil with low level of CA and inoculated with rhizosphere bacteria (LCWB); spiked soil added with middle level of CA and without rhizosphere bacteria (MCNB); spiked soil with middle level of CA and inoculated with rhizosphere bacteria (MCWB); spiked soil with high level of CA but no rhizosphere bacteria (HCNB) and spiked soil added with high level of CA and inoculated with rhizosphere bacteria (HCWB).
3.4 Plant source and preparation
Different plant sources were used in this study to determine whether there were differences in metal uptake between different types of reeds. One type was purchased non-contaminated reeds. Another was wild reeds collected from the AMD site.
3.4.1 Purchased non-contaminated reeds
The seeds and rhizomes of common reed Phragmites australis were purchased from Lorenz’s OK seeds, LLC (Okeene, Oklahoma). They were initially grown in commercial potting soil (Micracle-Gro lawn products, Inc) in pans (45 cm×25 cm×7.5 29 cm). Two centimeters of potting soil was placed in the bottom of each pan. This was followed by the rhizomes and another 2.5 cm of potting soil. The rhizomes were cultured in a greenhouse under natural light conditions. The luminance of light ranged from 1000 to 60000 Lux. The average temperature of the greenhouse was 22℃ and the humidity was 50%. Five hundred ml DI water was sprayed into each pan every day to maintain the soil moisture. After 30 days of growth in the potting soil, seedlings with similar biomass were transferred into spiked soil to initiate experiments. Prior to being transferred, the rhizomes of the reeds were rinsed with DI water to remove the attached potting soil.
3.4.2 Wild reeds
Several reeds were collected from sampling site E and then immediately transferred into spiked soil on September 5th, 2012. Since the reeds have grown in the sampling site for several years, the rhizomes and roots of the reeds were thicker, longer and stronger than that of the reeds cultured in greenhouses. The rhizomes and roots of the reeds were rinsed with DI water to remove the attached soils, prior to being planted into the spiked soils. Figure 3.3 presented the pictures of Phragmites australis propagated from uncontaminated rhizomes and reeds collected from the fields.
Some of the rhizomes of the reeds collected from site E were transferred into the commercial potting soils. After 30 days of growth in the potting soil, new seedlings
30 from the wild rhizomes with similar biomass were used to initiate the hydroponic experiments.
The non-contaminated reeds and wild reeds were cultured in spiked soil or hydroponic solution for 4 to 12 weeks. The chosen growth period was based on previous research (Ye et al., 1998). Every 4 weeks, part of reeds and rhizosphere soil were collected for analysis, which includes rhizosphere bacteria enumeration (section
3.8), soil or solution pH measure (section 3.12), soil mobile metal analysis (section
3.13), soil digestion (section 3.14), DCB extraction and plant digestion (section 3.15) and histological analysis (section 3.17). Every 4 weeks, rhizosphere bacteria were inoculated into the rhizosphere of rest of reeds (section 3.7).
a b
Figure 3.3 Pictures of Phragmites australis (a) propagated from purchased uncontaminated rhizomes (b) collected from the wild
3.5 Hydroponic experiments cultured with reeds propagated from wild rhizomes
In hydroponic experiments cultured with reeds propagated from wild rhizomes, the control group only contained nutrient materials which included 0.250 g N
(NH4NO3), 0.060 g Mg (MgSO4·7H2O), 0.109 g P (KH2PO4), and 0.207 g K (KH2PO4 31 and K2SO4) per liter solution. This was used to assess biomass growth of reeds. The treatment groups which contained nutrient materials and metals were separated into 3 different levels: low, middle and high level according to the concentrations of metals used. Table 3.1 listed the compositions of different hydroponic solutions. The metals concentrations of low level equals to that used in SAMD. The amounts of metals of high level equals to that in spiked soil. The pH of solutions of different treatment groups were adjusted to 3.5 by H2SO4, then rhizosphere bacteria or different amounts of CA were further added into part of the treatment groups. The amounts of CA amendments which listed in Table 3.2 were based on previous research (Mihalík et al.,
2010) and the concentrations of metals in solution.
Table 3.1 Compositions of solutions in hydroponic experiments Low level of metals Middle level of metals High level of metals (LM) (g/L) (MM) (g/L) (HM) (g/L)
0.39 Fe (FeSO4·7H2O) 21.67 Fe (FeSO4·7H2O) 42.94 Fe (FeSO4·7H2O)
0.03 Al (Al2(SO4)3·16H2O) 0.15 Al (Al2(SO4)3·16H2O) 0.27 Al (Al2(SO4)3·16H2O)
0.02 Mn (MnSO4·H2O) 0.04 Mn (MnSO4·H2O) 0.05 Mn (MnSO4·H2O)
0.20 Ca (CaSO4·2H2O) 0.72 Ca (CaSO4·2H2O) 1.24 Ca (CaSO4·2H2O)
0.10 Mg (MgSO4·7H2O) 0.35 Mg (MgSO4·7H2O) 0.59 Mg (MgSO4·7H2O)
0.05 Na (Na2SO4) 0.17 Na (Na2SO4) 0.28 Na (Na2SO4)
Table 3.2 Amounts of CA added in hydroponic experiments Treatment Low level of Middle level of High level of groups C6H8O7·H2O C6H8O7·H2O C6H8O7·H2O (LC) (g/L) (MC) (g/L) (HC) (g/L) LM 0.021 0.181 0.343 MM 1.061 9.020 17.859 HM 2.101 16.979 33.616
32 For solution with low levels of metals (LM), there were eight different treatment groups: solution without adding CA and rhizosphere bacteria (LMNCNB); solution inoculated with rhizosphere bacteria and without adding CA (LMNCWB); solution added with low level of CA and without inoculating with rhizosphere bacteria
(LMLCNB); solution added with low level of CA and inoculated with rhizosphere bacteria (LMLCWB); solution added with middle level of CA and without inoculating with rhizosphere bacteria (LMMCNB); solution added with middle level of CA and inoculated with rhizosphere bacteria (LMMCWB); solution added with high level of
CA and without inoculating with rhizosphere bacteria (LMHCNB); solution added with high level of CA and inoculated with rhizosphere bacteria (LMHCWB).
Similarly, there were also eight treatment groups for the solution with middle levels of metals (MM): MMNCNB, MMNCWB, MMLCNB, MMLCWB,
MMMCNB, MMMCWB, MMHCNB, MMHCWB; eight treatment groups for the solution with high levels of metals (HM): HMNCNB, HMNCWB, HMLCNB,
HMLCWB, HMMCNB, HMMCWB, HMHCNB, HMHCWB.
3.6 Hydroponic experiments cultured with reeds collected from site E
In hydroponic experiments cultured with reeds collected from site E, the control group also only contained nutrient materials as described in section 3.5. The treatment solution contained nutrient materials, middle level of metals (section 3.5) and different level of CA and rhizosphere bacteria.
33 There were nine different conditions: control group without adding CA and rhizosphere bacteria (control); treatment solution without inoculating bacteria and CA
(NCNB); added with 9.02 g/L CA but without inoculating bacteria and CA (WCNB); inoculated with acidophilic heterotrophs but without adding CA (NCAB); inoculated with acidophilic heterotrophs and added with 9.02 g/L CA (WCAB); inoculated with
Fe(II)OB but without adding 9.02 g/L CA (NCFB); inoculated with Fe(II)OB and added with 9.02 g/L CA (WCFB); inoculated with acidophilic heterotrophs and
Fe(II)OB but without adding CA (NCAFB); inoculated with acidophilic heterotrophs and Fe(II)OB and added with 9.02 g/L CA (WCAFB).
3.7 Rhizosphere bacteria: isolation and enrichment
The rhizosphere soil used to enrich rhizosphere bacteria were collected from the root zone of reeds grown at sampling point B. The rhizosphere soil was collected by a sterilized spatula, placed into sterilized glass jars and kept on ice for transport to the laboratory. The rhizosphere bacteria were isolated from soils and cultured in growth medium immediately after arriving in the laboratory. The bacteria isolated and enriched were the indigenous species capable of tolerating high concentrations of Fe,
Al and Mn. Approximately 5 g of soil was put into the 100 ml nutrient solution which contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 10 mM glucose, 0.25 g trypticase soy broth per liter, with a pH of 3.5 (Johnson, 1995b). Bacteria cells were separated from the soil by centrifugation (1000 rpm) for 15 minutes (IECentra-4B Centrifuge,
USA). They were then enriched in growth medium following standard subculture 34 techniques. The growth medium for mixed culture contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, 10 mM glucose, 10.736 g Fe
(FeSO4.7H2O) 0.068 g Al (Al2(SO4)3·16H2O), 0.013 g Mn (MnSO4·H2O) in one liter distilled (DI) water. The pH of the solution was adjusted to 3.5 by H2SO4. The bacteria were cultured on an incubation shaker (Lab-line instruments, Inc, USA) at
30℃, 125 rpm. The concentration of metals in the growth medium doubled every week until the concentration were identical with the concentration of Fe, Al, Mn in spiked soils. Fe(II)OB were enriched in growth medium which contained 14 mM
(NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, 25 mM FeSO4·7H2O in one liter DI water. Acidophilic heterotrophs were enriched in growth medium which contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, 10 mM glucose in one liter DI water. The pH of solution was adjusted to 3.5 by H2SO4.
3.8 Rhizobacteria inoculation
Parts of the reeds that were transferred to the spiked soil or hydroponic solution were inoculated with rhizosphere bacteria to assess the impacts of bacteria on growth and metals uptake of plants. First, bacteria cells were separated from 10 ml growth medium by centrifugation at 1000 rpm for 15 minutes. Cells were resuspended in 5 ml nutrient solution which contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 10 mM glucose, 0.25 g trypticase soy broth per liter. Then the cell suspensions were injected into the rhizosphere of the reeds in spiked soil or into solution by sterile syringe.
35 3.9 Plates counts for acidophilic heterotrophs and Fe(II)OB
Acidophilic heterotrophs were enumerated on WAYE medium (Johnson, 1995b), while Fe(II)OB were cultured on FETSB medium (Senko et al., 2008). One L WAYE medium contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, 10 mM glucose and 5 g agarose. WAYE medium were prepared as follows.
First 1.848 g (NH4)2SO4, 0.492 g MgSO4·7H2O, 0.25 g trypticase soy broth, and 1.8 g glucose was added into 700 ml DI water. The pH of the solution was adjusted to 3.5 by adding H2SO4. Next, DI water was added to make the final volume of the solution to 800 ml. The solution was autoclaved at 121℃ for 30 minutes. The gelling solution was prepared by adding 5 g agarose into 200 ml DI water. This was also autoclaved at
121℃ for 30 minutes. The two solutions were placed in a water bath to cool down to
50℃, and then combined into a final solution which was poured into sterilized Petri dishes to solidify.
One L FETSB medium contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, 25 mM FeSO4·7H2O, and 5 g agarose. FETSB medium were prepared as follows: 1.848 g (NH4)2SO4, 0.492 g MgSO4·7H2O, 0.25 g trypticase soy broth was added to 700 ml DI water. The pH of the solution was adjusted to 3.5 by adding H2SO4 and then added DI water to make the final volume of the solution to
775 ml. The solution was autoclaved at 121℃ for 30 minutes. The gelling solution was prepared by adding 5 g agarose into 200 ml DI water, then autoclaved (121℃, 30 minutes).The solutions were placed into water bath to cool down to 50℃ and mixed together. The ferrous sulfate solution was made by adding 1M FeSO4·7H2O into 800 36 ml DI water. The H2SO4 was used to adjust the pH of the solution to about 2.0 to dissolve FeSO4·7H2O. Next, DI water was added to make the final volume of solution to 1000 ml. The filter-sterilized 25 ml ferrous sulfate solution was added into the cooled mixed solution and poured into sterilized Petri dishes to solidify.
About 0.2 g soil from the rhizosphere of plants were suspended in 5 ml dilution solution which contained 14 mM (NH4)2SO4, 2 mM MgSO4·7H2O, 0.25 g trypticase soy broth, mixed by vortexing, and then serially diluted and spread on plates. The plates were incubated in dark. After 24 hours, the plates were placed in an inverted position to prevent the water droplets on the top of the plates to destroy the formed colony. Colony forming units (CFU) on the plates were counted four (WAYE) and six
(FETSB) weeks later (Leduc et al., 2002).
3.10 Soils incubation experiments
The method described by Senko et al. (2008) was used to measure Fe(II) oxidation kinetics in the rhizosphere soils of the reeds. Four g soils from the root zone were incubated with 40 ml synthetic acidic mine drainage (SAMD) in 125 ml erlenmeyer flask. The mouths of the flasks were sealed with foil. SAMD contained 7 mM FeSO4.7H2O, 5 mM CaSO4·2H2O, 4 mM MgSO4.7H2O, 1 mM Na2SO4, 0.5 mM
Al2(SO4)3·16H2O, 0.4 mM MnSO4·H2O and 0.1 mM (NH4)2Fe(SO4)2 (Senko et al.,
2008). The pH of SAMD was adjusted to 3.5 with H2SO4. In abiotic controls, biological activity was prevented by adding of 1.1 ml of formaldehyde (37 wt.%). The soils were incubated for 2 days. The pH of the incubation was determined after 6 h, 12 37 h, 24 h, 36 h and 48 h. At the same time, 0.1 ml solutions were sampled to determine the concentration of Fe(II) by ferrozine assay (Lovley and Philips, 1986) . The sample was mixed with five ml of 0.5 N HCl and centrifuged at 10000 RPM for five minutes.
Then 0.1 ml of the supernatant was added to 5 ml of ferrozine HEPES buffer solution
(Lovley and Phillips, 1987), which contained 1g ferrozine in 50 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (HEPES) at pH 7.0 (pH adjusted to 7.0 with NaOH). After being mixed for 15 s, the absorbance of the sample was measured by an ultraviolet–visible spectroscopy (Thermo spectronic 20D+) at
562 nm. The concentration of Fe(II) was calculated according to Fe(II) calibration curve.
3.11 Establish Fe(II) calibration curve
The Fe(II) calibration curve was established by measuring the absorbance of iron standard solution. Stock solution were prepared by dissolving 7.059 g
(NH4)2Fe(SO4)2·6H2O into 0.5 M hydrochloric acid. Serially diluted stock solution to prepare iron standard solutions, in which the concentration of Fe(II) was 2 mg/L, 5 mg/L, 10 mg/L and 20 mg/L. 0.1 ml of iron standard solution was transformed into 5 ml of Ferrozine HEPES buffer solution. After being mixed for 15 s, the A562 of the solution was determined. Figure 3.4 showed the Fe(II) calibration curve.
38 25 y = 97.354x - 1.4008 20 R2 = 0.9812 15
mg/L
2+ 10
Fe 5
0 0 0.05 0.1 0.15 0.2 0.25 Absrobance
Figure 3.4 Fe(II) calibration curve
3.12 CA biodegradation experiment
A CA biodegradation experiment was designed to determine whether or not CA solution can be consumed by the enriched rhizosphere bacteria. Rhizosphere bacteria was injected into filter-sterilized solution contained 2101 ppm, 17860 ppm or 33616 ppm C6H8O7·H2O in pre-sterilized erlenmeyer flasks (same concentration of CA added into spiked soil). The solution of control groups contained the same concentration of C6H8O7·H2O, but did not receive rhizosphere bacteria. All flasks were transferred to an incubation shaker (Lab-line orbit environ-shaker) at 30℃, 125 rpm for 1 month.
CA and NaOH calibration curve (Figure 3.5) was established to calculate the theoretical volume of 1 M NaOH needed to neutralize different amounts of CA. 1000 ppm, 2000 ppm, 5000 ppm and 10000 ppm standard C6H8O7·H2O solution were prepared and titrated with 1 M NaOH by using phenolphthalein as the indicator. The reaction equation was: C6H8O7·H2O +3NaOH →Na3C6H5O7+4H2O.
39 Four weeks later, the solutions with or without adding rhizosphere bacteria were titrated with 1 M NaOH. The actual volumes of NaOH used were compared with the theoretical volumes needed to determine whether CA was consumed by bacteria.
20 y = 0.0017x + 0.0779 2 15 R = 0.9999
10
1M NaOH1M (ml) 5
0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
C6H8O7.H2O ppm
Figure 3.5 CA and NaOH calibration curve
3.13 Measure pH, dissolved oxygen (DO) and conductivity
The pH of the soil was measured according the methods of Tan (1995). One M
KCl solution ratio of 1:1 was used for soil pH measurement. After mixing for 15 minutes, the pH of the solution was measured by pH meter (420A ATI Orion pH meter,
MA, USA). For hydroponic solution, the pH was measured directly with pH meter.
The DO of hydroponic solution was measured by DO meter (YSI Model 55), while the conductivity of solution was determined by (Mettler Toledo EL30).
3.14 Analysis of mobile metals in soils
The mobile metals were defined as the metals that were not tightly bound to soils which can be considered as the available and exchangeable fractions, while the total
40 metals are the summation of the bound metals and mobile metals (Maiz et al., 1997).
Mobile metals were extracted by 0.01 M CaCl2 solution followed the procedure (Maiz et al., 1997): Two gram of air dried soils were mixed with 20 ml 0.01 M CaCl2 in a 40 ml PRYEX centrifuge tube. The mixed solutions were agitated in a shaker for 2 h, and then were centrifuged at 2500 rpm for 15 min. Next, the supernatant was collected and kept in the refrigerator until analysis by inductively coupled plasma mass spectrometry (Perkins Elmer Plasma 400 Spectrophotometer ICP-MS).
3.15 Soil digestion: analysis of total metals in soils
Total metals in soil were determined based on the EPA acid digestion method
3050 (Carter, 1993): Two g of air dried soil was mixed with 10 ml of 1:1 HNO3, and heated at 95℃ for 15 minutes. Then five ml of concentrated HNO3 was added into it and refluxed at 95℃ for 30 minutes. Then five ml of concentrated HNO3 was added into the solution and refluxed for 30 minutes again. Next, 25 ml of 30% H2O2 was added into the solution slowly. After the peroxide reaction, 5 ml of concentrated HCl was further added into the solution. The solution was refluxed for 15 minutes without boiling. After cooling, the solution was filtered through a Whatman NO.42 filter paper and diluted with DI water to make the total volume to 50 ml.
3.16 DCB extraction and plant digestion
The roots and rhizomes of plants were gently washed by DI water to remove the soil on the surface, and then immersed in DI water overnight. Next, the remaining 41 plants were further separated into roots, rhizomes, stems, leaves, and flowers. Parts of the fresh roots or rhizomes were assigned to cold dithionite–citrate–bicarbonate (DCB) extraction techniques to determine the concentration of metal plaques on the surface of roots or rhizomes. Other plant tissues were air dried.
According to DCB extraction method (Taylor and Crowder, 1983a), 1 g fresh roots or rhizomes were agitated for 3 hr in solution which contained 40 ml of 0.3 M
Na3C6H5O7·2H2O, 5 ml of 1.0 M NaHCO3 and 3 g of Na2S2O4 at room temperature.
Next, the solution was collected, and the roots or rhizomes were rinsed 3 times with
10 ml DI water. Both of the original solution and rinsed solution were poured into amber bottle. The rinsed roots or rhizomes were then air dried as other plant tissues.
After drying, the tissues were weighed and crushed with mortar and pestle. The milled tissues were then digested according to the methods presented in Cutright et al. (2010): one g of plant tissues was soaked in 20 ml of nitric acid (70%) for six hours. The mixture was heated and boiled to 10 ml. Then, 4 ml of perchloric acid (70 %) was and the mixed solution was refluxed for 90 min. Finally, the solution was diluted to 20 ml with DI water. The solution was kept in the refrigerator until analyzed by ICP-MS.
3.17 ICP-MS method
Mixed standard solutions were prepared to calibrate ICP-MS, which contained 5 mg/L, 10 mg/L and 20 mg/L Fe, Mn and Al. The methods listed in the user’s manual were followed to run ICP-MS. The minimum detection limits of ICP-MS was 0.028
42 mg/L for Al, 0.015 mg/L for Fe, and 0.0016 mg/L for Mn. The solutions of samples needed to be diluted and filtered by 0.45 µm syringe filter before analysis.
3.18 Histological experiments
Three different sets of reeds used to do histological experiments. The first sets were reeds directly collected from AMD field (i.e. reeds from the wild) and analyzed immediately. The second sets were reeds collected from site E of AMD field on
September 5 and transferred into spiked soil in greenhouse and cultured until
November 28, 2012. Purchased uncontaminated reeds grown in clean soil in greenhouse were analyzed as the control. The third sets were reeds collected from site
E in August, 2012 and cultured in hydroponic solution for 12 weeks.
After harvesting, the fresh roots and rhizomes of reeds were cut into cross sections of 10 µm by cryostat (Bright OTF). A minimum of 10 sections were cut for each sample for reproducibility, as well as to enable for different staining techniques for Fe and Al. Tissue sections were analyzed prior to and after staining to assess which specific tissue section sequestered the metal. For Fe analysis, sections were stained by Perls’ prussian blue reactions. First, sections reacted with equal volumes of
2% HCl and 2% potassium ferrocyanide solutions for 10 minutes. Iron was released to protein by treatment of HCl and then reacted with potassium ferrocyanide to produce an insoluble blue compound (Bancroft and Gamble, 2008). Al analysis used the staining procure based on the approach of Walton (2004). First, sections were stained in 0.5% phloxine B for 3 min, then washed thoroughly with DI water to remove 43 excess stain. The washed sections were then put into 5% phosphotungstic acid for 1 min and washed thoroughly. Next, the sections were differentiated in 95% ethanol for
2 min, rinsed by water; then stained in 0.05% fast green FCF. After 3 min, the samples were washed in 1% glacial acetic acid for 1 min. The tissues that contained aluminum showed magenta (Walton 2004). The sections were then photographed with a light microscope (Olympus BX 60) equipped with a phtocamera (Olympus DP 11).
3.19 Statistical analysis
The experiment was two-factor (CA and bacteria) completely randomized design with subsamplings (or two-stage nested design). The main factors were CA with four levels (without CA, low, middle and high level of CA) and rhizosphere bacteria with two levels (with or without inoculating rhizosphere bacteria). Data on numbers of rhizosphere bacteria, metal plaque formation and metal uptake in reeds were analyzed with general linear model using the Minitab statistical package (Minitab 16). Data on metals concentrations in reeds collected from different sites were analyzed with one-way ANOVA. Differences between different sites, specific CA amendments and rhizosphere bacteria inoculation were identified by the Tukey Test at 5% probability.
44 CHAPTER IV
RESULTS AND DISCUSSIONS
4.1 Analysis of samples collected from field
Analysis was conducted on reeds and rhizosphere soil collected from the AMD site in different seasons. The main experiments included rhizosphere bacteria enumeration, soil incubation experiments, soil digestion, DCB extraction of plaque, plant digestion and histological analysis.
4.1.1 Enumeration of acidophilic heterotrophs and Fe(II)OB
In order to assess the growth of rhizosphere bacteria, microbes in the soils from the root zone of reeds were cultured on the WAYE and FETSB medium. Acidophilic heterotrophs were cultured on WAYE medium, while Fe(II)OB were cultured on
FETSB medium (Table 4.1).
Table 4.1 Rhizosphere bacteria in rhizosphere soil collected from different sites Sampling point Fe(II)OB (CFU/g) WAYE (CFU/g) A 5.50 ±0.25×103 b 2.33±0.05×105 a B 5.64 ±3.83×105 a 3.33±1.40×106 a C 3.88 ±0.94×104 ab 9.18±4.96×105 a D 1.49 ±1.15×105 ab 1.83 ±0.76×106 a E 1.40 ±1.08×105 ab 1.91 ±0.96×106 a The results were reported as average±standard deviation, n=10. Different letters in the same column indicate a significant difference at p<0.05
45 Fe(II)OB were determined by counting the red to orange colonies that grown on the FETSB medium (Senko et al., 2008; Lin et al., 2011). Fe(II)OB have been observed in the rhizosphere of reeds from all the sampling points. According to Weiss et al. (2003), Fe(II)OB were widespread in the root zone of wetland plants in thirteen wetlands and aquatic habitats in Virginia, Maryland, and West Virginia in USA.
Neubauer et al. (2005) also indicated that the rhizosphere were good habitats for
Fe(II)OB due to the interacting gradients of O2 and Fe(II), and the high inorganic C concentrations. The presence of Fe(II)OB in the rhizosphere of wetland plants were also conclusively demonstrated by Emerson et al. (1999). The numbers of Fe(II)OB in most of the sampling points were between 103-105 CFU/g, which was similar to the results of Weiss et al. (2003). They pointed out that Fe(II)OB in the rhizosphere of some wetland plants were abundant, which ranged from 3.4×102 to 1.2×106 CFU/g
(Weiss et al., 2003). The mean numbers of Fe(II)OB of different sampling points decreased in the following order B>D>E>C>A. Site A was expected to have the least numbers of Fe(II)OB as it was the control point, while site B which represented the source of AMD have the most numbers of Fe(II)OB. However, according to the statistical analysis, the numbers of Fe(II)OB in different sites were not significantly different from each other (p>0.05). This may due to the fact that the abundance of
Fe(II)OB may vary with sites and microsites, but little is know about the factors that control their distribution, abundance and activity (Neubauer et al., 2007).
The numbers of acidophilic heterotrophs in most of the sampling points were between 105-106 CFU/g, which are more than the number of Fe(II)OB. According to 46 the results of Sivaram (2010) who studied the acidophilic heterotrophs and Fe(II)OB in soil from different depth and locations in the same contaminated site, the overall numbers of microorganisms on FETSB medium were also less than that observed on the WAYE plates. According to the study of Leduc et al. (2002), acidophilic heterotrophs were also the most numerous groups compared with other quantified microorganisms such as Fe(II)OB, and sulfur oxidizing bacteria. The numbers of acidophilic heterotrophs in the sampling point B (3.33×106 CFU/g) which represented the source of AMD was the highest, while the numbers in sampling point A (2.33×105
CFU/g) which represented the control point was the lowest. The mean numbers of acidophilic heterotrophs in different sites decreased in the order B>E>D>C>A.
Acidophilic heterotrophs are highly diverse in AMD, but many factors can affect the number and distributions of microbes in AMD, such as temperature, ionic strength, pH and so on (Baker and Banifield, 2003).
Initial rhizosphere bacteria assessment indicated that sufficient microorganisms were present at site. Rhizosphere bacteria can be cultured to assess their potential to precipitate Fe and to enhance phytoremediation of metals. Rhizosphere bacteria used for phytoremediation were collected from B which have the most abundance of both acidophilic heterotrophs and Fe(II)OB.
4.1.2 Soil incubation experiments to assess Fe(II) oxidation kinetic rates
In soil incubation experiments, rhizosphere soils were incubated with synthetic
AMD in erlenmeyer flasks. To understand the role of rhizosphere Fe(II)OB in
47 formation of Fe plaque, the rates of Fe(II) oxidation in non-sterile soils collected from
the root zone of reeds were compared to Fe(II) oxidation rates in soils of which the
biological activity was inhibited by formaldehyde. Table 4.2, 4.3 and Figure 4.1
indicated the change of pH and Fe(II) concentration (mg/L) of sterile (abiotic) or
non-sterile (biotic) samples from different sampling points.
Table 4.2 pH and concentration of Fe(II) of sterile (abiotic) samples Sample A B C D E Time 0h pH 3.81±0.06 2.97±0.03 3.01±0.03 3.00±0.031 3.11±0.01 Fe(II) 7.33±1.67 11.68±0.98 10.28±0.89 11.00±1.43 9.80±0.42 6h pH 3.80±0.06 2.97±0.03 3.00±0.03 3.00±0.04 3.10±0.02 Fe(II) 7.23±1.75 11.39±0.88 9.96±0.81 10.87±1.43 9.73±0.31 12h pH 3.79±0.06 2.97±0.02 2.98±0.03 3.00±0.04 3.10±0.02 Fe(II) 7.20±1.72 11.26±0.70 9.86±0.84 10.80±1.43 9.73±0.48 24h pH 3.78±0.06 2.97±0.02 2.98±0.03 3.00±0.03 3.09±0.01 Fe(II) 7.10±1.80 11.19±0.65 9.80±0.80 10.70±1.43 9.70±0.35 36h pH 3.78±0.06 2.97±0.02 2.98±0.03 3.00±0.03 3.09±0.01 Fe(II) 7.00±1.80 11.03±0.65 9.76±0.81 10.67±1.43 9.73±0.48 48h pH 3.78±0.06 2.97±0.02 2.98±0.02 3.00±0.03 3.09±0.01 Fe(II) 7.00±1.80 10.96±0.52 9.73±0.83 10.70±1.49 9.70±0.34 The results were reported as average±standard deviation, n=3
Table 4.3 pH and concentration of Fe(II) of non-sterile (biotic) samples Sample A B C D E Time 0h pH 3.81±0.07 2.91±0.10 3.01±0.01 3.01±0.02 3.13±0.01 Fe(II) 7.72±0.92 14.24±1.57 12.50±0.20 13.01±0.61 11.22±0.15 6h pH 3.72±0.09 2.71±0.02 2.82±0.03 2.76±0.10 2.97±0.03 Fe(II) 5.80±0.52 8.23±0.38 9.30±0.76 8.34±1.23 8.27±1.08 12h pH 3.59±0.09 2.62±0.03 2.67±0.05 2.63±0.05 2.77±0.02 Fe(II) 5.33±0.12 4.12±0.10 4.34±1.38 3.92±0.13 4.25±0.20 24h pH 3.57±0.02 2.58±0.01 2.60±0.03 2.60±0.05 2.74±0.01 Fe(II) 4.42±0.98 1.88±0.20 2.46±0.49 2.52±0.84 3.38±0.01 48h pH 3.55±0.01 2.50±0.01 2.53±0.01 2.55±0.02 2.66±0.02 Fe(II) 2.62±0.11 0.94±0.01 1.39±0.15 1.49±0.31 2.04±0.06 The results were reported as average±standard deviation, n=3
48 10 3.85
3.80 8 3.75
6 3.70
(mg/l)
2+ 3.65 pH a
Fe 4 biotic Fe 3.60 abiotic Fe 2 3.55 biotic pH abiotic pH 0 3.50 0 0.5 1 1.5 2 Time(d)
Figure 4.1(a) Fe2+ concentration and pH of soil collected from sampling point A on March 16, 2012. Error bars represented one standard deviation, n=3.
16 3.05 3.00 14 2.95 12 2.90 2.85 10 2.80
(mg/L) 8 2.75
pH 2+ 2.70 b Fe 6 2.65 biotic Fe 4 2.60 abiotic Fe 2.55 2 biotic pH 2.50 abiotic pH 0 2.45 0 0.5 1 1.5 2 Time (d)
Figure 4.1(b) Fe2+ concentration and pH of soil collected from sampling point B on March 16, 2012. Error bars represented one standard deviation, n=3.
49 14 3.05 3.00 12 2.95 10 2.90 2.85 8 2.80
(mg/L) pH c
2+ 2.75 6 Fe 2.70 biotic Fe 4 2.65 abiotic Fe 2.60 2 biotic pH 2.55 abiotic pH 0 2.50 0 0.5 1 1.5 2 Time(d)
Figure 4.1(c) Fe2+ concentration and pH of soil collected from sampling point C on March 16, 2012. Error bars represented one standard deviation, n=3.
14 3.05 3.00 12 2.95 10 2.90 2.85 8 2.80
(mg/L)
pH
2+ 6 2.75 Fe 2.70 d 4 2.65 biotic Fe 2.60 2 abiotic Fe 2.55 biotic pH 0 2.50 abiotic pH 0 0.5 1 1.5 2 Time(d)
Figure 4.1(d) Fe2+ concentration and pH of soil collected from sampling point D on March 16, 2012. Error bars represented one standard deviation, n=3.
12 3.15 3.10 10 3.05 3.00 8 2.95 2.90 e
(mg/L) 6
pH
2+ 2.85 biotic Fe Fe 4 2.80 2.75 abiotic Fe 2 2.70 biotic pH 2.65 abiotic pH 0 2.60 0 0.5 1 1.5 2 Time(d)
Figure 4.1(e) Fe2+ concentration and pH of soil collected from sampling point E on March 16, 2012. Error bars represented one standard deviation, n=3.
50 There were not significant differences in the initial pH and Fe(II) concentration between sterile (abiotic) and non-sterile (biotic) samples, However, the pH and the amount of Fe(II) in non-sterile (biotic) samples decreased with time while the pH and
Fe(II) concentration in the sterile (abiotic) samples did not vary too much with time.
In Figure 4.1(a), the pH of biotic samples of site A which represented the control point decreased from 3.81 to 3.55, while the amount of Fe(II) decreased from 7.72 mg/L to 2.62 mg/L after 2 days. However, the pH in abiotic samples of A only reduced from 3.81 to 3.78 and the level of Fe(II) only varied from 7.34 mg/L to 7.00 mg/L after 48 hrs. The decreasing trends of pH and Fe(II) concentration of non-sterile
(biotic) samples of other sampling points were more evident and dramatic than that of site A. In Figure 4.1(b), the Fe(II) concentration varied from 14.24 mg/L to 0.94 mg/L in non-sterile samples of site B after 2 days, and the pH decreased from 2.91 to 2.50 accordingly. Figures 4.1(c), 4.1(d) and 4.1(e) also showed that removal of Fe(II) was observed in non-sterile incubations of other sampling points, and the pH decreased concomitantly with Fe(II). However, Fe(II) concentration and pH did not change too much in abiotic incubations. The results suggested that the removal of Fe(II) may be mainly mediated by Fe(II)OB instead of abiotic chemical reaction. The change of pH and Fe(II) concentrations was based on the difference in Fe(II)OB population density from each locations. It was not surprising that sampling point A which represented the control point depicted the least variation, while the sampling point B which had the most Fe(II)OB showed the most dramatic change in pH and Fe(II) concentrations.
According to previous research, the rate of chemically Fe(II) oxidation was low at low 51 pH and Fe(II)OB mediated between 45 and 90% of Fe(II) oxidation in laboratory studies (Emerson and Revsbech 1994; Sobolev and Roden 2001). However, the Fe(II) oxidation rate in real field which will be more variable than completely controlled lab experiment based on synthetic media.
From Figure 4.1(a) to 4.1(e), we can observe that pH and Fe(II) concentration decreased sharply in the first 6 hrs and did not vary too much from 1.5 days to 2 days.
The shape of the curves indicated that the oxidation of Fe(II) in the rhizosphere soils may be first order reaction. The First order Fe(II) oxidation rate constants (k) were calculated using the followed equation (5) (Senko et al., 2008):
In[Fe(II)t]=-kt + In[Fe(II)initial] (5)
The kinetic rates of different sampling points were 0.692±0.222 mg/L/d for sampling point A, 1.922±0.038 mg/L/d for B, 1.507±0.143 mg/L/d for C, 1.661±0.194 mg/L/d for D and 1.245±0.053 mg/L/d for E. Previous research has also measured the rate of Fe(II) oxidation. Kirby et al. (1999) also pointed out that first order rate law can be applied to Fe(II) oxidation and the rate from 10-7 to 10-5 mol/L/s were reported.
Previous research also suggested that Fe(II)OB may mediate rhizosphere Fe(II) oxidation and plaque formation (Neubauer et al., 2007; Weiss et al., 2003). Results of iron kinetic experiment also demonstrated that Fe(II)OB existing in rhizosphere can oxidize Fe(II) to Fe(III). The results of iron kinetic experiments also showed that first order rate constants of Fe(II) oxidation decreased in the order: B>D>C>E>A. The B point which possessed the most numbers of Fe(II)OB developed the highest first order rate constants of Fe(II) oxidation, while the A point which had the lowest numbers of 52 Fe(II)OB showed the lowest first order rate constants of Fe(II) oxidation. It was not surprising that rates of Fe(II) oxidation relate to the abundance of Fe(II)OB. The mean numbers of Fe(II)OB in E point were higher than that in C point, but the C point possessed higher Fe(II) oxidation rate constant. This may be attributed to the heterogeneous ecological conditions of microorganisms in fields. In real fields, the physical, chemical, nutritional, and ecological conditions for microorganisms may vary from the micrometer to beyond the kilometer scales (Madsen, 1998). The rhizosphere Fe(II) oxidation rates can also be affected by plant activity and other factors such as pH, temperature, the availability of O2, Fe(II) and Fe(III), etc.
(Neubauer et al., 2007).
4.1.3 Soil digestion and soil pH in field
pH and total/mobile metals concentrations in rhizosphere soil collected on March
16, 2012 were analyzed. The data were presented in Table 4.4 and Figure 4.2.
Table 4.4 pH and total metals in rhizosphere soils collected in March, 2012. Point A B C D E Metal pH 2.86±0.49 1.96±0.25 2.05±0.41 1.99±0.70 1.97±0.03 a a a a a Mn 2.45±0.16 0.04±0.00 0.05±0.01 0.08±0.02 0.01±0.00 a b b b b Fe 13.78±0.48 413.63±7.75 266.99±10.36 388.22±21.6 390.24±13.90 c a b a a Al 1.76±0.03 1.39±0.03 1.67±0.02 2.70±0.024 0.31±0.03 b c bc a d Error bar represented the standard deviation of triplicate samples. Different letters in the same row indicate a significant difference at p<0.05
53 The pH of rhizosphere soil was very low (<3), and decreased in the order of B～D～
E～C～A. It was not surprising since one of the typical characteristic of AMD was low pH (Herlihy et al., 1990). The oxidation of dissolved Fe(II) and hydrolysis of
Fe(III) which produced H+ was the main reason to cause the low pH of soil (Wieder et al., 1990). As discussed in section 4.1.1, the mean numbers of Fe(II)OB of different sampling points also decreased in the following order B>D>E>C>A. It also suggested that Fe(II)OB played an important role in oxidation of Fe(II) and hydrolysis of Fe(III) and cause the decrease of pH.
100 a a 80
a a 60 b Mn b b b b Fe 40 Al c a a c 20 b b b
mg Mn,Fe,Al/kg soil Mn,Fe,Al/kg mg
0 A B C D E
Figure 4.2 Mobile metals in rhizosphere soils collected on March 16, 2012. Error bar represented the standard deviation of triplicate samples. Different letters on the same metal indicate a significant difference at p<0.05.
The total Fe, Al, Mn in rhizosphere soils was Fe>>Al>Mn for all the sampling points.
The total Fe concentration was approximately 1-2 orders of magnitudes than total Al and Mn. The level of total Fe in soils of B, D and E point which were close to the source of surface AMD was higher. The total Fe concentration in soils of A point which was not on the pathway of AMD was the lowest. The order of total Mn levels 54 in different points was B～D～E>C>A. The highest level of total Al was in soils of D point, which was about 2.70 mg/g soil. The total Al concentration in the rhizosphere soils changed in the order of D>A～C～B>E. The level of total Mn in soils of A point was about 2.45 mg/g soil, which was more than ten times higher than that in other points. This may due to the fact that soil was not a homogeneous media and many factors such as pH and redox potential may affect the concentrations of metals in soil
(Hansel and Fendorf, 2001)
The concentrations of mobile Fe, Al and Mn in soils were much lower than the total metals. This was attributed to the fact that metals in soil existed in different forms which had different bioavailability, such as simple ions in soil solution, linked to organic matters, coprecipitated with oxides and carbonates and so on (Maiz et al.,
1997). The mobile Fe was even thousand times lower than the total Fe in rhizosphere soil. It may indicate that most of Fe was present in the form of Fe precipitation caused by the oxidation ability of Fe(II)OB. The mobile Fe and Al in different points changed in the order of B～D>E～C>A and D>A～B～C>E, which were similar to the trends of total Fe and Al in soil. The levels of mobile Mn in different points were C～D>
A～B～E, which were not similar to the trends of total Mn in soils (A～B>C～D～
E). The difference of metal concentrations in different sampling points may attribute to the change of flow paths. The microbial activity and the plant uptake may also change the metal concentrations in soil as AMD move across the site. As referred above, different environmental factors such as pH, redox potential and the presence of organic acid also influence the mobility of metals in soil (Chen et al., 2008). 55 4.1.4 DCB extraction of reeds collected from field
Fe, Mn and/or Al plaque can be formed on the surface of root systems of aquatic plants, such as cattail and bulrush, and limit the mobility of metals to above ground plant tissue (Karathanasis and Johnson, 2003). Greipsson and Crowder (1992) also indicated that iron plaque may act as a ‘barrier’ for other elements uptake in plants.
However, other researchers reported that the plaque may enhance the metal uptake by plants (Greipsson, 1994). Zhang et al. (1998) also pointed out Fe plaque can increase the accumulation of essential and nonessential metals. The exact function of the plaque for metal accumulation in different plant species was still not clear. The Fe plaque often unevenly distributed on the root on the roots or rhizomes and the amount of Fe plaque was site specific (St-Cyr and Crowder, 1989).
The amount of metals plaque on roots and rhizomes of reeds collected from different sites were analyzed. Figure 4.3 to 4.5 showed the levels of metal plaque on reed collected on March, 2012, January, 2013 and May, 2013. Although the reports about Mn plaque were not as wide as Fe plaque, the demonstration of the presence of
Mn plaque on the roots of plants was not new. Mn oxide plaque was observed on the roots of Oryza sativa by Bacha and Hossner (1977). Mn plaque was also reported on the roots of other aquatic plants such as cattail (Karathanasis et al, 2003). The Mn concentrations in the rhizosphere soils of other sampling points were much lower than that in the soils of point A. Thus it was not surprising that Mn plaque has been observed only on the surface of reeds collected from A. Batty et al. (2002) also found that Mn plaque produced on the roots of plants only at elevated concentration. 56 0.05 a 0.04 0.04 a 0.03 a 0.03 0.02 rhizomes 0.02 roots
mg(Mn)/g(biomass) 0.01 0.01 0.00 A B C D E
50
40 a
a b 30 a a b rhizomes 20 c b roots mg(Fe)/g(biomass) 10 d e b 0 A B C D E
0.25 a 0.20 a b 0.15 a c
0.10 rhizomes c c roots
mg(Al)/g(biomass) b 0.05 b b c
0.00 A B C D E
Figure 4.3 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on March 16, 2012. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
57 0.10
0.08 a a 0.06 a rhizomes 0.04 roots
mg(Mn)/g(biomass) 0.02
0.00 A B C D E
50
40 a a a b b 30 c b rhizomes 20 d c roots
mg(Fe)/g(biomass) 10 e d
0 A B C D E
a 0.20 a a a 0.15 c 0.10 b b rhizomes b b c b roots mg(Al)/g(biomass) 0.05
0.00 A B C D E
Figure 4.4 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on January 9, 2013. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
58 a 0.10
0.08 a a 0.06 rhizomes 0.04 roots
mg(Mn)/g(biomass) 0.02
0.00 A B C D E
60
50 a a a 40 b b b b b 30 rhizomes c c 20 roots
mg(Fe)/g(biomass) d 10 c 0 A B C D E
0.25 a a 0.20 a a 0.15 c
b rhizomes 0.10 b b b roots
mg(Al)/g(biomass) c b 0.05
0.00 A B C D E
Figure 4.5 (a) Mn (b) Fe and (c) Al plaque on roots/ rhizomes of reeds collected on May 20, 2013. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
59 Fe plaque were observed on the surface of rhizomes and roots from all the sampling points on March, 2012, January, 2013 and May, 2013. The amount of iron plaque on plants of different sampling points decreased in the order B～D～E>C～A, while the total and mobile Fe in different points also changed in the similar order B～
D> E～C>A. Reeds grown in soil with highest Fe concentration have most Fe plaque on the roots or rhizomes. Batty and Younger (2003) also reported that the color of Fe plaque increased with the increase of Fe supply. The amounts of Fe plaque may be related to the Fe concentrations in media. It’s reported that the oxidizing activity of roots and the action of Fe(II)OB caused the formation of iron plaque (Neubauer et al.,
2007). The results of the iron kinetic experiment (section 4.1.2) also demonstrated that Fe(II)OB existing in rhizosphere can oxidize Fe(II) to Fe(III) and the first order rate constants of Fe(II) oxidation decreased in the order: B>D>C>E>A. The B point with the most amount of Fe(II)OB and highest Fe(II) oxidation rate possessed the highest concentration of iron plaque, while the C point which had the lowest numbers of Fe(II)OB and the lowest first order rate constants of Fe(II) oxidation. It may also suggest that the amount of iron plaque relates to the abundance and activity of
Fe(II)OB. Crowder and Macfie (1986), as well as St-Cyr and Crowder (1988) also pointed out that the extent of the iron plaque on the roots was site dependent and varied with each microsite. Other factors such as temperature and pH also influence the formation of Fe plaque (Wieder et al., 1990)
Al was also found on the surface of roots or rhizomes of reed from all the sampling points. The amount of Al plaque on plants of different sampling points 60 decreased in the order and D～A>B～C～E, while the total and mobile Al in different points also changed in the similar order D>A～B～C>E. The higher Al levels in soils, the more Al plaque formed on the surfaces of reeds. The existence of Al-phosphate plaque on the roots of plants was reported extensively (Karathanasis et al., 2003).
Compared to Fe plaque, the concentrations of Al plaque was lower. This maybe attributed to the lower Al concentrations in soil. Besides, Al mobility was higher under lower pH conditions which may also limit the formation of Al plaque
(Karathanasis and Johnson, 2003).
The Fe and Mn plaque on reeds collected in May, 2013 was slightly higher than that on reeds collected in Jan, 2013 and March, 2012. The Mn plaque was 0.08±0.02 mg/g on roots of reeds collected from point A in May, 2013, which was higher than that on reeds collected from the same point in Jan, 2013 and March, 2012 (0.06±0.01 mg/g and 0.04±0.00 mg/g, respectively). The Fe plaque was 38.51±2.23 mg/g on roots of reeds collected from point B in May, 2013, while it was 30.27±1.84 mg/g and
23.02±2.68 mg/g on reeds collected from point B in Jan, 2013 and March, 2012. This was most probably due to the fact that the sources of AMD have not been cut off and
AMD are still flowing to the field. Thus the Mn and Fe metals are being oxidized and precipitated into plaque on the root system of reeds. However, the increase of Al plaque was less obvious than Mn and Fe plaque. The Al plaque was 0.17±0.01 mg/g on roots of reeds collected from point D in May, 2013, which was similar to that on rhizomes collected from point D in Jan, 2013 and March, 2012 (0.17±0.01 mg/g and
0.19±0.02 mg/g, respectively). The mine water discharge often contained very low 61 concentration of P which may limit the formation of Al-phosphate, which was the main component of Al plaque (Batty et al., 2002).
4.1.5 Plant digestion of reeds collected from field
The concentrations of Mn, Fe and Al in different organs of reeds were determined to investigate the ability of reeds to accumulate these metals from AMD. Figures 4.6 to 4.8 show the distribution of metals in reeds collected from different sampling points in March, 2012, Jan, 2013 and May, 2013.
The concentration of metals in plants was associated with the metal concentrations in rhizosphere soils. Fe concentrations were the highest among the other metals studied. It was not surprising as Fe had the highest concentrations in the soils (413.63±0.7.75 mg/g). The concentration of Al in rhizosphere soils of all the sampling points was greater than Mn, and plants also uptake more Al than Mn. The Fe concentration in the underground organs of reeds from different sampling points was
B～D>E～C～A while the mobile Fe concentration in soils was B～D> E～C>A.
The similar phenomenon was also observed by Batty and Younger (2003) who pointed out that Fe uptake by plants may increase with the increase of Fe supply.
Similarly, the roots contained more Al from sampling points with more Al. The Al levels in roots of reeds were D～A>C～B>E which was similar to the order of total and mobile Al in soil. These results were in agreement with previous studies.
According to Deng et al. (2004), elements in the underground tissues of wetland plants showed strong positive correlations with the sediment elements. 62 a
a 0.7 rhizomes roots 0.6 stems 0.5 a flowers 0.4 a 0.3 b a a 0.2 a
mg(Mn)/g(biomass) a a c c a b c a b a c b a c 0.1 c 0 A B C D E
b rhizomes 25 roots a 20 a stems a flowers 15 a a a b 10 b a b a c
mg(Fe)/g(biomass) b 5 a a a b b b c a b b b 0 A B C D E
c rhizomes 4 roots stems 3 a flowers
2 a a
a mg(Al)/g(biomass) 1 a c a a a a a b c b b c a b a c b a 0 A B C D E
Figure 4.6 (a) Mn (b) Fe and (c) Al in reeds collected on March 16, 2012. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
63 a
rhizomes 0.3 a a a roots 0.25 b stems a 0.2 a flowers b a 0.15 b c c a b \ b d 0.1 b b b b
mg(Mn)/g(biomass) b 0.05 0 A B C D E
b rhizomes 30 roots 25 a stems 20 a flowers a 15 b b b 10 a c a c mg(Fe)/g(biomass) c b b 5 d a b a b c c 0 A B C D E
c rhizomes 3 roots 2.5 a stems a a 2 a a flowers b b a a b b c b b 1.5 c c c d b 1 c mg(Al)/g(biomass) c c 0.5 d d e 0 A B C D E
Figure 4.7 (a) Mn (b) Fe and (c) Al in reeds collected on January 9, 2013. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
64 a rhizomes 0.3 roots a a 0.25 a stems a a 0.2 b leaves a a 0.15 a b b b c b c 0.1 d b b b b
mg(Mn)/g(biomass) d 0.05 0 A B C D E
b rhizomes 30 roots
25 a stems a 20 b leaves a 15 b a c 10 a c a b b mg(Fe)/g(biomass) a b a b 5 c c b c c b 0 A B C D E
c
rhizomes 4 roots a stems 3 b leaves b a c 2 a b c b a a b b c b a mg(Al)/g(biomass) 1 b b b d c c d 0 A B C D E
Figure 4.8 (a) Mn (b) Fe and (c) Al in reeds collected on May 20, 2013. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05.
65 The levels of mobile Mn in different points were C～D>A～B～E, which were also similar to orders of Mn in roots and rhizomes of reeds. Besides, reeds uptake less
Mn from A point which possessed the most total Mn in rhizosphere soil. It may suggest that the Mn plaque formed on the root surface can decrease the amount of Mn entering the root of reed. Mn plaque has also been shown to have an inhibitory effect on copper uptake by plants (Batty et al., 2002).
The metals were unevenly distributed in plant tissues. The Mn concentration in underground parts was not significantly different (p>0.05) from that in aboveground tissues. As expected, the Fe concentration in root and rhizome were significantly higher (p<0.05) than that in shoots. Batty and Younger (2003) cultured reeds in nutrient solution containing iron and the concentrations of Fe in roots were much higher than that in shoots. Bonanno and Giudice (2010) investigated the accumulation of heavy metals such as Cd, Mn, Ni, and Zn in reeds and they indicated that the subsurface organs of reeds were the primary areas of metal accumulation. Lesage et al.
(2007) also indicated the Fe concentration in stems Bonanno (2011) also showed that high concentrations of Al in roots and low mobility through tissues. In our study, however, aboveground organs were the main organs for Al storage. Pervious studies reported that the translocation of metals from roots to 66 shoots depends on various biological or chemical/physical factors and changed according to environmental conditions such as temperature, pH, water ion content, salinity conditions, availability of heavy metals and so on (Liang and Wong, 2003; Demirezen and Aksoy, 2004; Grisey et al., 2012). The metal concentrations in reeds in Jan, 2013 were also higher than that in reeds in March, 2012. For instance, the metals levels were 0.13±0.01 mg Mn/g, 19.41±1.65 mg Fe/g and 0.99±0.20 mg Al/g in roots of reeds collected from B in Jan, 2013. The metals levels were 0.09±0.03 mg Mn/g, 13.48±0.93 mg Fe/g and 0.08±0.01 mg Al/g in roots reeds collected from B in March, 2012. It was not surprising, since the reeds kept growing in the field and then can accumulate more metals into the biomass. The metals concentrations in the aboveground tissues of reeds collected in May, 2013 were higher than that in reeds collected in Jan, 2013. The metals concentrations were 0.09±0.02 mg/g Mn, 5.25±1.13 mg/g Fe and 1.25±0.23 mg/g Al in stems of reeds collected from site B in May, 2013, while the metal levels were 0.07±0.01 mg/g Mn, 4.63±0.67 mg/g Fe and 0.64±0.16 mg/g Al in stems of reeds collected from site B in Jan, 2013. However, the amounts of metals in the underground organs of reeds collected in May, 2013 were lower than that in reeds collected in Jan, 2013. For example, the roots of reeds collected from site C accumulated 0.21±0.03 mg/g Mn, 7.73±1.53 mg/g Fe and 1.16±0.12 mg/g Al in Jan, 2013, while the roots contained 0.17±0.02 mg/g Mn, 5.48±1.63 mg/g Fe and 0.71±0.12 mg/g Al in May, 2013. This may due to the fact that the propagation and growth of reeds in May enabled more metals to be transferred into the shoots, thus it may decrease the storage of metals in 67 roots and rhizomes of reeds. According to the reports of Duman (2007), higher levels of metals were stored in underground parts of reeds in autumn and winter. The metabolic rates of plants were lower in winter while the growth of plants during spring and summer may increase the metals and nutrients transport. Taylor and Crowder (1983b) also indicated that spring growth of cattail can cause the mobilization and transport of mineral nutrients from rhizome to aerial tissues. Besides, the change of season and weather may cause the variation in metal accumulation and translocation in reeds, since environmental factors such as metal concentrations, interactions between metals and other elements may differ in different seasons. 4.1.6 Histological experiments of reeds collected from field Roots and rhizomes are first steps in metal uptake and translocation. Therefore, histological experiments were conducted to determine the specific tissues that contained Fe and Al in roots and rhizomes of reeds. 4.1.6.1 Cross sections of fresh root and rhizome before staining Figure 4.9 showed the basic rhizome structures of reed before staining and Figure 4.10 contained the basic root structures of reed prior to staining. Identification of the specific root sections based on the assistance from Dr. Ott (Biology Department, University of Akron) and previous literature (Reale et al., 2012; Baldantoni et al., 2009; Beyer et al., 2009). 68 1mm 200µm EP C CC V V b c a Figure 4.9 Cross sections of rhizome prior to staining (a) rhizomes at 1 mm scale (b) 10 µm cross section of depiction (c) vascular bundle of rhizomes. epidermis (EP); cortex (C); vascular bundle (V); central cylinder (CC) 1mm S b a EX 200µm 200µm X C P A c EN d Figure 4.10 Cross sections of non-stained Phragmites australis root (a) root at 1 mm scale (b) 10 µm cross section of root (c) subsection of the root (d) part of root. stele (S), exodermis (EX), aerenchyma (A), pith (P), endodermis (EN), cortex (C), xylem (X). 4.1.6.2 Fe staining for reeds collected from field Figure 4.11 to 4.15 showed the representative images of stained root. Figure 4.17 to 4.21 rhizomes sections of reeds collected from AMD site in Jan, 2013. 69 200µm 200µm a b Figure 4.11 Fe stained cross section of root of reed collected from site A in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. Figure 4.16 and 4.22 presented the representative images of stained roots and rhizomes collected in May, 2013. A blue hue indicated the tissues contained iron. 200µm 200µm a b Figure 4.12 Fe stained cross section of root of reed collected from site B in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.13 Fe stained cross section of root of reed collected from site C in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 70 200µm 200µm a b Figure 4.14 Fe stained cross section of root of reed collected from site D in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.15 Fe stained cross section of root of reed collected from site E in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b 200µm 200µm 200µm e c d Figure 4.16 Fe stained cross sectione of root of reeds collected from (a) site A (b)site B (c) site C (d) site D and (e) site E in May, 2013 71 No structural difference was observed between samples from different locations or collected at different times. The roots consisted of exodermis and inner aerenchyma separated from the stele by endodermis (Baldantoni et al., 2009). Figure 4.11 to 4.16 indicated that most of iron was sequestered in the exodermis of roots. This is in agreement with other research that found that reeds can separate and store toxic matters in the tissues outside the endodermis and then prevented or reduced the translocation to other sites within the plants (Baldantoni et al., 2009). Soukup et al. (2002) also reported that the exodermis of reeds can be an effective barrier to restrict the penetration of iron into root tissues. The existence of exodermis and endodermis may play an important role in the protection of the root tissues against of toxic metals (Siqueira-Silva et al., 2012). The intensity of blue hue in the exodermis of roots of reeds collected from site B was the strongest; while the color of reeds collected from site A was the lightest. The color and areas of blue hue in the exodermis of roots of reeds decreased as the following order: B～D>E～C～A. The phenomenon was in accord with the quantitative data obtained from digested biomass. As was discussed in section 4.1.3 and 4.1.5, Fe concentration in soils and roots of reeds was in the order B～D>E～C～A. 72 1mm 200µm a b Figure 4.17 Fe stained cross section of rhizome of reeds collected from site A collected in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection that to clearly show the sequestration of iron. 1mm 200µm a b Figure 4.18 Fe stained cross section of rhizome of reeds collected from site B in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection. 1mm 200µm a b Figure 4.19 Fe stained cross section of rhizome of reeds collected from site C in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection. 73 1mm 200µm a b Figure 4.20 Fe stained cross section of rhizome of reeds collected from site D in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection. 1mm 200µm a b Figure 4.21 Fe stained cross section of rhizome of reeds collected from site E in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 200µm 200µm a b 200µm 200µm 200µm c d e b Figure 4.22 Fe stained cross section of rhizome of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013 74 Figure 4.17 to 4.22 showed that Fe depicted by a blue hue was unevenly distributed in the epidermis, cortex and central cylinder of rhizomes. Compared to cross sections of roots, the blue hue was less obvious in the cross sections of rhizomes. This due to the fact that the amounts of iron contained in the rhizome were less than that in the roots. Blue stain associated with Fe was found around the vascular bundles, which were the transport systems of plants. It was observed that the blue color around the vascular bundles of reeds from site B was the deepest. The intensity of blue hue around the vascular bundles of rhizomes decreased as order: B>D>E～C～A. This trend was same with the change of Fe concentration in soils in AMD site which also decreased in the order: B～D>E～C～A. Our previous data also indicated that reeds grown in soil with more Fe accumulated more Fe into the rhizomes. As such, more Fe may be transported through vascular bundles. 4.1.6.3 Al staining for reeds collected from field The representative images of stained root sections of reeds collected from AMD site contained in Figure 4.23 to 4.28. Figure 4.29 to 4.34 presented the representative pictures of stained rhizomes of reeds. Magenta indicated the tissues contained Al. 75 200µm 200µm a b Figure 4.23 Al stained cross section of root of reed collected from site A in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.24 Al stained cross section of root of reed collected from site B in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.25 Al stained cross section of root of reed collected from site C in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.26 Al stained cross section of root of reed collected from site D in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. 76 200µm 200µm a b Figure 4.27 Al stained cross section of root of reed collected from site E in Jan, 2013, (a) magnified image of stele (b) magnified image of exodermis. Unlike Fe that was mainly stored in the exodermis and endodermis of roots, Al was found in the steles of roots. This may attribute to the different tolerance, accumulation and translocation mechanism of plants to different metals (Bonanno and Giudice, 2010). 200µm 200µm a b 200µm 200µm 200µm c d e Figure 4.28 Al stained cross section of root of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013 The magenta hue in the exodermis and steles of roots of reed from site D was the strongest (Figure 4.26; 4.28 d). The color in the exodermis of roots collected from site 77 E appeared to be the lightest and even the pith did not turn magenta after staining (Figure 4.27; 4.28 e). Previous data in section 4.1.5 showed that Al concentration in the roots of reeds decreased in the order D～A>C～B>E. These results supported our histological observation phenomena, since the intensity of magenta hue in the roots of reeds also changed according to the order: D～A>C～B>E. 1mm 200µm a b Figure 4.29 Al stained cross sections of rhizomes of reeds collected from site A in Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm a b Figure 4.30 Al stained cross sections of rhizomes of reeds collected from site B Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 78 1mm 200µm a b Figure 4.31 Al stained cross sections of rhizomes of reeds collected from site C Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm a b Figure 4.32 Al stained cross sections of rhizomes of reeds collected from site D Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm a b Figure 4.33 Al stained cross sections of rhizomes of reeds collected from site E Jan, 2013, (a) full view of the cross section with image (b) a magnified subsection 79 200µm 200µm a b 200µm 200µm 200µm c d e Figure 4.34 Al stained cross section of rhizome of reeds collected from (a) site A (b) site B (c) site C (d) site D and (e) site E in May, 2013 Similar to Fe, Al was also sequestered in the epidermis, cortex and central cylinder of rhizomes as showed in Figure 4.29 to 4.34. No anatomical differences have been found in the rhizomes from different sites in different times. The magenta hue was apparent in the tissue close to vascular bundles. It was observed that the intensity of magenta hue around the vascular bundles of rhizomes collected from site D and A was stronger than that from other locations. The color was the lightest of rhizomes collected from site E. The magenta color around the vascular bundles of rhizomes decreased in the order of D～A>C～B>E. It was in accord with our quantitative data from plant digestion, which indicated that Al concentration was the highest in rhizomes of reeds collected from site D and was the lowest of site E in AMD site. 80 4.1.7 Summary of field experiments Initial rhizosphere bacteria assessment indicated that sufficient Fe(II)OB and acidophilic heterotrophs were present at the site. Fe(II)OB oxidized Fe(II) to Fe(III), which was in the form of first order reaction and decreased pH in soil. The first order rate constants of Fe(II) oxidation decreased in the order: B >D> C> E>A which was in accord with the abundance of Fe(II)OB of different sampling points. The amount of Fe plaque related to the abundance of Fe(II)OB and Fe amounts of in soil. Similarly, the higher Al levels in soils, the more Al plaque formed on the root surfaces of reeds. However, Mn plaque was observed only on the surface of reeds collected from site A where the Mn concentration was the highest. The amounts of Fe and Mn plaque increased with time. However, the increase of Al plaque was less obvious than Mn and Fe plaque which may be attributed to the low amounts of phosphate in soil which inhibited the formations of Al-phosphate plaque. The concentration of metals in plants was associated with the metal concentrations in rhizosphere soils. Fe concentrations were the highest in plants, since more Fe existed in soil than Al and Fe. The Fe concentrations in soil were 413.63±7.75 mg/g which were much higher than Mn (0.04±0.00 mg/g) and Al (1.39±0.03 mg/g), while the Fe amounts in roots (13.43±5.98 mg/g) were also higher than Mn (0.09±0.03 mg/g) and Al (0.08±0.01 mg/g). The metals were unevenly distributed in plant tissues. The Mn concentration in underground parts was not significantly different (p>0.05) from that in aboveground tissues. The Fe concentration in root and rhizome were significantly higher than that in shoots. 81 However, aboveground organs were the main organs for Al storage. Various biological, chemical, physical and environmental conditions such as temperature, pH, water ion content, salinity conditions and availability of heavy metals may affect the translocation of metals in plants. The metal concentrations in reeds increased with time and changed with season. More metals were found in the underground parts of reeds in winter while the growth of plants during spring and summer increased the metals in aboveground tissues. The results of histological experiments indicated that most of iron was sequestered in the exodermis and endodermis of roots. Fe depicted by a blue hue was unevenly distributed in the epidermis, cortex and central cylinder of rhizomes. Al was found in the exodermis, endodermis as well as the steles of roots. Al depicted by the magenta hue was also sequestered in the epidermis, cortex and central cylinder of rhizomes. The intensity of stained color in the roots and rhizomes of reeds were related to the amounts of Fe and Al in tissues. The more Fe or Al in biomass, the strong intensity the color was. 4.2 CA biodegradation experiment Table 4.5 contained the volume of NaOH actually consumed and theoretical needed to neutralize C6H8O7·H2O solution. The theoretical value was calculated according to C6H8O7·H2O and NaOH calibration curve. 82 Table 4.5 Volume of 1M NaOH to neutralize C6H8O7·H2O solution C6H8O7·H2O volume of 1M actually consumed for actually consumed solution NaOH solution for solution with (ppm) theoretically without rhizosphere rhizosphere needed bacteria (ml) bacteria (ml) (ml) A: 2101 3.7 3.5 0.1 B:17860 30.4 30.2 20.5 C: 33616 57.2 57.1 39.8 As expected, CA, “a biodegradable chelator”, can be consumed by rhizosphere bacteria. After 4 weeks, the amounts of CA did not reduce in solution without rhizosphere bacteria. For those inoculated with bacteria, 98% of the CA was consumed in solution A, 33% in solution B and 30% was consumed in solution C. It was in agreement with previous research. Römkens et al. (2002) conducted greenhouse experiments to study the effect of 0.01 M CA on microbial activity in soils and they pointed out that CA was a substrate for bacterial growth and was microbially degraded within a few days after addition. Muhammad et al. (2009) and Chen et al. (2006) also pointed out that CA can be easier to be biodegraded, compared with other chelators such as EDTA. Applied non-biodegradable or least biodegradable chelating agents in phytoremediation may cause the leaching of metals into the ground water (Santos et al., 2006). Thus biodegradable chelators CA have gotten greater social acceptance and been widely used in phytoremediation. However, low level of CA was easily and quickly degraded by microorganisms which may affect its effect on improving phytoremediation. Middle and high level of CA can exist for longer time which may have long term effect on increasing metal uptake in plants and enhancing phytoremediation efficiency. 83 4.3 Reeds cultured in spiked soil Experiments such as rhizosphere bacteria enumeration, DCB extraction and plant digestion were also conducted for reeds cultured in spiked soil. The following sections presented the results of spiked soil experiments. 4.3.1 Acidophilic heterotrophs and Fe(II)OB in spiked soil Rhizosphere soil was collected into presterilized bottle during plant harvesting. Acidophilic heterotrophs were enumerated on WAYE medium, while Fe (II) oxidizing bacteria were cultured on FETSB medium. Table 4.6 showed the number of bacteria in rhizosphere soil cultured with reeds for 4, 8 and 12 weeks. Table 4.6(a) Numbers of rhizosphere bacteria in soils cultured with reeds for 4 weeks Treatment Reeds WAYE (CFU/g soil) FETSB (CFU/g soil) Clean soil Purchased 1.75 ±0.50×104 h / Wild 1.75 ±0.25×104 e / NCNB Purchased 2.78 ±0.23×105 g 1.90 ±0.05×105 c Wild 3.03 ±0.02×105 d 2.25±0.01×105 c NCWB Purchased 2.60±0.10×106 g 2.33±0.03×106 a Wild 6.23±1.23×106 cd 6.83±0.53×106 a LCNB Purchased 3.80±0.20×105 f 1.58±0.08×105 c Wild 6.35±0.15×105 d 2.70±0.35×105 c LCWB Purchased 1.60 ±0.10×106 e 1.45±0.05×106 b Wild 4.53 ±0.53×106 cd 4.50±0.35×106 b MCNB Purchased 3.43 ±0.08×106 d 1.38±0.03×105 c Wild 6.55 ±1.05×106 cd 2.53±0.13×105 c MCWB Purchased 1.33 ±0.04×107 c 2.43±0.03×105 c Wild 1.58 ±0.02×107 c 4.28±0.13×105 c HCNB Purchased 4.15 ±0.00×107 b 1.30±0.10×105 c Wild 8.78 ±0.48×107 b 3.18±0.23×105 c HCWB Purchased 1.01 ±0.00×108 a 1.63±0.10×105 c Wild 1.21 ±0.05×108 a 3.45±0.05×105 c / indicated not detected; results were reported as average±standard deviation, n=10. Different letters for the same type of reeds indicated a significant difference at p<0.05. 84 Table 4.6 (b) Numbers of rhizosphere bacteria in soils cultured with reeds for 8 weeks Treatment Reeds WAYE (CFU/g soil) FETSB (CFU/g soil) Clean soil Purchased 1.75 ±1.25×104 d / Wild 2.25 ±0.75×104 e / NCNB Purchased 2.63 ±0.87×105 c 2.03 ±0.03×105 c Wild 3.35 ±0.15×105 d 2.33±0.13×105 d NCWB Purchased 4.13±1.38×106 c 2.80±0.20×106 a Wild 7.23±0.73×106 cd 8.48±0.13×106 a LCNB Purchased 3.20±0.80×105 c 2.83±0.18×105 c Wild 4.58±0.08×105 d 3.55±0.15×105 d LCWB Purchased 3.23 ±0.73×106 c 2.45±0.20×106 a Wild 5.40±0.40×106 d 5.08±0.03×106 b MCNB Purchased 4.23 ±2.25×105 c 2.53±0.08×105 c Wild 3.63 ±0.63×106 d 3.08±0.02×105 d MCWB Purchased 1.52 ±0.13×107 b 1.20±0.05×106 b Wild 1.64 ±0.01×107 bc 4.53±0.23×106 b HCNB Purchased 0.83 ±4.83×106 c 1.38±0.18×105 c Wild 2.65 ±0.40×107 b 3.75±0.05×105 d HCWB Purchased 7.25 ±0.30×107 a 1.53±0.13×106 b Wild 1.23 ±0.03×108 a 3.63±1.78×106 c / indicated not detected; results were reported as average±standard deviation, n=10. Different letters for the same type of reeds indicated a significant difference at p<0.05. Acidophilic heterotrophs and Fe(II)OB were important groups of bacteria at AMD site. Acidophilic heterotrophs and Fe(II)OB were found in all the spiked soils, but no Fe(II)OB was observed in clean soil. It’s not surprising to note that soil inoculated with rhizosphere bacteria possessed more Fe(II)OB and acidophilic heterotrophs than soil without adding rhizosphere bacteria. For instance, the soil LCNB cultured with purchased reeds have 3.80±0.20×105 CFU/g soil acidophilic heterotrophs, and 1.58±0.08×105 CFU/g soil Fe(II)OB while the soil LCWB grown with purchased reeds possessed 1.60 ±0.10×106 CFU/g soil acidophilic heterotrophs and 1.45±0.05×106 CFU/g soil Fe(II)OB after 4 weeks. For soil cultured with wild reeds, the soil NCNB have 3.03 ±0.02×105 CFU/g soil acidophilic heterotrophs, and 85 2.25±0.01×105 CFU/g soil Fe(II)OB while the soil NCWB possessed 6.23±1.23×106 CFU/g soil acidophilic heterotrophs, and 6.83±0.53×106 CFU/g soil Fe(II)OB after 4 weeks. Table 4.6(c) Numbers of rhizosphere bacteria in soils cultured with reeds for 12 weeks Treatment Reeds WAYE (CFU/g soil) FETSB (CFU/g soil) Clean soil Purchased 2.75 ±0.75×104 e / Wild 4.00 ±0.50×104 f / NCNB Purchased 2.45 ±0.05×105 d 1.60 ±0.01×105 c Wild 3.15 ±0.15×105 e 5.98±0.13×105 d NCWB Purchased 6.45±1.55×106 c 5.03±0.43×106 a Wild 1.25±0.10×107 cd 1.03±0.03×107 a LCNB Purchased 3.50±0.50×105 d 3.45±0.20×105 c Wild 4.45±0.05×105 e 4.20±0.10×105 d LCWB Purchased 6.35 ±0.85×106 c 4.68±0.38×106 a Wild 9.30±0.55×106 cde 8.35±0.25×106 b MCNB Purchased 4.55 ±0.45×105 d 2.45±0.10×105 c Wild 4.13 ±0.38×106 de 3.40±0.15×105 d MCWB Purchased 1.63 ±0.13×107 b 3.08±0.18×106 b Wild 1.78 ±0.18×107 c 6.30±0.20×106 c HCNB Purchased 6.28 ±0.23×106 c 1.35±0.15×105 c Wild 3.23 ±0.23×107 b 1.03±0.03×105 d HCWB Purchased 9.75 ±0.10×107 a 3.38±0.38×106 b Wild 1.46 ±0.03×108 a 6.13±0.18×106 c / indicated not detected; results were reported as average±standard deviation, n=10. Different letters for the same type of reeds indicated a significant difference at p<0.05. The similar trends were found in soil after 8 and 12 weeks. For spiked soil cultured with reeds for 4 weeks, the numbers of heterotrophs were higher in soils treated with CA than that without adding CA. Our results of CA biodegradation experiments indicated that CA could be biodegraded by rhizosphere bacteria. Chen et al. (2006) also pointed out that CA can be used as carbon source by many bacteria. That may explain why the soil initially added with higher level of CA had more 86 heterotrophs. However, the numbers of acidophilic heterotrophs did not increase or even slightly decrease in spiked soil NCNB, LCNB, MCNB, HCNB after 8 and 12 weeks. For example, the numbers of heterotrophs were 3.43±0.08×106 CFU/g soil in soil MCNB cultured with purchased reeds after 4 weeks, and the numbers changed to 4.23±2.25×105 CFU/g soil and 4.55±0.45×105 CFU/g soil after 8 and 12 weeks. For soil MCNB cultured with wild reeds for 4 weeks, the numbers of heterotrophs were 6.55±1.05×106 CFU/g soil. Then the population of heterotrophs became 3.63 ±0.63×106 CFU/g soil and 4.13±0.38×106 CFU/g soil after 8 and 12 weeks. This may due to the fact that CA has been consumed by the heterotrophs and there were less additive carbon source for heterotrophs. Then the main nutrients to maintain the population and activity of rhizosphere bacteria were the plant root exudates (Abhilash et al., 2011; Khan, 2005). The numbers of acidophilic heterotrophs did not decrease, and even slightly increase in spiked soil NCWB, LCWB, MCWB, HCWB after 8 weeks. For example, the numbers of heterotrophs were 1.33±0.04×107 CFU/g soil in soil MCWB cultured with purchased reeds after 4 weeks, and the numbers changed to 1.52±0.13×107 CFU/g soil and 1.63±0.13×107 CFU/g soil after 8 and 12 weeks. For soil MCWB cultured with wild reeds for 4 weeks, the numbers of heterotrophs were 1.58±0.02×107 CFU/g soil. Then the population of heterotrophs became 1.64 ±0.01×107 CFU/g soil and 1.78±0.18×107 CFU/g soil after 8 and 12 weeks. This may due to the fact that new rhizosphere bacteria were inoculated into the spiked soil every 4 weeks. 87 It was worthwhile to note that spiked soil without amending CA had more Fe(II)OB than soil added with CA. For instance, the numbers of Fe(II)OB in soil HCWB grown with wild reeds were 3.45±0.05×105 CFU/g soil after 4 weeks, while the population were 6.83±0.53×106 CFU/g soil in soil NCWB grown with the same kind of reeds for the same time period. The trend was similar for spiked soil cultured with purchased reeds. For example, the numbers of Fe(II)OB in soil HCWB grown with purchased reeds were 1.63±0.10×105 CFU/g soil after 4 weeks, while the population were 2.33±0.03×106 CFU/g soil in soil NCWB grown with purchased reeds for 4 weeks. Harrison (1984) reported that glucose, pyruvate or some organic impurities may inhibit the growth of Fe(II)OB. Marchand and Silverstein (2003) also pointed out that the addition of organic electron donor may promote reducing conditions favorable to iron reduction. As a organic acid, CA may also inhibit the growth of Fe(II)OB. So the spiked soil added with higher level of CA had lower number of Fe(II)OB in our study. The population of Fe(II)OB were higher in spiked soil cultured with wild reeds than that with purchased reeds. This may due to that the rhizosphere bacteria and wild reeds were all collected from the AMD site. The rhizosphere bacteria may better adapt to the rhizosphere environment of wild reeds than the purchased reeds. For spiked soil cultured with the same kind of reeds, the magnitudes of Fe(II)OB in spiked soil NCNB, LCNB, MCNB, HCNB did not change too much among 4, 8 and 12 weeks. For instance, the numbers of Fe(II)OB were 1.58±0.08×105 CFU/g soil in soil LCNB cultured with purchased reeds after 4 weeks, and the numbers changed to 88 2.83±0.18×105 CFU/g soil and 3.45±0.20×105 CFU/g soil after 8 and 12 weeks. For soil LCNB cultured with wild reeds for 4 weeks, the number of Fe(II)OB were 2.70±0.35×105 CFU/g soil. Then the population became 3.55±0.15×105 CFU/g soil and 4.20±0.10×105 CFU/g soil after 8 and 12 weeks. Compared with soil NCNB, LCNB, MCNB, HCNB cultured with purchased and wild reeds, the numbers of Fe(II)OB in spiked soil NCWB, LCWB, MCWB, HCWB increased more after 8 and 12 weeks. For example, the numbers of Fe(II)OB in soil NCNB grown with wild reeds changed from 2.25±0.01×105 CFU/g soil to 2.33±0.13×105 CFU/g soil and 5.98±0.13×105 CFU/g soil after 4, 8 and 12 weeks, and the numbers of Fe(II)OB in soil NCWB grown with wild reeds increased from 6.83±0.53×106 CFU/g soil to 8.48±0.13×106 CFU/g soil and 1.03±0.03×107 CFU/g soil after 4, 8 and 12 weeks. The numbers of Fe(II)OB in spiked soil LCWB, MCWB and HCWB also increased after 8 and 12 weeks. Two possible reasons may explain this phenomenon. On one hand, the spiked soils were inoculated with new rhizosphere bacteria every 4 weeks. On the other hand, the CA which can inhibit the growth of Fe(II)OB was consumed by heterotrophs and then the growth of Fe(II)OB may be faster. It was in agreement with the results of Marchand and Silverstein (2003). They indicated that the heterotrophs can relieve and reduce the inhibitory effect of glucose to Fe(II)OB and the presence of heterotrophs can reduce the glucose concentrations and permit iron oxidation. The results of population of Fe(II)OB were in accord with the results discussed in the section of soil metals: more metals precipitated and less mobile metals existed in the spiked soil possessed higher numbers of Fe(II)OB and 89 reeds grown in the spiked soil possessed more Fe(II)OB formed more Fe and Mn plaque on the surface of root systems. At the same time, the plants in soils had less mobile metals accumulated less metals in their tissues. 4.3.2 pH of soil Table 4.7 shows the pH of soil before and after CA amendments. Four, 8 and 12 weeks after culturing the reeds, the pH of soil were also measured, which was showed in Table 4.8. Table 4.7 pH of spiked soil before and after adding CA Treatment 8/29/2012 8/31/2012 Clean soil 5.20±0.02 5.23±0.02 NCNB 3.30±0.06 3.30±0.05 NCWB 3.26±0.02 3.27±0.01 LCNB 3.22±0.01 3.10±0.04 LCWB 3.25±0.03 3.09±0.01 MCNB 3.30±0.06 2.92±0.06 MCWB 3.24±0.02 2.95±0.06 HCNB 3.29±0.02 2.65±0.03 HCWB 3.27±0.02 2.67±0.04 The results were reported as average±standard deviation, n=3 Table 4.8 pH of spiked soil under different treatments 4 weeks 4 weeks 8 weeks 8 weeks 12 weeks 12 weeks Treatment after after after after after after purchased wild reeds purchased wild reeds purchased wild reeds reeds reeds reeds Clean soil 5.14±0.11 5.13±0.06 5.19±0.05 5.17±0.03 5.19±0.04 5.16±0.02 NCNB 2.12±0.04 2.10±0.04 2.08±0.02 2.09±0.03 2.06±0.01 2.07±0.01 NCWB 1.99±0.01 1.95±0.04 1.93±0.03 1.90±0.03 1.90±0.02 1.87±0.01 LCNB 2.15±0.03 2.11±0.04 2.11±0.01 2.12±0.04 2.09±0.01 2.09±0.01 LCWB 2.06±0.03 2.00±0.02 2.02±0.01 1.99±0.03 1.97±0.02 1.96±0.02 MCNB 2.31±0.01 2.33±0.10 2.26±0.04 2.24±0.03 2.24±0.03 2.22±0.02 MCWB 2.25±0.02 2.20±0.07 2.18±0.04 2.15±0.04 2.13±0.02 2.11±0.00 HCNB 2.42±0.02 2.44±0.09 2.36±0.08 2.32±0.02 2.33±0.03 2.30±0.04 HCWB 2.39±0.05 2.36±0.05 2.29±0.04 2.26±0.03 2.25±0.03 2.22±0.01 The results were reported as average±standard deviation, n=3 90 The pH of clean soil did not change during the experiments, since no CA or metals were added into it. Since CA is an organic acid, it was not surprising to note that the pH of spiked soil decreased after adding CA. As shown in Table 4.7, the more CA added into the soil, the lower pH value the soil possessed. However, 4, 8 and 12 weeks later, the pH of soil added with high level CA and metals was the highest while the pH of soil without adding CA was the lowest. For example, the pH of soil NCNB cultured with purchased reeds for 4 weeks was 2.12±0.04 while the pH of soil HCNB was 2.42±0.02. Similar for soil cultured with wild reeds, the pH of soil NCNB was 2.10±0.04 and was 2.44±0.09 of soil HCNB 4 weeks later. For amended with the same level of CA, the pH of spiked soil inoculated with rhizosphere bacteria was lower than that without rhizosphere bacteria. For instance, the pH of soil LCNB cultured with purchased reeds for 4 weeks was 2.15±0.03 while the pH of soil LCWB was 2.06±0.03. The pH of soil LCNB cultured with wild reeds for 4 weeks was 2.11±0.04 while the pH of soil LCWB was 2.00±0.02. The pH of soil MCNB cultured with purchased reeds for 8 weeks was 2.26±0.04 which was higer than the pH of soil MCWB (2.18±0.04). The pH of soil MCNB cultured with wild reeds for 8 weeks was 2.24±0.03 while the pH of soil MCWB was 2.15±0.04. These phenomena may contribute to oxidation of dissolved Fe(II) and hydrolysis of Fe(III) as described in followed equations (6 and 7) (Stumm and Morgan, 1996), which may cause the decrease of pH. 2+ + 3+ Fe +0.25O2+H →Fe +0.5 H2O (6) 3+ + Fe +3H2O →Fe(OH)3+3H (7) 91 The rate of abiotic oxidation of Fe(II) (Equation 6) was very slow at low pH (Stumm and Morgan, 1996), but Fe(II)OB can accelerate Fe(II) oxidation at low pH (Johnson and Hallberg, 2005). Therefore the spiked soil possessed more Fe(II)OB and developed lower pH. The results of previous research also indicated that the Fe(II) oxidation rate was related to the population of Fe(II)OB, and the Fe(II) oxidation occurred concomitantly with a decrease in pH (Senko et al., 2008; Dempsey et al., 2001). The pH of spiked soil cultured with wild reeds was slightly lower that with purchased reeds, since the rhizosphere of wild reeds had more Fe(II)OB than the purchased reeds. CA may also provide protons and electrons to reduce Fe(Ⅲ) into Fe(II) and CA can dissolve of precipitated compounds (Marques et al., 2009). Thus the spiked soil added with higher level of CA contained more mobile metals and had higher pH values. Eight and 12 weeks later, the pH of soil also slightly decreased; however, the tendency was not as sharp as that of 4 weeks soil. For instance, the pH of soil MCNB cultured with wild reeds changed from 2.92±0.06 to 2.33±0.10 after 4 weeks. Then it became 2.24±0.03 and 2.22±0.02, after 8 and 12 weeks. The pH of soil MCNB cultured with purchased reeds changed from 2.92±0.06 to 2.31±0.01 after 4 weeks and then became 2.26±0.04 and 2.24±0.03 after 8 and 12 weeks. The phenomena were in accord with the results of our kinetic experiments which also indicated that that pH of soil decreased in the first 6 hrs and did not vary too much later. This may due to the fact that the rate of oxidation of Fe(II) by Fe(II)OB was first-order (Park and Dempsey, 2005), and then the Fe(II) concentration and the pH decreased sharply in the first several days, and then become relatively constant later. 92 4.3.3 DCB extraction of reeds cultured in spiked soil for 4, 8 and 12 weeks The formation of plaque around the roots of pants has been widely reported (Greipsson, 1994; Snowden and Wheeler, 1995). According to the results of Karathanasis and Johnson (2003), the plaque on the root surface of some aquatic plants such as cattails and reeds can limit the translocation of metals to above ground plant organs and protect the plants from toxic metals. However, some conflicting results also existed. For example, Ye et al. (1997a) reported that the growth of cattail was decreased by the formation of plaque on the roots when exposed to Zn, Pb and Cd. The formation of Fe plaque was considered as the mechanisms of the oxidizing activity of roots and the Fe(II)OB (Ye et al., 1997a; Batty et al., 2000). Other metal plaque such as Mn and Al has also been observed on the roots surface of plants (Bacga and Hossner 1997; Batty et al., 2002). However, the formation mechanisms and the exact functions of these plaque were little known and conflicting. In this study, the concentration of Fe, Al and Mn plaque on the surface of roots and rhizomes of purchased reeds and wild reeds cultured for 4 weeks, 8 weeks and 12 weeks were analyzed. Plaque had already formed on root systems and metal had accumulated into tissues of wild reeds when collected from AMD contaminated field. Table 4.9 included the background metal levels attached to the surface of root system of wild reeds. The background metal concentrations were determined by analyzing reeds grown in clean soil that were not amended with metals. In order to better assess the effect of CA and rhizosphere bacteria on the metal plaque formation, the metal plaque concentrations on wild reeds did not include the background metal 93 concentrations (i.e. reset the baseline). Figure 4.35 to 4.37 showed the concentrations of metal plaque formed on the root systems of reed over 12 weeks. Table 4.9 Background values of metal plaque on reeds cultured in spiked soil Metal Plaque on Plaque on (mg/g) roots rhizomes Mn 0.00±0.00 0.00±0.00 Fe 5.56±1.15 4.96±1.03 Al 0.01±0.00 0.02±0.01 Results were reported as average±standard deviation, n=3 0.10 a a 0.08 b a b b a 0.06 c c c c a a rhizomes b 0.04 a b roots mg(Mn)/g(biomass) 0.02 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 0.12 a a a a 0.10 b a a 0.08 b b b b b b c 0.06 c c rhizomes c a 0.04 b roots b mg(Mn)/g(biomass) 0.02 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.35 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 94 a a 0.12 a b a a a a 0.10 b a b 0.08 a a a a b b b b 0.06 rhizomes 0.04 roots mg(Mn)/g(biomass) 0.02 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil a 0.18 0.16 0.14 a a 0.12 b a a b a a a 0.10 c b b b rhizomes 0.08 c d d 0.06 roots mg(Mn)/g(biomass) 0.04 0.02 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.36 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. CA had a significant effect (p<0.05) on the inhibition of Mn plaque on the root systems of both types of reeds. The concentration of Mn plaque was lower with the increasing CA. For both purchased and wild reeds, the root and rhizomes plaque were the highest for reeds grown in soil without adding CA, and were the lowest for reeds cultured in soil added with high level of CA during all growth periods. The root Mn plaque was 0.10±0.02 mg/g and 0.14±0.03 mg/g for purchased and wild reeds 95 cultured in NCWB for 8 weeks, respectively, while no Mn plaque were detected on the root of purchased and wild reeds cultured in soil HCWB for 8 weeks . a 0.24 0.20 a a a 0.16 a a a a a a a a 0.12 a rhizomes a roots 0.08 a b mg(Mn)/g(biomass) 0.04 b 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 0.25 a a a 0.20 a b b a c a b b b a 0.15 c a a c a a b b a b b b c rhizomes 0.10 b b c c c roots mg(Mn)/g(biomass) 0.05 c 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.37 Mn plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. Rhizosphere bacteria did not have a significant effect (p>0.05) on the formation of Mn plaque on the root systems of both types of reeds. The amounts of Mn plaque formed on reeds treated with rhizosphere bacteria were not significantly different from those on reeds cultured in spiked soil added with the same level of CA but without adding rhizosphere bacteria. For example, the rhizome Mn plaque was 96 0.05±0.00 mg/g for wild reeds in MCNB which was similar to the rhizome Mn plaque amounts (0.05±0.01 mg/g) on wild reeds in MCWB cultured for 8 weeks. It may be attributed to that the formation mechanism of Mn plaque. Previous research indicated that Mn oxides were mainly biologically formed by Mn oxidizing bacteria in environment with at near-neutral pH but not in circumstances with low pH (Nealson et al., 1988). The formation of Mn plaque may mainly caused by coprecipitate with or adsorb by Fe plaque in environment with low pH (Batty et al., 2002). Coprecipitation of metals with Fe plaque and then immobilization of these metals on root surfaces has been reported for several wetland plants such as P. australis (St-Cyr and Crowder, 1990; Emerson et al., 1999). Mn was reported as the most abundant coprecipitation metal with Fe plaque and the ratio of Mn:Fe varied greatly from site to site (Ye et al., 2003). As referred above, metal plaque especially Fe plaque around the roots of pants has been widely reported. The concentrations of Fe plaque formed on the root systems of reed over 12 weeks were also analyzed and showed in Figure 4.38 to 4.40. Fe plaque was observed on the root systems of purchased and wild reeds cultured for different periods across all treatment conditions. The concentrations of Fe plaque were higher than Mn plaque for both the purchased reeds and wild reeds. It was not surprising since the soil contained much more Fe than Mn. For example, the Fe plaque was 30.55±3.32 mg/g on the roots of purchased reeds cultured in NCNB for 4 weeks while the Mn plaque was 0.05±0.01 mg/g for purchased reeds grown in MCNB for the same time period. 97 80 a 70 a 60 b b b a 50 c c c c b b d d b 40 b e b c d c d e rhizomes 30 e c d d roots mg(Fe)/g(biomass) 20 10 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 90 a 80 a a 70 b a b a a 60 b c b b c c b 50 d c c d c d d 40 e d e d rhizomes d f 30 f roots mg(Fe)/g(biomass) 20 10 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.38 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 98 90 a 80 a a a 70 b a 60 b b b c a 50 c b c b c b b d d c d c c 40 d d rhizomes d c 30 d d roots mg(Fe)/g(biomass) 20 10 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 120 a a a 100 b b a b 80 b b 60 d c d e c e c rhizomes f f c c 40 g roots mg(Fe)/g(biomass) 20 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.39 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 99 a a 100 a 80 a b b c b b a 60 b c b b c b d c b b d rhizomes 40 d roots mg(Fe)/g(biomass) 20 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil a b a 120 a b 100 b a a b b a b 80 c b c b c d 60 d c c d c c e rhizomes e 40 roots mg(Fe)/g(biomass) 20 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.40 Fe plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. During all the growth periods, the Fe plaque concentrations of wild reeds were higher than that of purchased reeds under the same treatment conditions. For example, the Fe plaque was 38.85±2.12 mg/g on the roots of purchased reeds cultured in MCNB while was 43.96±1.29 mg/g for wild reeds grown in MCNB for 12 weeks. The availability of oxygen in the rhizosphere changed spatially and temporally with 100 the root system (Weiss, 2003). The biomass and ages of roots and rhizomes of wild reeds were bigger and older than that of purchased reeds. Thus the wild reed may possess better developed aerenchyma tissues in the root system which can provide more oxygen and better environment for the formation of Fe plaque (Weis and Weis, 2004). CA had a significant effect on the formation of Fe plaque on reeds during all growth periods (p<0.05). The concentration of Fe plaque on roots and rhizomes of reeds grown in spiked soil added with high level of CA was consistently lower than that in spiked soil added with low level of CA or without adding CA. CA may play a role in reducing the precipitation of Fe. For instance, the root Fe plaque was 16.44±1.56 mg/g and 27.27±1.63 mg/g for purchased and wild reeds cultured in HCNB for 8 weeks. For reeds cultured in LCNB for 8 weeks, the root Fe plaque was 34.94±2.68 mg/g and 69.18±3.13 mg/g for purchased and wild reeds, respectively. The rhizosphere bacteria also had important influence to increase the formation of Fe plaque (p<0.05). As shown in Figure 4.39, the root Fe plaque was 37.51±1.34 mg/g and 79.73±2.52 mg/g for purchased and wild reeds cultured in NCNB for 8 weeks, respectively; while the root Fe plaque was 72.20±5.15 mg/g and 91.35±4.40 mg/g for purchased and wild reeds cultured in NCWB for 8 weeks, respectively. These results were supported by previous research. It was reported that CA may provide protons and electrons to reduce metals, such as reduction of Fe3+ to Fe2+ and increased the solubility and bioavailability of metals in soil (Jones et al., 1998). Chelating agents such as CA can also prevent precipitation and sorption of the metals in soil through 101 the formation of metal-chelate complexes with metals, thereby maintaining their availability for plant uptake (Marques et al., 2009). It has also been reported that CA can improve the bioavailability and solubility of metals in muti-metal contaminated soil, such as Cd, Cu and Pb (Kim and Lee, 2010). According to the results of Muhammad et al. (2009), CA can also improve the solubilization of metals Cd, Cu and Cr from soil. There are wide evidence demonstrating that rhizosphere Fe(II)OB play a key role in the formation of Fe plaque on roots surface of wetland plants (Weiss et al., 2003; Batty et al., 2002). It has been shown that the root system of wetland plants released O2 into soils that contained Fe(II). The O2 used Fe(II)OB as an electron acceptor to oxidize Fe2+ into Fe3+, leading the formation of rust-colored Fe plaque (Mendelssohn et al., 1995). For example, Fe(II)OB was observed on the roots of 92% of wetland plants collected from 13 different aquatic environments (Weiss et al., 2003). Fe(II)OB may contribute to between 45 and 90% of Fe(II) oxidation in laboratory studies (Weiss et al., 2003). With the increase of time, the amounts of Fe plaque also increased. For example, the rhizome Fe plaque was 29.66±3.93 mg/g for purchased reeds cultured in MCWB for 4 weeks, while the Fe plaque increased to 39.86±2.19 mg/g after 12 weeks. Similar trends were found for wild reeds. This may due to that the effectiveness of CA to mobilize metals may decrease with time due to the microbial degradation (Römkens et al., 2002). Our results of CA biodegradation experiment also indicated that CA can be consumed by rhizosphere bacteria. Especially, low level of CA was almost totally consumed by rhizosphere bacteria after 4 weeks. According to our 102 results of rhizosphere bacteria enumeration (section 4.3.1), Fe(II)OB were active in the rhizosphere of reeds which also mediated the increase of Fe plaque. Al plaque was also formed on the surface of root and rhizomes of both kinds of reeds. Figure 4.41 to 4.43 presented the concentrations of Al plaque on reeds cultured under different treatment conditions for different time periods. 0.80 a a 0.70 a a a a a a a 0.60 a a a a a a 0.50 a a 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 0.90 a a a a a b b a b a 0.80 b c a b b c b 0.70 b b b b c c b b 0.60 c c b 0.50 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.41 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 103 a 0.80 a a a a a a b a b a a b b b b b b c a 0.70 c a b b b c 0.60 b c b 0.50 a 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil 0.90 a a a a a b b b c a 0.80 b b b b a b c c b c b 0.70 c c b c c c d c d 0.60 b 0.50 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.42 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 104 a 0.90 b a a a 0.80 a b a a a a b b b a a a a 0.70 b b b b b b b b 0.60 a 0.50 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil a a b 0.90 a a a a 0.80 a a b a b b a a b b b b a b b c b 0.70 c 0.60 b 0.50 0.40 rhizomes 0.30 roots mg(Al)/g(biomass) 0.20 0.10 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.43 Al plaque on the roots and rhizomes of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. It was not new to find that Al plaque can be formed on the root systems of reeds (Batty et al., 2002). In our study, the concentrations of Al plaque were higher than Mn plaque but lower than Fe plaque, which mainly related to the metals concentrations in soil which contained Fe>>Al>Mn. Similar to Fe and Mn plaque, CA also had a significant effect on the formation of Al plaque (p<0.05). For both purchase reeds and wild reeds, the roots and rhizomes of reeds cultured in spiked soil added with highest 105 level of CA had lowest concentration of Al plaque, while the reeds grown in soil without CA developed most Al plaque during all the growth period. For example, the root Al plaque was 0.50±0.05 mg/g for purchase reeds cultured in HCNB for 8 weeks, and was 0.61±0.08 mg/g for roots cultured in LCNB for the same growth period. Similar for wild reed, the root Al plaque was 0.50±0.03 mg/g on reeds cultured in HCNB for 8 weeks and was 0.62±0.02 mg/g for reeds grown in LCNB (Figure 4.42). It suggested that CA can also improve the solubility of Al in soil and restrain the formation of Al plaque. According to results of White et al. (2003), addition of CA can increased the aqueous concentration of some inorganic elements, such as Al, Fe, Mn, and P in soil. The rhizosphere bacteria did not play an important role in the formation of Al plaque as it did on Fe plaque (p>0.05), since the Al plaque concentration on both purchased and wild reeds inoculated with rhizosphere bacteria was not much different from that without bacteria during all the growth periods. For instance, the rhizome Al plaque for purchased reeds cultured in MCNB for 8 weeks (0.62±0.04 mg/g) was similar to the amounts of Al plaque on rhizome of reeds grown in MCWB for the same period (0.59±0.05 mg/g). This maybe attributed to the different formation mechanism between Al plaque and Fe plaque. It also suggested that the relationship between Al plaque was not close to Fe and Mn plaque. These findings were in agreement with some previous studies. It is reported that Al plaque was caused by the precipitation of Al phosphate on the roots surface of plants and the concentration of Al 106 deposit on root surface of reeds were not positively or negatively by the presence of Fe or Mn plaque (Batty et al., 2002). 4.3.4 Plant digestion of reeds cultured in spiked soil for 4, 8 and 12 weeks The concentrations of metals in the tissues of purchased reeds and wild reeds cultured for 4 weeks, 8 weeks and 12 weeks under different treatment conditions were analyzed. Figure 4.44 to Figure 4.52 present the concentrations of Mn, Fe and Al in rhizomes, roots, stems and leaves of purchased reeds and wild reeds. Metals were not detected in the purchased reeds grown in clean soil which was not spiked with metals. However, metals were found in the wild reeds cultured in clean soil during the growth period. This was due to the background metal concentration that had already accumulated in the wild reeds prior to being collected from the AMD site. Table 4.10 included the background metal accumulated in wild reeds. In order to better assess the effect of CA and rhizosphere bacteria on the metal uptake, the metal concentrations accumulated in wild reeds (Figure 4.44-4.52) did not include the background metal concentrations. Table 4.10 Background values of metal accumulations in reeds cultured in spiked soil Metal Metal in Metal in Metal in Metal in (mg/g) roots rhizomes stems leaves Mn 0.03±0.01 0.02±0.01 0.02±0.01 0.01±0.00 Fe 2.01±0.18 0.99±0.19 0.20±0.01 0.04±0.01 Al 0.05±0.01 0.04±0.01 0.03±0.01 0.02±0.01 Results were reported as average±standard deviation, n=3 107 a a b rhizomes 0.18 a c a a b b b 0.16 c c roots d c 0.14 d d stems 0.12 a a a a leaves e b b 0.1 b b c c b a 0.08 b a b a 0.06 c b c a a c a b d mg(Mn)/g(biomass) c c b b 0.04 b c b d d d b d 0.02 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB b 0.35 rhizomes a roots 0.3 a a a stems 0.25 b b b b b c b leaves 0.2 c b c c d c a d c d b e a 0.15 c a c c c b b e d c d c 0.1 d d d e d mg(Mn)/g(biomass) e e e d 0.05 e e 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.44 Mn concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 108 a 0.25 a a a rhizomes a b b 0.2 b a a roots b c b c a a c a a b b a b b stems 0.15 b c b b a leaves c 0.1 b a a a b a b a a b b mg(Mn)/g(biomass) b b b b b 0.05 b b b b b 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB b leaves 0.2 a a a 0.16 b b 0.12 b 0.08 c c c c mg(Mn)/g(biomass) 0.04 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Figure 4.45 Mn concentration (a) in the tissues of purchased reed and (b) in the leaves of wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=3. Different letters on the same plant organs indicate a significant difference at p<0.05. 109 a rhizomes 0.25 a roots a a a a stems 0.2 a a a b a b a a a a leaves 0.15 b b a b a b b a 0.1 a a a b b a a b b a b b c a b b c mg(Mn)/g(biomass) b c b c d b c 0.05 d d d b d 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB b a rhizomes b 0.35 a roots a 0.3 b a stems c b b c b c 0.25 c c d c a leaves d d e c d e d d b 0.2 e e a a f f c b 0.15 e b b d d d c e c e 0.1 d f mg(Mn)/g(biomass) d f d d 0.05 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.46 Mn concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 110 a 120 rhizomes a roots 100 a b b stems 80 b c a leaves a b b 60 b c d 40 d d c d d d mg(Fe)/g(biomass) d a a a b b b b b b a a a b 20 c c c c c c c c c 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil b rhizomes 140 roots 120 stems a a 100 a a leaves 80 b a a 60 b b a c b b c c c 40 c c d d mg(Fe)/g(biomass) d d c a b d e e d d b b c c a c a 20 e f e f d e e f c d a b 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.47 Fe concentration in the tissues of (a) purchased reeds and (b) wild reeds cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on same plant organs indicate a significant difference at p<0.05. 111 a 120 rhizomes roots 100 a a stems 80 a a leaves a a 60 a a b b b b 40 c b c c b mg(Fe)/g(biomass) a b a 20 b b b c b b b c b a c a a a c c 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil b leaves a 5 a b 4 b b c 3 c c c d d d 2 d 1 mg(Fe)/g(biomass) 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.48 Fe concentration (a) in the tissues of purchased reed an (b) in the leaves of wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=3. Different letters on the same plant organ indicate a significant difference at p<0.05. 112 a rhizomes 120 roots 100 a a stems 80 a a a leaves a b a b a b 60 b b b c c c c d c d c 40 c d c d mg(Fe)/g(biomass) c c a b a a 20 b d b d b d b d b a c a b 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil b rhizomes 140 roots 120 a a b b c stems 100 b leaves 80 a a b a c c b 60 d c b d d c d 40 c c c mg(Fe)/g(biomass) d d d b b d c a a c 20 e e f e e e e e c d b a 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.49 Fe concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=2. Different letters on same plant organs indicate a significant difference at p<0.05. 113 a rhizomes 0.9 a a a a a b b roots 0.8 b b c b c stems 0.7 d b c d 0.6 d e d d c leaves 0.5 e e e 0.4 b a a d c c c b c a b 0.3 d d d b c a d b a a e e b e b c mg(Al)/g(biomass) 0.2 c c c c 0.1 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil b rhizomes 2.5 a roots a b b a 2 b a stems c a leaves c b a a 1.5 b a a a b b b b 1 d d d b d b b b c c b a a mg(Al)/g(biomass) c c b c 0.5 c d c d d d 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.50 Al concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 4 weeks. Error bar represented one standard deviation, n=3. Different letters on the same plant organ indicate a significant difference at p<0.05. 114 a a rhizomes 1.2 a roots 1 a stems b a a 0.8 b b a c b b c b leaves c b c b 0.6 c b c c c a a a a 0.4 c b b b a b a a a c c a c a b b b a mg(Al)/g(biomass) c b b 0.2 b b 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil b leaves a 0.6 a 0.5 b b c 0.4 b c c 0.3 d d 0.2 d d mg(Al)/g(biomass) 0.1 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Figure 4.51 Al concentration (a) in the tissues of purchased reed and (b) in the leaves of wild reed cultured in spiked soil for 8 weeks. Error bar represented one standard deviation, n=2. Different letters on the same plant organ indicate a significant difference at p<0.05. 115 a rhizomes 1.2 roots 1 stems a a a a b leaves 0.8 b c b c c b b d b d 0.6 d d c c d d 0.4 a a b b a a b a mg(Al)/g(biomass) c b c b b a a b c b b d d b c 0.2 d d 0 Clean soil NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB b a rhizomes 5 roots a a 4 a stems leaves 3 b b b 2 b b b b b a a b b b b b mg(Al)/g(biomass) b b c c b a a 1 d c d c c c b 0 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB soil Figure 4.52 Al concentration in the tissues of (a) purchased reed and (b) wild reed cultured in spiked soil for 12 weeks. Error bar represented one standard deviation, n=2. Different letters on same plant organs indicate a significant difference at p<0.05. Compared with the purchased reeds, the wild reeds cultured under the same treatment conditions accumulated more metals into their plant tissues. This maybe attributed to the fact that the wild reeds grown in contaminated sites had already adapted to the environments, and then can perform better at metal uptake. According to other experiments that studied the heavy metal tolerance of plants, populations surviving in metals contaminated sites may possess genetically-based tolerances compared with “normal” populations of the same species (Antonovics et al., 1971). 116 Macnair (1993) also indicated that the tolerant ecotypes are less affected by the toxic materials than normal ones of the same species. For both purchased reeds and wild reeds cultured under all the treatment conditions for 4, 8 and 12 weeks, the total concentration of metals in plants was Fe>>Al>Mn. This may relate to the concentration of metals in spiked soil which contains much more Fe than Al and Mn (about 42943 ppm Fe, 272 ppm Al and 51 ppm Mn). This trend was similar to the finding of Batty and Younger (2004). They indicated that the concentration of metals (Fe, Al, Ni and Ca) in reeds collected from wetlands contained high levels of metals was significantly higher than that grew in wetlands with low levels of metals. For both purchased reeds and wild reeds, the concentration of metals in plants tissues varied with treatment conditions. CA had significant effect on the uptake of Mn, Fe and Al in underground parts of both type of reeds cultured for different time period (p<0.05). The roots and rhizomes of reeds grown in spiked soil added with higher level of CA contained higher concentration of Mn than that in reeds grown in spiked soil without adding CA. For purchased reeds cultured for 12 weeks, the concentration of Mn in rhizomes of reeds increased from 0.09±0.03 mg/g in spiked soil NCNB to 0.14±0.02 mg/g in spiked soil HCNB. For wild reeds cultured for 12 weeks, the concentration of Mn in roots of reeds increased from 0.18±0.01 mg/g in spiked soil NCNB to 0.32±0.01 mg/g in spiked soil HCNB. The Al concentrations in roots of purchased reeds grown for 12 weeks increased from 0.50±0.02 mg/g in LCNB to 0.74±0.02 mg/g in HCNB. For wild reeds cultured 12 weeks, the Al 117 concentrations of rhizome grown in soil LCNB changed from 0.79±0.06 mg/g to 3.89±0.72 mg/g in HCNB. This trend was more obvious for Fe. Purchased reeds cultured for 12 weeks decipted an increased Fe concentration in roots from 32.75±2.79 mg/g in NCNB to 68.94±2.79 mg/g in MCNB. Similarly, for wild reeds grown for 12 weeks, the Fe concentration in roots increased from 39.52±1.21 mg/g in NCNB to 96.99±5.75 mg/g in HCNB. This was in accord with previous research that CA can increase the bioavailability of heavy metals in soils and enhance the metals accumulation in belowground parts of plants (Alidoust et al., 2009; Sinhal et al., 2010 and Quartacci et al., 2005). Wang et al. (2006) indicated that more Ni and Cd were accumulated in the roots of Brassica juncea L in soil treated with 5.0 mM/kg CA than that added with 2.5 mmol/kg CA. CA also significantly increased the concentrations of metals in stems and leaves of reeds (p<0.05). For instance, the Mn concentrations in leaves of purchased reeds grown in soil NCNB for 8 weeks changed from 0.02±0.00 mg/g to 0.08±0.02 mg/g in soil HCNB; while the Mn concentrations in leaves of wild reeds changed from 0.04±0.00 mg/g to 0.15±0.01 mg/g. The Al concentrations in leaves of wild reeds grown in soil LCNB for 8 weeks changed from 0.20±0.02 mg/g to 0.50±0.03 mg/g in soil HCNB. Similar trends were also observed for Fe. The Fe concentrations in stems of purchased reeds grown in soil NCNB for 12 weeks changed from 2.55±0.64 mg/g to 6.94±0.37 mg/g in soil HCNB; while the Fe concentrations in stems of wild reeds changed from 2.63±0.26 mg/g to 10.50±0.60 mg/g. The study of Mihalík et al. (2010) showed that 5 mM/kg soil of CA increased the accumulation of U in the above ground 118 parts of sunflower and willow. Sinhal et al. (2010) also found that 10 and 20 mg/L CA enhanced the uptake of Zn, Cu, Pb and Cd in roots, stems and leaves of marigolds. It was also worthwhile to indicate that the rhizosphere bacteria played a significant role in decreasing the uptake of Mn and Fe in both type of reeds (p<0.05). Reeds grown in soil inoculated with rhizosphere bacteria accumulated less Fe and Mn into both blew and above ground tissues. For instance, the Fe concentration in leaves of purchased reeds grown in NCNB for 8 weeks was 2.18±0.01 mg/g, which was more (p<0.05) than that in leaves of purchased reeds grown in NCWB for the same time period (1.37±0.05 mg/g). The Mn concentration in roots of wild reeds grown in NCNB for 12 weeks was 0.18±0.01 mg/g, which was more (p<0.05) than that in roots of wild reeds grown in NCWB for the same time period (0.10±0.01 mg/g). As discussed in section of metals plaque (4.3.3), the inoculated rhizosphere Fe(II)OB play an important role in forming Fe plaque on the root surface of plants. It has also been widely reported that the Fe plaque can function as a barrier to the uptake of other metals into plant tissues (Batty et al., 2002). However, other researchers such as Greipsson (1994) indicated that Fe plaque may enhance the uptake of iron by rice and lessen the toxic of zinc. According to our results, Fe plaque formed by Fe(Ⅱ)OB can not prevent but can inhibit the Fe and Mn accumulation in reeds. The concentration of Al in reeds in spiked soils inoculated with bacteria was not too different from that in spiked soil without adding bacteria (p>0.05). For example, the Al concentrations were 0.08±0.01 mg/g in leaves of purchased reeds grown soil NCNB for 8 weeks while the concentrations were 0.06±0.02 mg/g in purchased reeds 119 grown in soil NCWB for 8 weeks. For wild reeds, the Al concentrations were 0.12±0.01 mg/g in leaves grown in soil NCNB for 8 weeks while the concentrations were 0.11±0.02 in purchased reeds grown in soil NCWB for 8 weeks. This may due to the fact that, as discussed in the DCB extraction (section 4.3.3), the concentration of Al plaque was also not different in soil inoculated with or without rhizosphere bacteria. The study of Batty et al. (2002) also indicated that the amounts of Al in roots of reeds were not affected by the presence of Fe and Mn plaque. The shoot concentrations of metals in reeds were calculated. The translocation factors of metals in purchased and wild reeds were also shown in Table 4.11 and 4.12. Table 4.11(a) Shoot concentration and translocation factor of purchased reeds cultured in different treatment conditions for 4 weeks Shoot Shoot Shoot Treat Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.02±.00 2.39±.21 0.09±.00 0.15±.02 0.09±.01 0.22±.03 NCNB cd c e b a a 0.01±.00 1.39±.48 0.10±.01 0.14±.04 0.08±.04 0.24±.04 NCWB d c e b a a 0.03±.00 2.56±.50 0.13±.02 0.21±.04 0.08±.01 0.27±.03 LCNB bcd c cde ab a a 0.02±.00 1.52±.00 0.12±.00 0.15±.00 0.08±.00 0.28±.00 LCWB cd c de b a a 0.03±.00 6.40±.00 0.17±.00 0.21±.00 0.08±.00 0.28± MCNB bc ab bc ab a .00 a 0.02±.00 5.01±.19 0.15±.02 0.17±.01 0.08±.00 0.28±.04 MCWB bcd b cd b a a 0.05±.00 8.04±.51 0.20±.01 0.36±.03 0.09±.01 0.30±.01 HCNB a a ab a a a 0.04±.00 7.07±.27 0.23±.01 0.26±.04 0.09±.01 0.32±.00 HCWB b a a ab a a 120 Table 4.11(b) Shoot concentration and translocation factor of purchased reeds cultured in different treatment conditions for 8 weeks Shoot Shoot Shoot Treat Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.02±.00 2.38±.41 0.10±.01 0.22±.04 0.08±.00 0.24±.02 NCNB c cd cd bcd a a 0.01±.00 1.58±.07 0.10±.02 0.15±.01 0.08±.02 0.25±.03 NCWB d d d d a a 0.03±.00 2.41±.02 0.13±.02 0.20±.00 0.07±.00 0.27±.03 LCNB c cd bcd bcd a a 0.02±.00 1.52±.06 0.12±.01 0.14±.02 0.07±.01 0.27±.04 LCWB cd d bcd cd a a 0.04±.00 5.21±.80 0.16±.00 0.24±.00 0.08±.01 0.28±.01 MCNB b ab ab bc a a 0.03±.00 4.24±.24 0.16±.01 0.19±.01 0.07±.00 0.29±.01 MCWB c bc abc bcd a a 0.07±.00 6.21±.14 0.22±.00 0.35±.01 0.08±.00 0.31±.02 HCNB a a a a a a 0.04±.00 5.63±.24 0.21±.01 0.25±.01 0.08±.01 0.30±.02 HCWB b ab a b a a With the increase of CA, the shoot concentrations of metals in both purchased and wild reeds increased (p<0.05). For instance, the shoot concentration of Mn in purchased reeds increased from 0.02±0.00 mg/g in NCNB to 0.05±0.00 mg/g in HCNB after 4 weeks; while the shoot concentration of Fe and Al increased from 2.39±0.21 mg/g to 8.04±0.51 mg/g, 0.09±0.00 mg/g to 0.20±0.01 mg/g, respectively. For wild reeds, the shoot concentration of Mn increased from 0.05±0.00 mg/g in NCNB to 0.15±0.01 mg/g in HCNB after 4 weeks; while the shoot concentration of Fe and Al increased from 1.71±0.11 mg/g to 7.12±0.49 mg/g, 0.21±0.00 mg/g to 1.06±0.17 mg/g, respectively. 121 Table 4.11(c) Shoot concentration and translocation factor of purchased reeds cultured in different treatment conditions for 12 weeks Shoot Shoot Shoot Treat Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.02±.00 2.38±.42 0.10±.00 0.14±.05 0.07±.01 0.25±.00 NCNB bc c c b a a 0.02±.00 1.57±.10 0.10±.00 0.12±.03 0.07±.01 0.23±.00 NCWB c c c b a a 0.03±.00 2.50±.08 0.13±.00 0.20±.00 0.07±.01 0.27±.02 LCNB bc c bc ab a a 0.02±.01 1.61±.08 0.14±.02 0.16±.05 0.07±.00 0.28±.03 LCWB bc c bc b a a 0.04±.00 5.36±.01 0.17±.00 0.22±.00 0.08±.00 0.28±.00 MCNB b ab b ab a a 0.02±.00 4.65±.00 0.17±.00 0.15±.00 0.08±.00 0.27±.01 MCWB bc b b b a a 0.07±.01 6.04±.13 0.23±.00 0.35±.04 0.08±.00 0.31±.01 HCNB a a a a a a 0.04±.00 5.39±.14 0.23±.00 0.22±.02 0.09±.00 0.30±.01 HCWB bc ab a ab a a / indicated blow detection limits; error bar represented the standard deviation of triplicate samples. Different letters for each column indicated a significant difference at p<0.05. Besides, the rhizosphere bacteria also significantly reduced the shoot concentrations of Fe and Mn in both type of reeds (p<0.05). For instance, the shoot concentration of Mn in purchased reeds reduced from 0.03±0.00 mg/g in LCNB to 0.02±0.00 mg/g in LCWB after 4 weeks; while the shoot concentration of Fe and Al reduced from 2.56±0.50 mg/g to 1.52±0.00 mg/g, 0.13±0.02 mg/g to 0.12±0.00 mg/g, respectively. For wild reeds, the shoot concentration of Mn reduced from 0.07±0.01 mg/g in LCNB to 0.04±0.00 mg/g in LCWB after 4 weeks; while the shoot concentration of Fe reduced from 2.34±0.04 mg/g to 1.58±0.07 mg/g. These findings 122 further proved that metal plaque formed by rhizosphere bacteria can inhibit the metal uptake in plants. Table 4.12 Shoot concentration and translocation factor of wild reeds cultured in different treatment conditions for 4 and 12 weeks Shoot Shoot Shoot Treat week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.05±.00 1.71±.11 0.21±.00 0.34±.00 0.05±.00 0.25±.02 4 def c b a a a NCNB 0.05±.01 2.31±.22 0.31±.01 0.28±.04 0.06±.00 0.21±.00 12 cde de d bc c b 0.02±.00 0.88±.01 0.20±.00 0.27±.02 0.04±.00 0.25±.01 4 f c b a a a NCWB 0.02±.00 0.98±.05 0.30±.00 0.22±.03 0.04±.00 0.21±.01 12 e e d c bc ab 0.07±.01 2.34±.04 0.33±.01 0.47±.03 0.07±.00 0.35±.04 4 cd c b a a a LCNB 0.07±.00 2.75±.12 0.44±.01 0.42±.03 0.07±.00 0.30±.00 12 cd d c abc abc ab 0.04±.00 1.58±.07 0.33±.05 0.35±.01 0.06±.00 0.37±.07 4 ef c b a a a LCWB 0.04±.00 2.18±.05 0.45±.01 0.30±.02 0.07±.01 0.30±.01 12 de de c abc abc ab 0.08±.00 6.46±.61 0.53±.03 0.45±.02 0.08±.00 0.46±.00 4 c ab b a a a MCNB 0.12±.01 8.33±.54 0.59±.06 0.50±.00 0.12±.02 0.35±.03 12 b ab b ab ab ab 0.05±.01 4.70±.34 0.51±.00 0.38±.00 0.08±.01 0.43±.00 4 de b b a a a MCWB 0.08±.01 5.34±.24 0.59±.02 0.44±.08 0.08±.01 0.35±.00 12 bc c b abc abc ab 0.15±.01 7.12±.49 1.06±.17 0.59±.10 0.08±.01 0.70±.24 4 a a a a a a HCNB 0.19±.01 9.40±.53 1.21±.01 0.61±.06 0.10±.01 0.39±0.06 12 a a a a abc a 0.12±.00 5.54±.27 0.97±.04 0.47±.02 0.06±.00 0.62±.00 4 b ab a a a a HCWB 0.16±.00 6.81±.03 1.22±.01 0.55±.01 0.13±.01 0.37±.00 12 a bc a ab a a 123 For purchased reeds, the TF of Mn were significantly elevated by CA (p<0.05), however, the TF of Fe and Al were not influenced by CA (p>0.05). This may be due to the fact that plants had different tolerant mechanisms to different metals and different metals possessed different mobility (Taylor and Crowder, 1983b). For wild reeds, the TF of all these three metals were significantly increased with the increase of CA (p<0.05). This may be explained by the fact that wild reeds had already adapted to the elevated metal levels. Ernst (2006) also indicated that plants growing in metal-enriched soils may develop some tolerance mechanisms but without avoiding uptake more metals into biomass. The concentrations of metals in wild reeds increased with time. For instance, the concentration of Mn, Fe and Al in rhizomes of wild reeds increased from 0.17±0.01 mg/g to 0.21±0.01 mg/g, 44.89±4.30 mg/g to 48.43±5.59 mg/g, 1.18±0.10 to 1.31±0.06 mg/g after growing in MCNB after 4 and 12 weeks, respectively. The concentrations of metals in the shoots of wild reeds also increased with time. The shoot concentration of Mn, Fe and Al in wild reeds increased from 0.15±0.01 mg/g to 0.19±0.01 mg/g, 7.12±0.49 mg/g to 9.40±0.53 mg/g, 1.06±0.17 to 1.21±0.01 mg/g after growing in HCNB for 4 and 12 weeks. Compared with wild reeds, the concentrations of metals in below ground organs of purchased reeds cultured for 12 weeks were not too different from those cultured for 4 and 8 weeks. For instance, the concentration of Mn, Fe and Al in rhizomes of purchased reeds increased from 0.08±0.01 mg/g to 0.10±0.02 mg/g, 26.83±2.98 mg/g to 29.48±3.73 mg/g, 0.39±0.03 to 0.41±0.02 mg/g after growing in 124 LCNB after 4 and 12 weeks. There was also only a slight difference between the shoot concentrations of metals in purchased reeds cultured for 12 weeks and that in purchased reeds cultured for 4 and 8 weeks. For instance, the shoot concentrations of Mn, Fe and Al were 0.05±0.00 mg/g, 8.04±0.51 mg/g and 0.20±0.01 mg/g in purchased reeds cultured in HCNB for 4 weeks; while the concentrations were only 0.07±0.01 mg/g, 6.04±0.13 mg/g, and 0.23±0.00 mg/g after 12 weeks. This may be attributed to the toxicity of metals to reeds. Before the initiation of experimental treatments, all seedlings showed good growth and the leaves of reeds were green. After culturing in the spiked soil for several weeks, the leaves turned yellow, the growth of shoot stunted. The accumulation of metals may be inhibited with the cease in growth of reed seedlings. It also reported that the growth of reed seedlings was inhibited in solution with high level of Fe concentration and reeds exhibited visual symptoms including shoot die-back and mottling of leaves which may relate to possible iron toxicity (Batty and Younger, 2003). According to previous research, about 1100-1600 mg/kg Fe in leaves could cause the toxicity to wetland plants and the threshold may be different for different plant species with different ages (Batty and Younger, 2003). Compared to the purchased reeds, the wild reeds showed more tolerance to the metals in soil and accumulated more metals from soil. As discussed above, this may due to that the wild reeds possessed some adaption mechanisms to the soil contaminated with heavy metals. For both purchased and wild reeds cultured for 4, 8 and 12 weeks, most of Fe, Al and Mn were the sequestrated in the roots and rhizomes of reeds. According to Table 125 4.11 and 4.12, the TF of all these three metals were less than 1. It also suggested that portion of metals can be transported into the aboveground organs of reeds, however, roots and rhizomes were the main tissues for metal storage. These findings were also supported by previous studies. Sawidis et al. (1995) indicated that a large amount of heavy metals were preferentially accumulated in the roots and rhizomes of reeds. According to Bonanno and Giudice (2010), the concentration of Mn, Cu and Cr in reeds grown in a wetland decreased in the order of root>rhizome>shoot. Baldantoni et al. (2009) determined the concentration of some heavy metals such as Cr, Cu, Fe and Mn in the leaves and roots of reeds collected from three sites in the Lake Averno in Italy. They pointed out that although translocation occurred, metal concentrations were more than one order of magnitude lower in the leaves than that in the roots. Batty et al. (2002) conducted hydroponic experiments and the results showed that the concentration of Al was higher in roots than that in shoots. Baldantoni et al. (2009) also pointed out that reeds can immobilize the toxic elements in the roots to protect rhizomes which are the only persistent part of the plant. Although reeds may not possess the advantage to transfer metals from roots and rhizomes into aboveground tissues, it has extensive roots and rhizomes and shows vigorous vegetative growth (Ye et al., 1997b) which are also of prime importance for plants used to clean contaminated soil. Besides, phytoremediation technology includes different categories based on the mechanisms for cleaning the polluted medium. Phytoextraction which means plants absorb contaminants from soil through the root systems and transfer them to harvestable shoots (Hemen, 2011) are not the 126 only phytoremediation technology. Phytostabilization and phytofiltration also play an important role in clean contaminated medium. Besides, transferring of metals into shoot biomass may cause accumulated metals pass into the food chain by herbivores feeders which could also be a disadvantage (Ye et al., 1997a). Reed also has many other advantages: such as strong adaptability to different climatic conditions; ability to grow in hydroponic or terrestrial habitats; high production of biomass, rapid growth rate (Baldantoni et al., 2009), which make it a good candidate for phytoremediation technique. 4.3.5 Metals concentrations in spiked soil In this study, soil was spiked on 8/3/2012 and left to “age” until 8/28/2012. Then different levels of CA were added into soil on 8/29/2012. Two days after adding CA (8/31/2012), the seedlings of reeds were transferred from potting soil into spiked soil and rhizosphere bacteria were inoculated into part of the soil. Table 4.13 and 4.14 showed the concentrations of total and mobile metals in spiked soil before and after adding CA. Two days after adding CA into spiked soil, the mobile metal fraction increased with the increasing of concentration of CA. CA significantly (p<0.05) increased the amounts of mobile Mn, Fe and Al in soil. For instance, the mobile Fe in spiked soil cultured with wild reeds increase from 20684.02±817.93 mg/kg to 38467.33±1048.52 mg/kg after adding with high level of CA. Muhammad et al. (2009) also showed that the concentration of soluble metals in soil increased sharply in the first several days after adding of CA. 127 Table 4.13(a) Metals in spiked soil cultured with purchased reeds on 8/29/2012 Treat Metals Mn Fe Al Clean Total 40.44±5.02b 8750.13±505.54b 63.41±3.03b soil Mobile 1.25±0.25b 23.50±5.50b / NCNB Total 93.62±6.13a 50549.85±1567.25a 305.82±18.35a Mobile 24.96±4.92a 20807.81±628.62a 97.25±4.58a NCWB Total 93.67±6.27a 50374.12±1185.41a 324.70±12.29a Mobile 24.95±4.97a 20244.44±776.96a 94.83±4.91a LCNB Total 93.66±6.20a 51364.19±974.76a 330.93±6.08a Mobile 24.98±4.99a 21588.65±1596.64a 102.42±7.48a LCWB Total 106.21±6.26a 51294.31±3396.70a 349.85±12.63a Mobile 27.45±7.47a 20563.83±1144.07a 107.29±12.42a MCNB Total 106.09±12.43a 52682.22±1021.29a 324.55±12.81a Mobile 29.95±9.96a 19834.34±3458.26a 102.33±12.41a MCWB Total 112.40±12.60a 51297.74±314.76a 312.22±37.78a Mobile 24.98±5.00a 19565.63±2944.74a 92.42±7.52a HCNB Total 106.13±6.36a 52819.86±2612.38a 330.84±5.87a Mobile 27.45±2.46a 21012.81±637.91a 99.81±9.84a HCWB Total 99.92±0.06a 52449.68±1387.45a 324.71±37.28a Mobile 27.44±7.47a 21599.56±1207.28a 109.76±10.02a Table 4.13(b) Metals in spiked soil cultured with wild reeds on 8/29/2012 Treat Metals Mn Fe Al Clean soil Total 39.45±8.00b 9090.15±453.22b 66.65±3.27b Mobile 2.00±0.50b 26.23±2.24b / NCNB Total 106.09±6.15a 52650.09±4244.05 a 318.27±18.46a Mobile 24.96±5.02a 21717.66±784.22a 102.33±7.60a NCWB Total 99.91±12.45a 52624.30±4247.27a 330.95±18.61a Mobile 27.46±7.46a 20154.11±1185.75a 94.86±9.89a LCNB Total 99.90±0.01a 52024.34±865.56a 337.17±12.51a Mobile 27.49±2.50a 21615.91±1030.15a 99.96±5.00a LCWB Total 106.07±6.37a 52048.99±2936.05a 355.89±19.16a Mobile 29.95±4.99a 19924.51±1128.65a 104.83±9.97a MCNB Total 112.43±12.48a 53361.50±2299.53a 331.05±6.28a Mobile 29.98±5.01a 18282.02±2745.90a 102.43±2.54a MCWB Total 106.06±18.82a 52724.38±4058.11a 318.15±19.03a Mobile 24.97±4.99a 18195.26±3168.03a 89.902±4.98a HCNB Total 118.71±6.27a 54387.63±2800.93a 337.38±12.55a Mobile 22.49±7.50a 19542.20±168.01a 102.45±12.52a HCWB Total 99.94±0.03a 56473.55±840.24a 337.07±31.32a Mobile 29.98±4.98a 21246.87±1132.01a 109.92±14.92a 128 Table 4.14 (a) Metals in spiked soil cultured with purchased reeds on 8/31/2012 Treat Metals Mn Fe Al Clean Total 40.97±7.01b 8710.71±599.57 b 63.69±2.28 b soil Mobile 1.00±0.50b 24.74±2.25 d / NCNB Total 93.66±6.34a 51564.43±1361.92a 330.90±5.89a Mobile 24.96±5.03b 20708.07±524.12c 97.29±2.32c NCWB Total 106.17±6.22a 50841.26±2140.61a 324.74±12.40a Mobile 27.45±2.50b 20034.14±1032.15c 97.32±2.48c LCNB Total 106.21±18.76a 51705.28±1902.74a 343.62±18.68a Mobile 27.48±7.50b 21714.15±265.96c 104.91±4.95c LCWB Total 106.13±6.25a 51817.83±3506.05a 330.89±18.71a Mobile 27.48±2.51b 20906.14±424.33c 109.91±4.95c MCNB Total 106.11±6.22a 53093.94±2332.59a 318.34±6.31a Mobile 32.42±7.48ab 32539.26±1435.80b 221.97±2.47b MCWB Total 106.05±18.70a 53107.68±2169.27a 318.16±31.14a Mobile 29.96±0.04 ab 31733.99±656.01b 212.20±2.20b HCNB Total 99.91±0.03a 52596.04±1074.37a 324.70±12.38a Mobile 37.46±2.51a 39308.77±1618.93a 242.24±17.55a HCWB Total 105.98±6.21a 52986.10±918.36a 342.89±18.63a Mobile 39.92±0.07a 39082.42±1177.58a 234.49±4.59a Table 4.14(b) Metals in spiked soil cultured with wild reeds on 8/31/2012 Treat Metals Mn Fe Al Clean Total 43.95±4.51b 9009.15±37.88b 70.42±2.03b soil Mobile 0.75±0.25a 21.98±6.49d / NCNB Total 99.89±12.40a 53073.53±1163.63a 324.70±12.78a Mobile 27.46±2.52a 20684.02±817.93c 92.34±2.40c NCWB Total 93.62±6.23a 53386.02±3263.68a 337.02±12.44a Mobile 24.96±0.01a 20618.05±1072.48c 94.84±9.95c LCNB Total 99.97±12.47a 51192.79±2742.01a 362.40±12.58a Mobile 26.96±4.98a 20084.91±1279.06c 102.38±2.54c LCWB Total 106.04±18.89a 54161.38±2766.96a 330.50±6.80a Mobile 22.48±2.50a 20279.84±900.34c 107.42±2.50c MCNB Total 106.20±18.72a 52470.86±2643.32a 324.86±12.42a Mobile 29.97±4.99a 30148.03±989.27b 217.29±2.51b MCWB Total 106.19±6.26a 52726.71±4450.42a 312.32±25.04a Mobile 32.46±2.51a 28466.86±1607.01b 204.74±14.88b HCNB Total 106.10±6.21a 54138.22±504.40a 343.28±6.13a Mobile 34.96±0.02a 37196.97±582.01a 229.73±10.13a HCWB Total 106.13±6.21a 50964.40±1606.04a 337.14±12.58a Mobile 34.95±4.98a 38467.33±1048.52a 229.72±4.87a 129 Widely reports showed that CA can enhance the solubility of metals in soil (Najeeb et al., 2011; Mihalík et al., 2010). Lozano et al. (2011) pointed out that the more CA applied to the soil the more U solubilization achieved. In our study, the extents to which CA increased the mobility of metals were different for Mn, Fe and Al. This may due to the fact that different types of complexes formed between metals and CA may influence the bioavailability of metals. Chen et al. (2003) conducted adsorption and hydroponic experiments to study the role of CA on the phytoremediation of heavy metal contaminated soil. They indicated that the amount of lead or cadmium adsorbed by soil decreased with the increasing of the concentration of CA and the decrease rates were different for different metals. Chelating agents such as CA can be used to increase the bioavailability of heavy metals in soil by different mechanisms (Komárek et al., 2007). Chelating agents can prevent precipitation and sorption of the metals in soil through the formation of metal-chelate complexes with metals, thereby maintaining their availability for plant uptake (Marques et al., 2009). Chelating agents can also bring metals into solution by dissolution of precipitated compounds and desorption of sorbed chemicals (Marques et al., 2009). CA may also provide protons and electrons to reduce metals, such as reduction of Fe3+ to Fe2+ and increases the solubility and bioavailability of metals in soil (Jones et al., 1998). After 4, 8, 12 weeks of culturing the reeds, the soil were collected and air dried. The total and mobile metals in soil have also been analyzed, which were showed in Table 4.15 and Table 4.16. 130 Table 4.15(a) Metals in soil (mg/kg) cultured with purchased reeds for 4 weeks Treat Mn Fe Al Clean soil Total 37.94±2.95b 8632.13±377.04b 60.66±1.67b Mobile 0.75±0.25c 20.96±1.03f / NCNB Total 65.54±10.32a 36302.24±1373.71a 234.07±20.42a Mobile 29.91±3.49ab 16765.16±1082.33cd 93.46±4.08b NCWB Total 73.69±7.38a 33787.16±2191.11a 242.87±18.78a Mobile 24.96±3.53b 13073.72±1082.22e 102.33±5.62b LCNB Total 65.53±10.35a 36324.24±1261.84a 246.50±18.39a Mobile 28.67±4.14ab 17366.54±1082.69c 100.96±7.42b LCWB Total 68.61±6.28a 35935.13±2226.35a 255.71±18.60a Mobile 26.18±2.16ab 14070.43±1107.14de 104.73±6.12b MCNB Total 80.11±5.41a 34598.83±5140.60a 245.52±16.95a Mobile 31.17±2.20ab 29033.82±1041.30b 219.43±3.31a MCWB Total 74.85±8.76a 38550.66±2165.98a 240.19±16.49a Mobile 29.92±0.05ab 27601.65±984.62b 215.69±7.44a HCNB Total 78.06±13.65a 38247.99±367.32a 249.75±15.13a Mobile 33.70±2.13a 37580.90±1400.60a 238.88±7.51a HCWB Total 74.89±8.84a 38157.25±1413.88a 243.37±18.63a Mobile 32.46±4.35ab 36850.21±978.33a 229.69±19.57a Table 4.15(b) Metals in soil (mg/kg) cultured with purchased reeds for 8 weeks Treat Mn Fe Al Clean soil Total 32.21±2.23b 8178.04±771.15b 56.19±6.27b Mobile 0.75±0.25b 20.75±0.25d / NCNB Total 59.31±5.40a 29736.59±2343.00a 193.54±27.21a Mobile 21.24±4.15a 10226.19±200.30c 83.70±9.56 b NCWB Total 59.33±5.40a 27843.04±1496.81a 212.32±8.78a Mobile 19.98±3.53a 8325.31±899.35c 81.18±5.44b LCNB Total 59.33±13.61a 29938.39±2635.66a 215.47±10.40a Mobile 22.48±4.33a 10187.87±479.42c 82.41±5.62b LCWB Total 56.21±6.26a 29544.83±2130.76a 221.72±10.40a Mobile 21.24±2.17a 9690.67±556.39c 81.21±2.17b MCNB Total 68.69±6.23a 28191.23±2143.03a 212.31±8.75a Mobile 24.99±3.53a 19137.96±1702.52a 126.18±17.10a MCWB Total 62.44±8.81a 31041.48±1375.35a 215.41±5.33a Mobile 22.49±2.50a 14044.76±806.04b 131.17±18.84a HCNB Total 68.67±10.81a 31734.03±1134.82a 215.39±13.64a Mobile 28.73±4.14 a 19639.57±567.02a 147.39±15.98a HCWB Total 65.58±10.36a 31119.96±629.28a 215.46±10.33a Mobile 27.48±2.50a 18311.73±1939.28a 152.41±10.96a 131 Table 4.15(c) Metals in soil (mg/kg) cultured with purchased reeds for 12 weeks Treatment Mn Fe Al Clean soil Total 30.14±0.23b 8025.97±385.39b 54.56±2.27b Mobile 0.75±0.25b 16.99±2.50d 0.50±0.00c NCNB Total 48.41±2.70a 24720.07±2081.36a 174.90±15.29a Mobile 17.49±2.50a 8310.29±151.75c 74.95±6.10b NCWB Total 46.84±5.40a 23368.43±870.08a 177.98±10.35a Mobile 16.24±2.17a 7000.29±450.71c 77.46±2.50b LCNB Total 49.98±8.84a 24825.68±945.64a 178.04±10.37a Mobile 18.74±4.14a 8263.66±237.50c 73.69±4.16b LCWB Total 43.71±6.25a 24659.74±1111.28a 181.09±6.24a Mobile 14.99±3.54a 7156.65±262.48c 74.95±3.54b MCNB Total 56.20±6.23a 25061.26±1468.25a 190.47±10.36a Mobile 21.24±2.17a 15093.51±1573.62a 106.21±11.40a MCWB Total 53.08±5.41a 24946.37±1204.76a 187.35±8.81a Mobile 17.49±2.50a 11077.62±730.78b 112.45±13.44a HCNB Total 56.22±6.24a 26010.28±1005.52a 190.55±16.27a Mobile 23.73±4.14a 15603.20±504.21a 123.66±13.41a HCWB Total 53.09±5.41a 25813.77±1673.90a 196.74±13.62a Mobile 21.14±5.45a 14183.36±1446.81a 190.49±16.22a Table 4.16(a) Metals (mg/kg) in soil cultured with wild reeds for 4 weeks Treatment Mn Fe Al Clean soil Total 35.72±7.28b 7723.10±1105.60d 53.18±9.18b Mobile 0.75±0.25 b 20.22±0.22d / NCNB Total 68.73±6.24a 35140.83±799.13c 231.19±13.98a Mobile 22.46±2.50a 15062.03±3064.30c 97.32±7.46c NCWB Total 71.73±10.30a 31992.06±2197.76c 243.28±18.61a Mobile 19.98±3.54a 11030.24±894.43d 97.38±9.00c LCNB Total 71.81±10.37a 36232.97±1757.55bc 252.87±16.15a Mobile 27.46±2.49a 17441.25±1071.79c 97.35±5.60c LCWB Total 71.82±10.35a 35010.64±1848.20c 265.42±35.78a Mobile 23.71±2.16a 13889.25±854.98cd 99.84±6.09c MCNB Total 87.42±17.64a 35946.68±6582.64bc 256.02±28.58a Mobile 28.72±7.40a 25467.83±1554.16b 214.72±17.72 ab MCWB Total 74.94±8.85a 35134.35±1878.63c 227.93±20.48a Mobile 27.48±4.33a 24782.49±482.72b 207.37±5.57b HCNB Total 93.65±6.20a 43799.70±4067.31a 280.98±25.86a Mobile 27.48±5.57a 34243.84±1005.98a 233.57±6.41a HCWB Total 81.20±13.98a 45720.22±2527.48ab 274.82±31.88a Mobile 27.48±2.50a 34097.23±1056.41a 231.05±9.02 ab 132 Table 4.16(b) Metals (mg/kg) in soil cultured with wild reeds for 8 weeks Treatment Mn Fe Al Clean soil Total 32.21±2.23b 7178.69±228.20d 53.69±3.72b Mobile 0.75±0.25b 20.75±0.25f 0.50±0.00d NCNB Total 56.19±6.21a 24524.95±1078.76abc 190.43±10.38a Mobile 18.74±2.16a 8838.86±385.85de 76.20±6.49c NCWB Total 49.86±8.82a 22675.22±679.32c 196.71±13.61a Mobile 15.00±3.53a 7378.06±192.99e 77.48±7.50c LCNB Total 56.21±10.8a 25053.35±948.38abc 199.88±17.65a Mobile 17.79±2.50a 12237.28±475.46 b 76.20±8.20c LCWB Total 59.31±5.40a 23866.92±1324.16 bc 202.92±35.37a Mobile 14.99±0.00a 10201.51±203.04 cd 72.48±4.32c MCNB Total 65.59±10.37a 26772.35±1369.31 ab 212.38±8.81a Mobile 21.24±5.45a 16785.55±615.92 a 97.46±16.77bc MCWB Total 62.45±8.82a 25472.51±618.80 abc 206.10±6.28a Mobile 19.99±6.12a 11562.11±1374.05 bc 109.97±11.72ab HCNB Total 68.68±6.24a 27081.23±1197.66a 209.16±13.61a Mobile 23.74±4.14a 17016.96±666.80a 126.18±10.83a HCWB Total 65.59±5.42a 26071.63±1546.98ab 212.40±8.83a Mobile 22.49±2.50a 15892.81±634.94a 126.20±8.94a Table 4.16(c) Metals (mg/kg) in soil cultured with wild reeds for 12 weeks Treatment Mn Fe Al Clean soil Total 29.99±1.00b 6991.95±7.40b 52.48±3.51b Mobile 0.50±0.00b 19.25±1.25 d / NCNB Total 44.66±5.81a 17637.44±1496.86a 137.40±17.66a Mobile 9.99±1.77a 5049.20±295.86c 66.21±6.50c NCWB Total 39.04±5.17a 16904.50±1344.93a 128.04±16.21a Mobile 8.12±2.07a 4592.74±375.59c 67.45±5.59c LCNB Total 42.61±5.18a 17952.25±1798.37a 140.52±18.46a Mobile 10.62±2.07a 5352.76±796.72c 68.70±4.16c LCWB Total 40.60±5.41a 16654.66±1737.59a 146.78±10.35a Mobile 9.37±1.08a 4707.75±48.07c 69.07±7.07bc MCNB Total 48.41±2.70a 19835.68±2808.60a 140.54±18.44a Mobile 11.87±2.07a 8003.47±562.22ab 86.18±7.38ab MCWB Total 45.28±5.18a 18025.41±1926.47a 137.41±17.66a Mobile 9.37±2.72a 6794.41±865.38b 91.19±8.94a HCNB Total 49.96±4.42a 20146.99±4335.06a 137.38±8.84a Mobile 13.12±2.07a 8679.75±341.98a 98.69±2.17a HCWB Total 46.85±3.14a 19696.43±2936.80a 124.93±15.30a Mobile 12.49±3.06a 7352.52±463.37ab 99.92±3.51a 133 The concentration of mobile and total metals in clean soil did not change too much after 4 or 8 weeks. As expected, the concentrations of total and mobile metals in spiked soil cultured with reeds for 12 weeks were less than that planted with reeds for 4 and 8 weeks. For soil cultured with reeds of the same period, the concentrations of total and mobile metals in spiked soil cultured with wild reeds were less than that in spiked soil planted with purchased reeds. For example, the mobile Mn, Fe and Al were 23.73±4.14 mg/kg, 15603.20±504.21 mg/kg and 123.66±13.41 mg/kg in soil HCNB cultured with purchased reeds for 12 weeks (Table 4.15c). While the mobile Mn, Fe and Al were 13.12±2.07 mg/kg, 8679.75±341.98 mg/kg, and 98.69±21.7 mg/kg in soil cultured with wild reeds for 12 weeks (Table 4.16c). The decrease of the concentration of metals may relate to the metal uptake by reeds. As discussed in section of plant digestion (section 4.3.4), the wild reeds accumulate more metals into their plant tissues than purchased reeds. Reeds cultured for 12 weeks also had more metals into their biomass than reeds of 4 and 8 weeks. After culturing reeds for 4, 8 and 12 weeks, mobile metals in spiked soil added with middle and high level of CA were still higher (p>0.05) than that in soil without adding CA. For example, the concentrations of mobile Mn, Fe and Al were 21.24±4.15 mg/kg, 10226.19±200.30 mg/kg and 83.70±9.56 mg/kg in soil NCNB cultured with purchased reeds for 8 weeks (Table 4.15b), while the concentrations were 24.99±3.53 mg/kg, 19137.96±1702.52 mg/kg and 126.18±17.10 mg/k in soil MCNB cultured with the same kind of reeds for the same time period (Table 4.15b). However, the concentrations of mobile Mn, Fe and Al in soil added with low level of 134 CA were not significantly different (p>0.05) from that in soil without adding CA after 8 or 12 weeks. For example, the concentrations of mobile Mn, Fe and Al were 9.99±1.77 mg/kg, 5049.20±295.86 mg/kg and 66.21±6.50 mg/kg in soil NCNB cultured with wild reeds for 12 weeks. They were similar to the concentrations of mobile metals in soil LCNB with the wild reeds for the same time period which were 10.62±2.07 mg/kg, 5352.76±796.72 mg/kg, 68.70±4.16 mg/kg (Table 4.16c). This may be due to that the consumption of bacteria can cause the effect of CA to decrease with time (Römkens et al., 2002). According to the results of Muhammad et al. (2009), the mobility of Cu in soil increased sharply in the first several days of application of CA, and then the concentrations tended to be relatively constant later. It was observed that the mobile Fe in spiked soil inoculated with bacteria were less than that in the soil of same age and added the same level of CA but without adding bacteria. The rhizosphere bacteria played an important role to decreased the mobility of Fe in soil (p<0.05) during all the growth period. The concentrations of mobile Fe were 12237.28±475.46 mg/kg in soil LCNB cultured with wild reeds for 8 weeks, while the concentrations were 10201.51±203.04 mg/kg in soil LCWB cultured with wild reeds for 8 weeks (Table 4.16b). As discussed in the section of DCB extraction (4.3.3), the plants in spiked soil inoculated with bacteria had more Fe plaque on the root surface. Both of these results proved that the rhizosphere bacteria played a significant role in precipitating Fe and decrease the mobility of Fe. In addition to the metals formed plaque on the root/rhizome surface of plants; accumulated into plants, adsorbed into soil matrix; some metals salted out and 135 precipitated on the surface of soil due to H2O uptake by plants and Fe(II)OB bacteria activity. Figure 4.53 showed the fate of metals in the spiked soil experiments. The concentrations and amounts of metals precipitated on soil and lost into the bottom of pans as leachate have also been analyzed to calculate the mass balance of metals (Table 4.17 and 4.18). The quantified rate (“% quantified”) in Table 4.17 and 4.18 indicated the metal contents that have been successfully traced. The mass balance losses may be due to the fact that some metals were hard to trace such as those being adsorped by bacterial cells. Also, soil is not a homogenous media which may cause inaccuracy for mass balance. However, the error range in this study was 10%-20%, which was similar to previous research and in an acceptable range (Yoon et al., 2003). 136 Flower CA-M M M M Stem M CA-M M M CA-M Leave M M CA-M M CA-M CA Soil - M Surface M CA-M M Rhizome M M M MP B M M B Root B M M-S B M M CA M M MP B M CA-M M M M-B CA CA-B M CA-M M M-S M M CA-M M M M M Figure 4.53 The fate of metals in spiked soil experiments M: metal, MP: metals plaque, B: bacteria, M-S: metals bound to soil, M-B, metals adsorbed by bacteria cells, CA: citric acid, CA-M: citric acid-metal complex, CA-B: citric acid consumed by bacteria, Surface M: metals salt out and precipitate on the surface of soil. 137 Table 4.17(a): Mass balance for Mn in spiked soil cultured with purchased reeds for 12 weeks Treat Original Plaque Into Precipitate Leachate Remaining % amounts (mg) plants (mg) (mg) in soil Quantified in soil (mg) (mg) NCNB 29.66 0.20 0.26 0.23 0.17 24.21 84.5% NCWB 29.66 0.34 0.23 0.30 0.18 23.42 82.5% LCNB 29.67 0.33 0.39 0.22 0.10 24.99 87.7% LCWB 28.11 0.39 0.33 0.28 0.13 21.86 81.8% MCNB 34.34 0.32 0.53 0.17 0.07 28.10 85.0% MCWB 31.22 0.25 0.39 0.20 0.15 26.54 88.2% HCNB 34.34 0.03 0.53 0.12 0.17 28.11 84.3% HCWB 32.79 0.06 0.51 0.15 0.17 26.54 83.6% Table 4.17(b): Mass balance for Fe in spiked soil cultured with purchased reeds for 12 weeks Treat Original Plaque Into Precipitate Leachate Remaining % amounts (mg) plants (mg) (mg) in soil Quantified in soil (mg) (mg) NCNB 14868.3 122.4 61.3 464.8 95.6 12360.0 88.1% NCWB 13921.5 214.6 51.4 830.2 89.5 11684.2 92.4% LCNB 14969.2 134.6 99.4 481.8 92.5 12412.8 88.3% LCWB 14772.4 208.3 72.8 841.3 91.1 12329.9 91.7% MCNB 14095.6 139.3 195.8 407.7 97.1 12218.3 94.9% MCWB 15520.7 144.3 161.2 477.5 96.7 12004.8 86.0% HCNB 15867.0 76.01 177.3 350.7 99.3 13005.1 86.4% HCWB 15560.0 104.2 174.9 440.8 98.9 12906.9 88.2% Table 4.17(c): Mass balance for Al in spiked soil cultured with purchased reeds for 12 weeks Treat Original Plaque Into Precipitate Leachate Remaining % amounts (mg) plants (mg) (mg) in soil Quantified in soil (mg) (mg) NCNB 96.77 2.02 0.71 0.23 0.15 87.45 93.6% NCWB 106.16 2.02 2.30 0.22 0.18 88.99 88.3% LCNB 107.74 2.14 1.47 0.25 0.22 89.02 86.4% LCWB 110.86 2.12 1.62 0.17 0.27 90.55 85.4% MCNB 106.16 2.43 1.35 0.22 0.25 95.24 94.7% MCWB 107.71 2.21 2.21 0.20 0.23 93.68 91.5% HCNB 107.70 1.85 2.70 0.18 0.17 95.27 93.0% HCWB 107.73 2.00 3.20 0.15 0.20 95.25 93.6% 138 Table 4.18(a): Mass balance for Mn in spiked soil cultured with wild reeds for 12 weeks Treat Original Plaque Into Precipitate Leachate Remaining % amounts (mg) plants (mg) (mg) in soil Quantified in soil (mg) (mg) NCNB 28.09 0.29 0.86 0.30 0.15 22.33 85.2% NCWB 24.98 0.61 0.63 0.37 0.17 19.52 85.2% LCNB 28.11 0.67 1.49 0.25 0.12 21.08 84.0% LCWB 29.66 0.58 0.80 0.30 0.12 20.30 74.5% MCNB 32.79 0.46 1.70 0.20 0.08 24.20 81.3% MCWB 31.23 0.56 1.36 0.22 0.13 22.64 79.8% HCNB 34.34 0.09 2.44 0.15 0.15 24.98 81.0% HCWB 32.80 0.30 2.27 0.17 0.13 23.42 80.2% Table 4.18(b): Mass balance for Fe in spiked soil cultured with wild reeds for 12 weeks Treat Original Into Precipitate Leachate Remaining % amounts Plaque plants (mg) (mg) in soil Quantified in soil (mg) (mg) (mg) NCNB 12262.4 340.3 158.2 470.5 95.6 8818.7 80.6% NCWB 11337.6 462.1 131.7 859.8 93.6 8452.3 88.2% LCNB 12526.7 355.0 177.0 494.3 89.7 8976.1 80.6% LCWB 11933.4 412.3 136.9 841.0 100.6 8327.3 82.3% MCNB 13386.2 258.0 344.1 440.8 99.2 9917.8 82.6% MCWB 12736.3 303.0 310.5 550.3 95.3 9012.7 80.7% HCNB 13540.6 150.9 388.9 406.0 90.4 10073.5 82.0% HCWB 13035.8 243.0 346.8 517.8 92.8 9847.2 84.7% Table 4.18(c): Mass balance for Al in spiked soil cultured with wild reeds for 12 weeks Treat Original Plaque Into Precipitate Leachate Remaining % amounts (mg) plants (mg) (mg) in soil Quantified in soil (mg) (mg) NCNB 95.22 3.73 5.41 0.27 0.23 68.70 82.3% NCWB 98.36 4.11 7.11 0.25 0.20 64.02 77.0% LCNB 99.94 4.12 7.71 0.22 0.18 70.26 82.5% LCWB 101.46 4.08 6.89 0.17 0.25 73.39 83.6% MCNB 106.19 3.96 10.35 0.20 0.22 70.27 80.0% MCWB 103.05 4.03 10.66 0.18 0.15 68.70 81.2% HCNB 104.58 3.52 26.80 0.23 0.20 68.69 95.1% HCWB 106.20 3.90 27.65 0.28 0.17 68.69 88.9% 139 To better understand the transport and transformation of metals under different treatment conditions, the quantified rate of metals were calculated. As showed in Table 4.17 and 4.18, the quantified rates of metals were the highest in clean soil. The quantified rates of metals in spiked soil ranged from 70% to more than 90% for Mn, Fe and Al, which reflected that the results clearly explain the fate of metals. With the increase of CA, less metal plaque formed on the root surface of reeds. Also, less metal precipitated on the surface of soil and more metal accumulated into the plant tissues. These phenomena further proved that the CA can increase the solubility of metals in soil and enhance the metal accumulation in reeds. With the increase of CA, more metals accumulated into the plant tissues. However, most of metals still remained in soil. Reed is a rapid growth perennial macrophyte with a profuse tuberous root system and reasonably high biomass in the field (Baldantoni et al., 2009; Ye et al., 1997a). With enough growth periods, more metals could be accumulated into the plant tissues and less metal would remain in soil. 4.3.6 Histological experiments for Fe in reeds cultured in greenhouse Histological experiments were conducted to determine the specific tissue for Fe storage in reeds cultured in clean soil or spiked soil in greenhouse. The results may further explain the effect of CA and rhizosphere bacteria on metal uptake in different types of reeds. The following sections showed the representative staining images. According to the staining procedure, the tissues contained iron would turn blue after staining. 140 4.3.6.1 Fe staining for purchased uncontaminated reeds grown in clean soil Figure 4.54 and 4.55 showed the stained root sections of uncontaminated reeds cultured in clean soil. No blue color was found in the root or rhizomes sections of purchased uncontaminated reeds grown in clean soil. It is not surprising that the sections were not depicted, since our quantitative data in section 4.3.4 also showed that the root and rhizome of uncontaminated reed did not contain Fe. 200µm 200µm a a b Figure 4.54 Fe stained cross section of root of uncontaminated reed grown in clean soil, (a) magnified image of stele, (b) magnified image of exodermis 1mm 200µm m a b Figure 4.55 Fe stained cross section of rhizome of uncontaminated reed grown in clean soil, (a) full view of the cross section with image (b) a magnified subsection 141 4.3.6.2 Fe staining for wild reeds cultured in clean and spiked soil Figure 4.56 showed the stained root sections of wild reeds cultured in clean soil Figure 4.57 to 4.64 showed the representative pictures of stained root sections of wild reeds cultured in spiked soil. 200µm 200µm a b Figure 4.56 Fe stained cross section of root of wild reed grown in clean soil, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.57 Fe stained cross section of root of reed grown in spiked soil HCNB (a) magnified image of stele (b) magnified image of exodermis. 142 200µm 200µm a b Figure 4.58 Fe stained cross section of root of reed grown in spiked soil HCWB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.59 Fe stained cross section of root of reed grown in spiked soil MCNB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.60 Fe stained cross section of root of reed grown in spiked soil MCWB, (a) magnified image of stele (b) magnified image of exodermis. 143 200µm 200µm a b Figure 4.61 Fe stained cross section of root of reed grown in in spiked soil LCNB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b a Figure 4.62 Fe stained cross section of root of reed grown in spiked soil LCWB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.63 Fe stained cross section of root of reed grown in spiked soil NCNB, (a) magnified image of stele (b) magnified image of exodermis. 144 200µm 200µm a b Figure 4.64 Fe stained cross section of root of reed grown in spiked soil NCWB, (a) magnified image of stele (b) magnified image of exodermis. Although the clean soil was not spiked, the root and rhizomes of reeds grown in clean soil contained iron. As mentioned in earlier section (4.1.5), this may due to that the reeds were collected from contaminated field and Fe was already accumulated into the reeds. Figure 4.56 showed that Fe mainly sequestered in the exodermis of root of reeds grown in clean soil. Figure 4.57 to Figure 4.64 were cross sections of reeds grown in spiked soil with different amounts of CA or rhizosphere bacteria. Both the exodermis and the stele of roots contained Fe. That was the greatest difference exhibited to the roots of reeds directly collected from the field. Root tissue from reeds collected from the field (i.e. wild reeds) and subjected to immediate staining did not have Fe in the stele. This may be associated with the total amounts of Fe in the roots. Our previous data in section 4.3.4 showed that the roots of reeds transferred into spiked soil in greenhouse contained more Fe than that in reeds harvested from the field. This might have enabled more iron enter into the exodermis and steles of roots. In addition, the color was the deepest in the root section of reeds treated with high level of CA while the color was the lightest of roots in spiked soil without adding CA. 145 The blue hue mainly concentrated in the pith of root grown in soil with low level of CA, as showed in Figure 4.61 to 4.64. With the increase of CA the blue hue extended to the whole stele of roots, as presented in Figure 4.57 to Figure 4.60. This phenomenon may also be associated with the mobile metals in soil and the total amounts of Fe in plants. As expected, mobile metal fraction increased with an increase of CA (Mihalík et al., 2010; Alidoust et al., 2009). This was corroborated by soil with higher level of CA had more Fe in the plant biomass. For soil amended with the same level of CA, Fe concentration was less in the roots inoculated with rhizosphere bacteria than that in soil without inoculated with rhizosphere bacteria. This was in accord with the results of histological observations. For instance, the blue hue was more obvious in the pith of root in LCNB than that in LCWB. Similarly, the intensity of blue hue was stronger in the stele of root in MCNB than that in MCWB. Figure 4.65 showed the stained rhizome sections of wild reeds cultured in clean soil Figure 4.66 to 4.73 showed the representative pictures of stained rhizome sections of wild reeds cultured in spiked soil. 1mm 200µm m a b Figure 4.65 Fe stained cross section of rhizome of reeds in clean soil, (a) full view of the cross section with image (b) a magnified subsection that to clearly show the sequestration of iron. 146 1mm 200µm m a a b Figure 4.66 Fe stained cross sections of rhizomes of reeds in spiked soil HCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b . Figure 4.67 Fe stained cross sections of rhizomes of reeds in spiked soil HCWB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.68 Fe stained cross sections of rhizomes of reeds in spiked soil MCNB, (a) full view of the cross section with image (b) a magnified subsection 147 1mm 200µm m a b Figure 4.69 Fe stained cross sections of rhizomes of reeds in spiked soil MCWB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.70 Fe stained cross sections of rhizomes of reeds in spiked soil LCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.71 Fe stained cross sections of rhizomes of reeds in spiked soil LCWB, (a) full view of the cross section with image (b) a magnified subsection 148 1mm 200µm m b a Figure 4.72 Fe stained cross sections of rhizomes of reeds in spiked soil NCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m b a Figure 4.73 Fe stained cross sections of rhizomes of reeds in spiked soil NCWB, (a) full view of the cross section with image (b) a magnified subsection Since the reeds initially grown in AMD site, metals were already accumulated in the plant tissues (see section 4.1.5). That is why the rhizomes of reeds growing in clean soil also contained Fe. However, the blue hue of rhizomes in clean soil was not as prominent as that in rhizomes cultured in spiked soil, which was in accord with the results of plant digestion in section 4.3.4. The quantitative data also showed that the amounts of Fe in rhizomes in clean soil were less than that in spiked soil. There were no structural differences between the rhizomes in greenhouse and rhizomes collected from the field. Blue hue which indicated Fe was observed in the cortex and central 149 cylinder of rhizomes, and was especially obvious in the tissue around vascular bundles. Similar to the stained root sections, the intensity and area of blue hue was the greatest in the rhizomes grown in soil treated with high level of CA. The blue hue was not very apparent in the rhizomes sections of reeds in soil without adding CA. These phenomena may relate to the Fe concentrations in the rhizomes of reeds. Quantitative data in section 4.3.4 also showed that rhizomes of reeds cultured in spiked soil treated with higher level of CA contained more Fe than the rhizomes grown in soil without adding CA. 4.3.7 Histological experiments for Al in reeds cultured in greenhouse Histological experiments were also conducted for Al in reeds cultured in clean soil or spiked soil in greenhouse. The following sections showed the representative Al staining images. 4.3.7.1 Al staining for purchased uncontaminated reeds grown in clean soil The roots and rhizomes of uncontaminated reeds were used to do histological staining for Al. Figure 4.74 and 4.75 showed the cross sections of roots and rhizomes after staining. 150 200µm 200µm a b Figure 4.74 Al stained cross section of root of uncontaminated reed grown in clean soil, (a) magnified image of stele, (b) magnified image of exodermis. 1mm 200µm m a b Figure 4.75 Al stained rhizomes cross sections of uncontaminated reed grown in clean soil, (a) a full view of the cross section with image (b) a magnified subsection According to the staining procedure for Al, tissue contained Al would turn magenta. As expected, no magenta was observed in the root and rhizome sections of uncontaminated reeds grown in clean soil. Quantitative data in section 4.3.4 also indicated that the root and rhizome of uncontaminated reed grown in clean soil did not contain Al. 4.3.7.2 Al staining for wild reeds cultured in clean and spiked soil Figure 4.76 showed the images of stained root sections of wild reeds cultured in clean. Figure 4.77 to 4.84 showed the typical images of stained root sections of wild reeds cultured in spiked soil. 151 200µm 200µm a b Figure 4.76 Al stained cross section of root of wild reed grown in clean soil, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.77 Al stained cross section of roots of reeds grown in spiked soil HCNB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.78 Al stained cross section of roots of reeds grew in spiked soil HCWB, (a) magnified image of stele (b) magnified image of exodermis. 152 200µm 200µm a b Figure 4.79 Al stained cross section of roots of reeds grew in spiked soil MCNB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.80 Al stained cross section of roots of reeds grew in spiked soil MCWB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.81 Al stained cross section of roots of reeds grew in spiked soil LCNB, (a) magnified image of stele (b) magnified image of exodermis. 153 200µm 200µm a b Figure 4.82 Al stained cross section of roots of reeds grew in spiked soil LCWB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.83 Al stained cross section of roots of reeds grew in spiked soil NCNB, (a) magnified image of stele (b) magnified image of exodermis. 200µm 200µm a b Figure 4.84 Al stained cross section of roots of reeds grew in spiked soil NCWB, (a) magnified image of stele (b) magnified image of exodermis. Al was mainly sequestered in the exodermis of roots of wild reeds cultured in clean soil and spiked soil without addition of CA, as showed in Figure 4.76 and 154 Figure 4.83 and 4.84. The intensity of magenta hue in exodermis increased with the increase of CA. The magenta hue even spread to the whole stele of roots in spiked soil added with high level of CA, which was presented in Figure 4.77 and Figure 4.78. Compared to CA, the rhizosphere bacteria did not have a significant effect on the Al sequester in roots. The intensity of magenta hue in roots inoculated with rhizosphere bacteria was not much different from that without inoculated with bacteria, as presented in Figure 4.77 to Figure 4.80. This phenomenon may be associated with the mobile Al in soil and the total amounts of Al in roots. Our quantitative data in section 4.3.5 indicated that CA increased the amounts of mobile Al in soil and enhanced the uptake of Al by roots. For soils amended with the same amounts of CA, the Al concentrations in root inoculated with rhizosphere bacteria was not much different from that without inoculated with bacteria. Figure 4.85 showed the images of stained rhizomes sections of wild reeds cultured in clean soil. Figure 4.86 to 4.93 showed the representative images of stained rhizomes sections of wild reeds cultured in spiked soil. 1mm 200µm m a b Figure 4.85 Al stained cross section of rhizome of wild reeds in clean soil, (a) full view of the cross section with image (b) a magnified subsection that to clearly show the sequestration of Al 155 1mm 200µm m a b Figure 4.86 Al stained cross sections of rhizomes of reeds in spiked soil HCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.87 Al stained cross sections of rhizomes of reeds in spiked soil HCWB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.88 Al stained cross sections of rhizomes of reeds in spiked soil MCNB, (a) full view of the cross section with image (b) a magnified subsection 156 1mm 200µm m a b Figure 4.89 Al stained cross sections of rhizomes of reeds in spiked soil MCWB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.90 Al stained cross sections of rhizomes of reeds in spiked soil LCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.91 Al stained cross sections of rhizomes of reeds in spiked soil LCWB, (a) full view of the cross section with image (b) a magnified subsection 157 1mm 200µm m a b Figure 4.92 Al stained cross sections of rhizomes of reeds in spiked soil NCNB, (a) full view of the cross section with image (b) a magnified subsection 1mm 200µm m a b Figure 4.93 Al stained cross sections of rhizomes of reeds in spiked soil NCWB, (a) full view of the cross section with image (b) a magnified subsection Magenta hue was observed in the rhizomes cross sections of reeds cultured under all the conditions, which was in accord with our previous data. No anatomical difference was observed in the rhizomes grown in different conditions. Similar to Fe, Al was observed in the cortex and central cylinder of rhizomes. The magenta hue was especially apparent in the tissue around vascular bundles. As expected, the magenta hue was the strongest in the rhizome grown in soil treated with high level of CA while the color was least apparent in the rhizomes grown in soil without adding CA. The more CA added into spiked soil, the more Al transported into the rhizomes and the stronger intensity of magenta hue was. Our data of plant digestion in section 4.3.4 158 showed that the Al concentrations in rhizomes decreased according to the order: HCNB～HCWB＞MCNB～MCWB＞LCNB～LCWB＞NCNB～NCWB＞clean soil. The intensity and area of magenta hue in the rhizomes appeared to change according to the same order. These results further proved that CA can increase Al uptake in reeds. 4.3.8 Summary of spiked soil experiments treated with purchased and wild reeds Fe(II)OB did not have important affect on the formation of Mn and Al plaque but significantly mediated the formation of Fe plaque and decreased the pH of soil. The amounts of metal plaque increased with time, however, most plaque formed in the first 4 and 8 weeks. This may be due to the fact that Fe oxidation was fast in the first several weeks. Fe plaque can not prevent but can inhibit the accumulation of Mn and Fe in purchased reeds and wild reeds, but had no important affect on Al uptake. As a organic material, CA increased the growth of heterotrophs and inhibited the growth of Fe(II)OB. The spiked soil added with higher level of CA had higher number of heterotrophs and lower number of Fe(II)OB. As such, CA inhibited the formation of Fe plaque. With the increase of CA, less Fe plaque formed on the root surface of reeds. CA can also increase the mobility of Fe, Mn and Al in soils and enhanced the metal accumulation and translocation in purchased reeds and wild reeds. Nevertheless, most of metals were stored in the belowground parts of reeds. The more CA added, the more metal accumulated into reeds. Compared with purchased reeds, wild reeds 159 cultured under the same treatment conditions accumulated more metals into their plant tissues and the metals amounts increased with time. This maybe attributed to the fact that the wild reeds grown in contaminated sites had already adapted to the hostile environments, and then can perform better at metal uptake. The histological experiments also proved that CA can increase the metal accumulation in reeds. low level of CA enhance Fe entering the pith of root, while high level of CA cause Fe extend to the whole stele of roots. CA also increased the amounts of Al enter the stele of roots. The amounts of Fe and Al in the tissue around vascular bundles of rhizomes were also elevated by Al. 4.4 Hydroponic experiments for reeds propagated from wild rhizomes Hydroponic culture is an important way to investigate the effect of CA and rhizosphere bacteria on the metal uptake by reeds. First, Phragmites australis is a typical aquatic plant. Second, a hydroponic experiment does not have the same mass transfer limitations as soil and therefore yield better understanding of the fate of metals in the solution and plants. In hydroponic experiments, the control group which only contained nutrient materials was used to assess biomass growth of reeds. The treatment groups which contained nutrient materials and metals were separated into 3 different levels: low level of metals (LM), middle level of metals (MM) and high level of metals (HM) according to the concentrations of metals used. The components of solution were presented in section 3.5. Reeds propagated from wild rhizome grown 160 in potting soil were cultured in the hydroponic solution for 4, 8 and 12 weeks and then harvested for analysis. 4.4.1 Rhizosphere bacteria in solution cultured with reeds propagated from wild rhizomes To better understand the interactions among CA, bacteria growth and metal uptake by plants, acidophilic heterotrophs and Fe(II)OB in solution were enumerated and presented in Table 4.19. As expected, solution inoculated with rhizosphere bacteria possessed more Fe(II)OB and acidophilic heterotrophs than solutions without adding rhizosphere bacteria during all growth periods. For example, the LMNCNB solution possessed 1.1±0.11×104 CFU/ml acidophilic heterotrophs and 9.00±0.50×102 CFU/ml Fe(II)OB, while the LMNCWB solution had 3.81±0.59×105 CFU/ml acidophilic heterotrophs and 2.00±0.10×104 CFU/ml Fe(II)OB after 4 weeks (Table 4.19a). Similar trends were found for rhizosphere bacteria in MM and HM solution. The MMLCNB solution had 1.62±0.18×104 CFU/ml acidophilic heterotrophs and 1.00±0.04×103 CFU/ml Fe(II)OB, while the MMLCWB solution contained 4.08±0.28×105 CFU/ml acidophilic heterotrophs and 3.00±0.09×104 CFU/ml Fe(II)OB after 4 weeks (Table 4.19b). The HMMCNB solution possessed 4.03±0.17×104 CFU/ml acidophilic heterotrophs and 2.00±0.07×103 CFU/ml Fe(II)OB, while the HMMCWB solution had 6.04±0.16×105 CFU/ml acidophilic heterotrophs and 3.00±0.09×104 CFU/ml Fe(II)OB after 4 weeks (Table 4.19c). 161 Table 4.19 (a) Numbers of rhizosphere bacteria in LM solution cultured with reeds for 4, 8 and 12 weeks Treatment Time WAYE (CFU/ml) FETSB (CFU/ml) Control 4 weeks 1.60 ±0.20×103 d / solution 8 weeks 2.90 ±0.50×103 d / 12 weeks 1.50 ±0.30×103 d / LMNCNB 4 weeks 1.11 ±0.11×104 c 9.00 ±0.50×102 d 8 weeks 9.10 ±1.10×103 c 5.00 ±0.60×102 de 12 weeks 4.22 ±0.18×103 c 4.00 ±0.20×102e LMNCWB 4 weeks 3.81 ±0.59×105 b 2.00 ±0.10×104 a 8 weeks 5.11 ±1.29×105 b 2.00 ±0.05×104 a 12 weeks 3.90 ±0.50×105 b 2.00 ±0.02×104 a LMLCNB 4 weeks 9.10 ±1.10×103 c 7.00 ±0.10×102 d 8 weeks 1.09 ±1.10×104 c 5.00 ±0.10×102 de 12 weeks 6.70 ±0.70×103 c 4.00±0.40×102 e LMLCWB 4 weeks 3.62±0.18×105 b 1.00 ±0.10×104 b 8 weeks 5.00 ±0.20×105 b 1.00 ±0.08×104 b 12 weeks 5.40 ±0.20×105 b 2.00 ±0.06×104 b LMMCNB 4 weeks 1.69 ±0.09×104 c 5.00 ±0.40×102 d 8 weeks 1.59 ±0.01×104 c 3.00 ±0.10×102 e 12 weeks 1.30 ±0.10×104 c 2.00 ±0.10×102 e LMMCWB 4 weeks 4.15 ±0.15×105 b 5.00 ±0.80×103 c 8 weeks 8.30 ±0.70×105 ab 8.00±0.50×103 c 12 weeks 1.13 ±0.03×106 a 9.00±0.50×103 c LMHCNB 4 weeks 4.15 ±0.65×104 c 3.00 ±0.30×102 d 8 weeks 3.73 ±0.47×104 c 1.00 ±0.20×102 e 12 weeks 3.23 ±0.37×104 c 9.00±1.00×101 e LMHCWB 4 weeks 6.70 ±0.90×105 a 2.00 ±0.07×103 cd 8 weeks 8.70±0.90×105 a 3.00 ±0.20×103d 12 weeks 1.27 ±0.09×106 a 3.00 ±0.20×103 d / indicated blow detection limits; results were average±standard deviation, n=10. It’s not surprising that the numbers of heterotrophs were higher in solution added with high level of CA than that without or only low level of CA. For instance, the LMNCWB solution possessed 3.81±0.59×105 CFU/ml acidophilic heterotrophs, while the LMHCWB solution had 6.70 ±0.90×105 CFU/ml acidophilic heterotrophs after 4 weeks (Table 4.19 a). 162 Table 4.19(b) Numbers of rhizosphere bacteria in MM solution cultured with reeds for 4, 8 and 12 weeks Treatment Time WAYE (CFU/ml) FETSB (CFU/ml) MMNCNB 4 weeks 1.35±0.15×104 c 2.00 ±0.03×103 d 8 weeks 1.18±0.18×104 c 1.00 ±0.06×103 de 12 weeks 1.01±0.21×104 e 1.00 ±0.04×103 e MMNCWB 4 weeks 3.73 ±0.47×105 b 3.00 ±0.10×104 a 8 weeks 4.53 ±0.47×105 b 4.00 ±0.10×104 a 12 weeks 5.02 ±0.18×105 c 5.00 ±0.20×104 a MMLCNB 4 weeks 1.62 ±0.18×104 c 1.00 ±0.04×103 d 8 weeks 1.34 ±0.06×104 c 1.00 ±0.09×103 de 12 weeks 1.17 ±0.03×104 e 1.00 ±0.06×103 e MMLCWB 4 weeks 4.08±0.28×105 b 3.00 ±0.09×104 b 8 weeks 4.61±0.21×105 b 3.00 ±0.08×104 b 12 weeks 7.40±0.60×105 b 3.00 ±0.04×104 b MMMCNB 4 weeks 2.32 ±0.12×104 c 1.00 ±0.09×103 d 8 weeks 1.81±0.21×104 c 8.00 ±0.40×102 e 12 weeks 1.62±0.22×104 e 7.00 ±0.60×102 e MMMCWB 4 weeks 4.60 ±0.20×105 b 1.00 ±0.10×104 c 8 weeks 5.10 ±0.01×105 ab 2.00 ±0.05×104 c 12 weeks 7.40 ±0.60×105 b 2.00 ±0.10×104 c MMHCNB 4 weeks 3.00 ±0.40×105 b 6.00 ±0.30×102 d 8 weeks 3.00 ±0.20×105 c 5.00 ±0.40×102 e 12 weeks 2.70 ±0.30×105 d 4.00 ±0.40×102 e MMHCWB 4 weeks 1.04 ±0.12×106 a 4.00 ±0.10×103 d 8 weeks 1.75 ±0.09×106 a 7.00 ±0.30×103 d 12 weeks 1.98 ±0.04×106 a 1.00 ±0.10×104 d / indicated blow detection limits; results were average±standard deviation, n=10. Similar trends were found in MM and HM solution. The more CA added, the more acidophilic heterotrophs were in the solution. This was due to that CA can be used as carbon source by many microorganisms (Chen et al., 2006). According to the results of Küsel et al. (2003), citrate can be consumed by rhizosphere bacteria within 6 to 11 days at pH 3. Francis (1998) also pointed out that CA can be metabolized intracellularly in bacteria by the enzymes. CA can also form metal-citrate complex 163 with metals, which may also be metabolized or accumulated by some heterotrophic bacteria (Francis, 1998). Table 4.19(c) Numbers of rhizosphere bacteria in HM solution cultured with reeds for 4, 8 and 12 weeks treatment time WAYE (CFU/ml) FETSB (CFU/ml) HMNCNB 4 weeks 1.17 ±0.17×104 d 3.00 ±0.09×103 d 8 weeks 9.80 ±1.80×103 d 3.00 ±0.10×103 d 12 weeks 9.20 ±1.20×103 c 3.00 ±0.09×103 d HMNCWB 4 weeks 3.12 ±0.28×105 cd 1.00 ±0.04×105 a 8 weeks 3.76 ±0.16×105 c 2.00 ±0.09×105 a 12 weeks 6.50 ±0.70×105 b 2.00 ±0.06×105 a HMLCNB 4 weeks 1.39 ±0.01×104 d 2.00 ±0.09×103 d 8 weeks 9.60 ±1.60×103 d 2.00 ±0.05×103 d 12 weeks 9.10 ±1.10×103 c 2.00 ±0.10×103 d HMLCWB 4 weeks 3.57±0.17×105 cd 5.00 ±0.70×104 b 8 weeks 4.05±0.45×105 c 1.00 ±0.08×105 b 12 weeks 8.40±0.60×105 b 1.00 ±0.06×105 b HMMCNB 4 weeks 4.03 ±0.17×104 d 2.00 ±0.07×103 d 8 weeks 3.57 ±0.17×104 d 1.00 ±0.07×103 d 12 weeks 3.29 ±0.09×104 c 1.00 ±0.10×103 d HMMCWB 4 weeks 6.04 ±0.16×105 bc 3.00 ±0.09×104 c 8 weeks 9.90±0.70×105 b 3.00 ±0.01×104 c 12 weeks 1.41±0.15×106 a 4.00 ±0.09×104 c HMHCNB 4 weeks 7.50 ±1.50×105 b 1.00 ±0.09×103 d 8 weeks 5.40 ±0.80×105 c 1.00 ±0.02×103 d 12 weeks 5.00 ±0.80×105 b 9.00 ±0.70×102 d HMHCWB 4 weeks 2.07 ±0.09×106 a 8.00 ±0.03×103 d 8 weeks 2.22 ±0.06×106 a 1.00 ±0.08×104 cd 12 weeks 2.85±0.11×106 a 1.00 ±0.09×104 d / indicated blow detection limits; results were as average±standard deviation, n=10. Fe(II)OB was found in the rhizosphere of reeds under all the treatment conditions. Previous research also reported the presence of lithotrophic Fe(II)OB in the rhizosphere of wetland plants (Neubauer et al., 2007). Contrary to acidophilic 164 heterotrophs, the numbers of Fe(II)OB decreased with the increase of CA. For example, the LMLCWB solution possessed 1.00±0.10×104 CFU/ml Fe(II)OB, while the solution LMHCWB had 2.00±0.07×103 CFU/ml Fe(II)OB after 4 weeks (Table 4.19 a). The MMNCWB solution possessed 3.00±0.10×104 CFU/ml Fe(II)OB, which was higher than the numbers of Fe(II)OB in MMHCWB solution after 4 weeks (4.00±0.10×103 CFU/ml). The numbers of Fe(II)OB in HMLCWB solution (5.00±0.70×104 CFU/ml) was also higher than the numbers of Fe(II)OB in HMHCWB solution (8.00±0.03×103 CFU/ml) after 4 weeks (Table 4.19c). This may due to the fact that organic acid can promote reducing conditions and inhibit the growth of Fe(II)OB (Marchand and Silverstein, 2003). Küsel et al. (2003) also reported that carbon source such as glucose and CA would reduce Fe(III). Further, the solution contained higher CA possessed higher numbers of acidophilic heterotrophs. Some heterotrophic acidophiles species may be involved in the reduction of Fe(III) (Küsel et al., 2003). The numbers of Fe(II)OB and acidophilic heterotrophs in solution without inoculating bacteria did not change too much or only slightly decreased with time. For example, the LMMCNB solution possessed 1.69±0.09×104 CFU/ml acidophilic heterotrophs and 5.00±0.40×102 CFU/ml Fe(II)OB after 4 weeks, but the numbers decreased to 1.30±0.10×104 CFU/ml and 2.00±0.10×102 CFU/ml after 12 weeks (Table 4.19a). The numbers of acidophilic heterotrophs and Fe(II)OB were 3.00±0.40×105 and 6.00 ±0.30×102 CFU/ml in MMHCNB solution after 4 weeks, which decreased to 2.70±0.30×105 CFU/ml and 4.00±0.40×102 CFU/ml after 12 165 weeks (Table 4.19b). The HMNCNB solution had 1.17±0.17×104 CFU/ml acidophilic heterotrophs after 4 weeks, but the numbers reduced to 9.20±1.20×103 CFU/ml after 12 weeks (Table 4.19c). This may due to the fact that CA was consumed by heterotrophs or complexed with metals and then there were less additive carbon sources for the growth of heterotrophs. The plant root exudates were the main nutrients to maintain the population rhizosphere bacteria (Abhilash et al., 2011; Khan, 2005; Küsel et al., 2003). Besides, the population of Fe(II)OB were related to the amount of Fe(II) present (Emerson et al., 1999; Neubauer et al., 2007). Positive correlation was found between numbers of FeOB and the amounts of Fe(II) (Chen et al., 2008). As more Fe(II) were oxidized into Fe3+ and less Fe(II) remained, the numbers of Fe(II)OB may decrease. Nevertheless, the numbers of rhizosphere bacteria in solution inoculated with bacteria did not decrease with time. For instance, the LMHCWB cultured with reeds for 4 weeks possessed 6.70±0.90×105 CFU/ml acidophilic heterotrophs and 2.00 ±0.07×103 CFU/ml Fe(II)OB, and the numbers of acidophilic heterotrophs and Fe(II)OB maintained at 1.27±0.09×106 CFU/ml and 3.00±0.20×103 CFU/ml in LMHCWB solution cultured with reeds for 12 weeks (Table 4.19a). The MMMCWB solution possessed 4.60±0.20×105 CFU/ml acidophilic heterotrophs and 1.00±0.10×104 CFU/ml Fe(II)OB after 4 weeks, and the numbers slightly increased after 12 weeks which were 7.40±0.60×105 CFU/ml for acidophilic heterotrophs and 2.00±0.10×104 CFU/ml for Fe(II)OB (Table 4.19b). The numbers of acidophilic heterotrophs and Fe(II)OB were 3.12±0.28×105 CFU/ml and 1.00±0.04×105 CFU/ml 166 in HMNCWB solution after 4 weeks, which did not decrease after 12 weeks ( 6.50±0.70×105 CFU/ml and 2.00 ±0.06×105 CFU/ml for acidophilic heterotrophs and Fe(II)OB, respectively). This may be attributed to that new rhizosphere bacteria were inoculated into the solution every 4 weeks, which can maintain and increase the numbers of rhizosphere bacteria. The rhizosphere was a complicated biochemical environment. Chemical reactions occurred in the solution can affect the transformation metals in rhizosphere, the activities of microorganism and plants also influenced the fate of metals and CA in root zone. Further research may be needed to investigate the reaction kinetics between metals and CA and to study the relationship among chelators, metal availability and microorganism activity in field. 4.4.2 pH change of solution cultured with reeds propagated from wild rhizomes As shown in Table 4.20, the pH of solution decreased after adding into CA. As anticipated, the more CA added into the solution, the lower the pH was. For instance, the pH of LMMCNB solution decreased from 3.48±0.02 to 3.00±0.02 after adding 1.061 g/LCA (Table 4.20a). The pH of MMMCNB solution reduced from 3.50±0.01 to 2.70±0.02 after adding 9.020 g/L CA (Table 4.20b). The pH of HMHCNB solution changed from 3.51±0.01 to 2.20±0.01 after addition of 33.616 g/L CA (Table 4.20c). It was not surprising, since CA was a kind of organic acid which can decrease the pH of solution. Chen et al. (2003) also found that the pH of soil solution decreased in the presence of CA. 167 Table 4.20 (a) pH of LM solution under different treatment conditions Initial pH After 4 weeks after 8 weeks after 12 weeks Treatment adding culturing culturing after CA reeds reeds culturing reeds Control 5.75±0.02 5.75±0.04 5.71±0.02 5.73±0.03 5.72±0.03 LMNCNB 3.49±0.01 3.50±0.01 2.60±0.02 2.50±0.02 2.47±0.02 LMNCWB 3.49±0.02 3.51±0.02 2.49±0.01 2.30±0.02 2.27±0.03 LMLCNB 3.49±0.02 3.50±0.03 2.58±0.01 2.49±0.05 2.47±0.01 LMLCWB 3.50±0.01 3.49±0.02 2.48±0.02 2.29±0.02 2.26±0.03 LMMCNB 3.48±0.02 3.00±0.02 2.96±0.02 2.93±0.02 2.91±0.02 LMMCWB 3.51±0.03 3.02±0.01 2.93±0.02 2.90±0.02 2.89±0.04 LMHCNB 3.50±0.01 2.79±0.01 2.73±0.01 2.73±0.01 2.71±0.03 LMHCWB 3.50±0.02 2.80±0.01 2.72±0.02 2.72±0.02 2.71±0.02 The results were reported as average±1 standard deviation, n=3 Table 4.20 (b) pH of MM solution under different treatment conditions Initial pH After 4 weeks after 8 weeks after 12 weeks Treatment adding culturing culturing after CA reeds reeds culturing reeds MMNCNB 3.50±0.01 3.50±0.00 2.14±0.01 2.12±0.02 2.10±0.01 MMNCWB 3.50±0.02 3.50±0.02 2.04±0.01 2.01±0.00 2.00±0.01 MMLCNB 3.50±0.01 3.30±0.01 2.15±0.00 2.13±0.02 2.11±0.00 MMLCWB 3.51±0.01 3.30±0.02 2.05±0.01 2.02±0.02 2.01±0.00 MMMCNB 3.50±0.01 2.70±0.02 2.22±0.01 2.19±0.00 2.18±0.01 MMMCWB 3.50±0.02 2.70±0.02 2.18±0.01 2.15±0.01 2.13±0.01 MMHCNB 3.50±0.01 2.40±0.00 2.28±0.01 2.24±0.00 2.22±0.02 MMHCWB 3.51±0.01 2.40±0.01 2.25±0.01 2.21±0.01 2.18±0.01 The results were reported as average±standard deviation, n=3 The pH of solution kept decreasing as the reeds grew in the solution over 4 weeks. The solution inoculated with Fe(II)OB had lower pH than the solution added with the same level of CA but without adding with Fe(II)OB. For example, the pH of LMLCNB solution decreased from 3.37±0.02 to 2.58±0.02, while the pH of LMLCWB solution reduced from 3.38±0.01 to 2.48±0.02 after 4 weeks (Table 4.20a). The pH of MMNCNB solution changed from 3.50±0.00 to 2.14±0.01, while the pH of 168 MMNCWB solution decreased from 3.50±0.02 to 2.04±0.02 after 4 weeks (Table 4.20b). The pH of HMMCNB solution changed from 2.50±0.02 to 2.11±0.01, while the pH of HMMCWB solution reduced from 2.50±0.00 to 2.08±0.02 after 4 weeks (Table 4.20c). This may due to the oxidation of Fe(II) by Fe(II)OB. Previous research indicated that the rhizosphere Fe(II)OB can use the oxygen released by plant roots to oxidize Fe(II) (Weiss et al., 2004). It was not surprising to find that the oxidation of and precipitation of Fe(II) can cause the decrease of pH (Akcil and Koldas, 2006). Otte et al. (1989) also indicated that the oxidation of Fe(II) can cause the lowering of pH in the rhizosphere of plants. According to the results of Begg et al. (1994), the pH in the rhizosphere of rice (Oryza sativa L.) decreased more than two units due to ferrous iron oxidation. Table 4.20 (c) pH of HM solution under different treatment conditions Initial pH After 4 weeks after 8 weeks after 12 weeks Treatment adding culturing culturing after CA reeds reeds culturing reeds HMNCNB 3.50±0.01 3.50±0.01 2.05±0.01 2.02±0.02 2.00±0.01 HMNCWB 3.51±0.01 3.51±0.02 1.98±0.02 1.95±0.01 1.93±0.00 HMLCNB 3.50±0.01 3.20±0.02 2.06±0.02 2.03±0.01 2.01±0.00 HMLCWB 3.50±0.01 3.20±0.01 2.00±0.01 1.97±0.01 1.95±0.00 HMMCNB 3.50±0.02 2.50±0.02 2.11±0.01 2.07±0.02 2.05±0.01 HMMCWB 3.50±0.02 2.50±0.00 2.08±0.02 2.04±0.00 2.02±0.02 HMHCNB 3.51±0.01 2.20±0.01 2.15±0.01 2.12±0.01 2.09±0.01 HMHCWB 3.51±0.02 2.20±0.02 2.13±0.01 2.08±0.00 2.05±0.01 The results were reported as average±standard deviation, n=3 The pH decrease trend of solution without adding CA or added with low level of CA were more dramatic than the solution added with middle or high level of CA. For example, the pH of LMNCNB solution decreased from 3.50±0.01 to 2.60±0.02, while 169 the pH of LMHCNB solution decreased from 2.79±0.01 to 2.73±0.01 after 4 weeks (Table 4.20a). Similar trends were observed for solution contained with middle and high level of CA. The pH of MMNCWB solution decreased from 3.50±0.02 to 2.04±0.01, while the pH of MMHCWB solution decreased from 2.70±0.02 to 2.18±0.01 after 4 weeks (Table 4.20b). The pH of HMLCNB solution decreased from 3.20±0.02 to 2.06±0.02, while the pH of HMMCNB solution decreased from 2.20±0.01 to 2.15±0.01 after 4 weeks (Table 4.20c). This may due to Fe(II) being able to form complex with CA and then less Fe2+ were oxidized into Fe3+. As such, the solution added with middle or high level of CA possessed higher pH than solution without adding CA. Küsel et al. (2003) indicated that the increased availability of carbon in the rhizosphere might cause the increase of pH during the vegetation period. Jones et al. (1996) also pointed out CA can cause the dissolution of Fe plaque, which may affect the pH of solution. The pH of solution did not change too much after 8 and 12 weeks. For example, the pH of LMMCNB solution only changed from 2.93±0.02 to 2.91±0.02; while the pH of LMLCNB solution changed from 2.49±0.05 to 2.47±0.01 after 8 and 12 weeks (Table 4.20a). The pH of MMNCNB solution changed from 2.12±0.02 to 2.10±0.01; while the pH of MMNCWB solution changed from 2.01±0.00 to 2.00±0.01 after 8 and 12 weeks (Table 4.20b). This may be related to the decrease of the Fe(II) oxidation rate. As reported by Neubauer et al. (2007), the rate of Fe(II) oxidation which followed first-order equation was fast in the first 4-5 weeks and then became relatively stable. The Fe(II) supply rate can also affect the Fe(II) oxidation rates 170 (Neubauer et al., 2007). The oxidation of Fe(II) and the subsequence hydrolysis to form oxide and or oxyhydroxides can generate protons to low the solution pH (Wieder et al., 1990). As the Fe(II) oxidation rate slowed down and the amounts of Fe(II) decreased, the pH of solution also decreased slowly. Since the pH of AMD is very low, it is essential to use plants that can survive in the acidic and hostile environment to clean AMD contaminated water or soil. Phragmites australis was reported to grow on the margins of lakes pH of 3 or below (Fyson, 2000), which make it a suitable candidate to restore AMD polluted media. 4.4.3 DCB extraction of reeds propagated from wild rhizome cultured in solution The concentrations of metal plaque formed on reeds cultured in solution with different levels of metals were analyzed. According to our previous study, metal plaque had already formed on reeds grown in the contaminated field (refer to section 4.1.4). In order to better assess the effect of CA and rhizosphere bacteria on the metal plaque formation (Figure 4.94-4.98), assessment of the metal plaque concentrations on reeds propagated from wild rhizome and cultured in metal solution did not include the background metal plaque concentrations shown in Table 4.21. Table 4.21 Background values of metal plaque on reeds propagated from wild rhizome Metal (mg/g) Plaque on roots Plaque on rhizome Mn 0.00±0.00 0.00±0.00 Fe 0.00±0.00 5.18±0.16 Al 0.00±0.00 0.01±0.00 Results were reported as average±standard deviation, n=3 171 The formation of Al phosphate was reported on the roots of some crop plants and wetland plants (Batty et al., 2002; Wheeler, 1994). In this study, Al plaque was not found on reeds cultured in LM, MM and HM solution. This may be related to the low concentrations of phosphate present in the solution, since previous study reported that the main component of Al plaque was Al phosphate (Batty et al., 2002). In addition, the insoluble Al oxyhydroxides can also not be formed under acidic conditions (Wieder et al., 1990). The pH of metal solution of this study were <3.5 (section 4.4.2), which may also inhibit the formation of Al plaque, since the mobility and bioavailability of Al were high in acidic water (Batty et al., 2002). There are extensive reports about the formation of Mn plaque on plants (Liu et al., 2010). However, Mn plaque was not found on the root system of reeds cultured in LM solution under all the treatment conditions during all growth periods. Nevertheless, Mn plaque was observed on the root system of reeds cultured in MM and HM solution, which was presented in Figure 4.94 and 4.95. This may be related to the Mn concentrations in solution. It was reported that Mn plaque was found on the roots of rice (Oryza sativa L.) and the Mn plaque concentrations increased with the Mn levels in solution (Batty et al., 2002; Crowder and Coltman, 1993).The Mn concentration was 0.02 g Mn/L, 0.04 g Mn/L and 0.05 g Mn/L in LM, MM and HM solution, respectively. So it was not surprising that Mn plaque were only found on the root system of reeds grown in MM and HM solution. 172 0.08 a 0.07 a a 0.06 a a a 0.05 b 0.04 b rhizomes 0.03 c roots 0.02 mg(Mn)/g(biomass) b 0.01 0.00 Control NCNB LCNB MCNB HCNB Solution 0.10 a 0.09 a a 0.08 a b 0.07 b b 0.06 c b 0.05 rhizomes 0.04 c roots 0.03 c mg(Mn)/g(biomass) 0.02 0.01 0.00 Control NCWB LCWB MCWB HCWB solution a b 0.14 a a a 0.12 a a a b a b a b b 0.10 c c b 0.08 c a b a rhizomes 0.06 b c c b roots 0.04 b mg(Mn)/g(biomass) 0.02 0.00 Clean NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB Soil Figure 4.94 Mn plaque on the roots and rhizomes of reeds cultured in MM solution for (a) 4 (b) 8 (c)12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 173 0.14 a a a 0.12 a 0.10 a 0.08 b rhizomes 0.06 b c roots 0.04 b mg(Mn)/g(biomass) 0.02 0.00 Control NCNB LCNB MCNB HCNB solution a 0.18 a a 0.16 a 0.14 0.12 b 0.10 b 0.08 b rhizomes b 0.06 b roots mg(Mn)/g(biomass) 0.04 0.02 0.00 Control NCWB LCWB MCWB HCWB solution 0.20 a a a a a 0.18 a a 0.16 a 0.14 b c b 0.12 b b 0.10 b rhizomes 0.08 b b roots 0.06 b mg(Mn)/g(biomass) 0.04 0.02 0.00 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.95 Mn plaque on the roots and rhizomes of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 174 The concentrations of Fe plaque on the reeds cultured under different conditions were showed in Figure 4.96 to 4.98. As expected, the amounts of Fe plaque were much higher than Mn plaque. Since the solution contained much more Fe than Mn. A ratio of 43:1 to 190:1 (Fe:Mn) in plaque on plants have been reported under laboratory conditions (Liu et al., 2005). This may also due to that Fe oxides precipitate at lower redox potentials than Mn oxides (Liu et al., 2005). Fe plaque has been commonly found on the root system of aquatic plants, such as Oryza sativa, Typha latifolia, Phragmites communis and Phragmites australis (Liu et al., 2004). As already mentioned, the aerenchyma in the roots of wetland roots can transfer oxygen into the substrate, which can oxidize Fe2+ to Fe3+ and then form Fe plaque (Crowder and Macfie, 1986) The oxides in Fe plaque often contained ferrihydrite, lepidocrocite and/or goethite (Weiss et al., 2004). Hansel and Fendorf (2001) also reported that Fe plaque contained ferrihydrite (ca. 63%), goethite (32%) siderite (5%) and that the Fe plaque formation can reach more than 10% root dry weight. Various factors such as the availability of Fe2+, the oxygenation capability of the root system, plant species, can affect the amounts of Fe plaque (Chen et al., 2008; Jiang et al., 2009). It was also reported that the extent of plaque formation depended on the concentrations of Fe2+ in solution (Taylor et al., 1984; Zhang et al., 1999). In this study, the amounts of Fe plaque on reeds cultured in HM and MM solution were also higher than those on reeds grown in LM solution (Figure 4.96 and 4.97). 175 8 a a 6 a a a 4 rhizomes b b b b roots mg(Fe)/g(biomass) 2 0 Control solution NCNB LCNB MCNB HCNB 8 a a a a 6 b 4 rhizomes b b b b roots mg(Fe)/g(biomass) 2 0 Control solution NCWB LCWB MCWB HCWB 8 a a a a 7 b a 6 a b a 5 c 4 rhizomes 3 b c c b c b c b roots mg(Fe)/g(biomass) 2 1 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.96 Fe plaque on the roots and rhizomes of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 176 30 a a 25 a a 20 a a b b 15 b rhizomes b 10 roots mg(Fe)/g(biomass) 5 0 Control solution NCNB LCNB MCNB HCNB 50 a a b 40 a a b c 30 b b c c rhizomes 20 roots mg(Fe)/g(biomass) 10 0 Control solution NCWB LCWB MCWB HCWB a 50 a a a a 40 b b b b c b c b b c b c c 30 c c d b d c d rhizomes 20 c roots mg(Fe)/g(biomass) 10 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.97 Fe plaque on the roots and rhizomes of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 177 50 a a a 40 a a 30 b b b b rhizomes 20 roots mg(Fe)/g(biomass) 10 0 Control solution NCNB LCNB MCNB HCNB a a b b a a 90 b b 80 70 60 b b 50 b 40 rhizomes c b 30 roots mg(Fe)/g(biomass) 20 10 0 Control solution NCWB LCWB MCWB HCWB a a a b b a b 90 b 80 70 b 60 b b b b b c 50 c b b 40 c c c rhizomes c c 30 roots mg(Fe)/g(biomass) 20 10 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.98 Fe plaque on the roots and rhizomes of reeds cultured in HM solution (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 178 It was not surprising that the rhizosphere bacteria played a significant role in enhancing the formation of Fe plaque (p<0.05). For example, the rhizome Fe plaque on reeds grown in solution LMLCNB was 4.63±0.36 mg/g, which was significantly lower (p<0.05) than that in solution LMLCWB for 12 weeks (6.03±0.57 mg/g) (Figure 4.96). The rhizome Fe plaque on reeds grown in solution MMNCNB was 27.17±4.32 mg/g, which was significantly lower (p<0.05) than that in solution MMNCWB for 12 weeks (36.55±3.11 mg/g) (Figure 4.97). Similarly, the root Fe plaque on reeds grown in solution HMNCNB (45.56±3.93 mg/g) was significantly lower (p<0.05) than that in solution HMNCWB for 12 weeks (81.47±5.16 mg/g) (Figure 4.98). Extensive reports indicated that rhizosphere was an ideal oxic-anoxic interface for the growth of Fe(II)OB which played a key role in mediating Fe(II) oxidation and plaque formation (Weiss et al., 2004). Vesk et al. (1999) also indicated that Fe plaque formation was associated with the oxidizing activity of the plant roots and associated microorganisms. Fe(II)OB in the rhizosphere of plants may directly influence plaque formation (Chen et al., 2008). However, the rhizosphere bacteria did not appear to have an important role in forming Mn plaque (p<0.05). The amounts of Mn plaque formed on the reeds treated with rhizosphere bacteria were not significantly different from those on reeds without inoculation bacteria. For example, the root Mn plaque on reeds cultured in solution MMNCNB was 0.08±0.02 mg/g, which was similar to that in solution MMNCWB for 12 weeks (0.09±0.02 mg/g) (Figure 4.94). The rhizome Mn plaque on reeds grown in 179 solution HMNCNB (0.16±0.02 mg/g) was not significantly different from that in solution HMNCWB for 12 weeks (0.15±0.02 mg/g) (Figure 4.95). This may due to that Mn plaque can not formed by microorganism under acidic conditions (Nealson et al., 1988). Fe was the main element and Mn was the secondary metal in plaque, since Mn plaque was mainly formed by co-precipitation with or adsorption by Fe plaque (Liu et al., 2005). CA played a significant role to decrease the formation of both Mn and Fe plaque on the root system of reeds (p<0.05). The Mn plaque on the roots and rhizomes of reeds grown in solution MMLCNB were 0.05±0.00 mg/g and 0.05±0.01 mg/g, which were significantly higher (p<0.05) than those on reeds cultured in solution MMHCNB for 4 weeks (0.01±0.00 mg/g and 0.01±0.01 mg/g). The Mn plaque on the roots and rhizomes of reeds cultured in HMNCWB for 8 weeks were 0.15±0.02 mg/g and 0.14±0.00 mg/g (Figure 4.95). They were significantly higher (p<0.05) than the Mn plaque amounts on reeds cultured in solution HMMCWB for 8 weeks, which were (0.06±0.01 mg/g and 0.05±0.01 mg/g). Similar for Fe plaque, the roots and rhizomes grown in solution that contained middle and high level of CA formed less Fe plaque than those cultured in solution treated with low level of CA or without CA. The Fe plaque on the roots and rhizomes of reeds grown in solution LMLCNB were 4.77±0.49 mg/g and 4.43±0.83 mg/g, which were significantly higher (p<0.05) than those on reeds cultured in solution LMMCNB for 4 weeks (Figure 4.96). The Fe plaque on the roots and rhizomes of reeds grown in solution MMLCNB were 19.64±2.76 mg/g and 19.41±1.31 mg/g, 180 which were significantly higher (p<0.05) than those on reeds cultured in solution MMMCNB for 4 weeks (12.05±1.59 mg/g and 12.06±1.25 mg/g). It was not novel to find that CA can increase metal mobility, such as Mn by chelation and/or decreasing pH (Karathanasis and Johnson, 2003; Crowder and Coltman, 1993). Citrate also had a high affinity for Fe and can also increase Fe solubility by forming complex (Jones, 1998). According to the results of Taylor et al. (1984), the supply of Fe such as Fe3+, Fe3+ chelate or Fe2+ chelate can effectively reduce the extent of plaque formation. Organic acids can also rapidly release Fe held in goethite and ferrihydrite (Jones et al., 1996), which may limit the formation of Fe plaque. Besides, as discussed in section of rhizosphere bacteria (section 4.4.1), CA can inhibit the growth of Fe(II)OB and then further decrease the formation of Fe plaque. The amounts of Fe plaque and Mn plaque increased with time. However, most of Fe and Mn plaque were formed in the first 4 or 8 weeks. For instance, the root Fe plaque concentration on reeds cultured in solution LMNCNB for 4 weeks was 4.98±0.65 mg/g, which was similar to that on reeds in solution LMNCNB for 12 weeks (5.08±0.88 mg/g). The rhizome Fe plaque concentration on reeds cultured in solution MMHCWB for 8 weeks was 15.67±1.91 mg/g, while that on rhizome in solution MMHCWB for 12 weeks were 20.23±2.85 mg/g (Figure 4.97). The amounts of root Mn plaque on reeds cultured in solution HMHCNB for 4 weeks (0.02±0.00 mg/g) was similar to the root Mn plaque on reeds in solution HMHCNB for 12 weeks (0.04±0.00 mg/g). As discussed in section 4.4.2, the biological Fe oxidation rate can be expressed in first-order equation (Senko et al., 2008). Neubauer et al. (2007) also 181 indicated that Fe(II) oxidation on Juncus effusus roots were fast in the first 5 weeks. Further, Mn plaque formation was positively correlated with Fe plaque (Liu et al., 2005). That may explain why most of Fe and Mn plaque were formed in the first 4 or 8 weeks. Based on the stability constants of complexes between metals and ligands and the methods provided by Morel and Hering (1994), the concentrations of free metal, inorganic and organically complexed metal in the hydroponic solution were calculated and shown in Table 4.22 to 4.24. According to the results, no Mn, Fe and Al citrate complex would be formed in LM solution added with low level of CA (0.021g CA/L). Thus the low level of CA did not have an effect to reduce the plaque formation in LM solution. When middle and high level of CA (1.061 g CA/L and 2.101 g CA/L) was added into the LM solution, most of Fe was complexed with phosphate and citrate (50% each) therefore there were less free Fe2+ to form Fe plaque. Fe plaque on wetland plants was found to be predominantly from Fe3+ and small amounts of free Fe2+ (Ye et al., 1997b). These findings were in accord with our results of plaque extraction (section 4.4.3) that much lower Fe plaque was formed on the roots of reeds cultured in solution added with middle and high level of CA than reeds grown in LM solution without adding CA or added with low level of CA. According to the calculation, CA can also effectively form complex with Mn and Fe in MM and HM solution. With the increase of CA, more Mn and Fe citrate complex could be formed. It further proved that our results of plaque extraction (section 4.4.3) that CA can significantly reduce the formation of metal plaque by forming citrate complex. 182 As shown in Table 4.22 to 4.24, most of phosphate formed complexes with Fe metal solution, but not Al. As mentioned above, the main components of Al plaque were Al-phosphate (Batty et al., 2002). So it was not surprising that no Al plaque were found on the root system of reeds cultured in solution with different levels of metals. Table 4.22(a) Concentrations of free inorganic and organically complexed Fe in LM solution 2+ Metal Free Fe FeSO4 Fe-phosphate Fe-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.048 12.1% 0.151 38.1% 0.197 49.7% 0.00 0.0% 0.021 0.048 12.1% 0.151 38.1% 0.197 49.7% 0.00 0.0% 0.181 0.00 0.0% 0.00 0.0% 0.198 50.0% 0.198 50.0% 0.343 0.00 0.0% 0.00 0.0% 0.198 50.0% 0.198 50.0% Table 4.22(b) Concentrations of free inorganic and organically complexed Mn in LM solution 2+ Metal Free Mn MnSO4 Mn-phosphate Mn-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.02 100.% 0.00 0.0% 0.0% 0.0% 0.00 0.0% 0.021 0.02 100.0% 0.00 0.0% 0.00 0.0% 0.00 0.0% 0.181 0.00 0.0% 0.00 0.0% 0.00 0.0% 0.02 100.0% 0.343 0.00 0.0% 0.00 0.0% 0.00 0.0% 0.02 100.0% Table 4.22(c) Concentrations of free inorganic and organically complexed Al in LM solution 3+ Metal Free Al Al2(SO4)3 Al-phosphate Al-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.00 0.0% 0.03 100.0% 0.00 0.0% 0.00 0.0% 0.021 0.00 0.0% 0.03 100.0% 0.00 0.0% 0.00 0.0% 0.181 0.00 0.0% 0.00 0.0% 0.00 0.0% 0.03 100.0% 0.343 0.00 0.0% 0.00 0.0% 0.00 0.0% 0.03 100.0% 183 Table 4.23(a) Concentrations of free inorganic and organically complexed Fe in MM solution 2+ Metal Free Fe FeSO4 Fe-phosphate Fe-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.31 1.43% 21.15 97.65% 0.20 0.92% 0.00 0.00% 1.061 0.30 1.38% 21.14 97.55% 0.19 0.88% 0.04 0.18% 9.020 0.27 1.24% 18.84 86.86% 0.19 0.88% 2.39 11.02% 17.859 0.24 1.11% 16.41 75.69% 0.19 0.88% 4.84 22.32% Table 4.23(b) Concentrations of free inorganic and organically complexed Mn in MM solution 2+ Metal Free Mn MnSO4 Mn-phosphate Mn-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.013 36.11% 0.023 63.89% 0.0% 0.0% 0.00 0.0% 1.061 0.013 36.11% 0.023 63.89% 0.00 0.0% 0.00 0.0% 9.020 0.007 18.92% 0.011 29.73% 0.00 0.0% 0.019 51.35% 17.859 0.004 11.11% 0.007 19.44% 0.00 0.0% 0.025 69.44% Table 4.23(c) Concentrations of free inorganic and organically complexed Al in MM solution 3+ Metal Free Al Al2(SO4)3 Al-phosphate Al-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.00 0.0% 0.15 100.0% 0.00 0.0% 0.00 0.0% 1.061 0.00 0.0% 0.15 100.0% 0.00 0.0% 0.00 0.0% 9.020 0.00 0.0% 0.15 100.0% 0.00 0.0% 0.00 0.0% 17.859 0.00 0.0% 0.15 100.0% 0.00 0.0% 0.00 0.0% Table 4.24(a) Concentrations of free inorganic and organically complexed Fe in HM solution 2+ Metal Free Fe FeSO4 Fe-phosphate Fe-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.32 0.74% 42.48 98.79% 0.20 0.47% 0.00 0.00% 2.101 0.32 0.75% 42.20 98.56% 0.20 0.47% 0.10 0.23% 16.979 0.29 0.68% 37.86 88.25% 0.20 0.47% 4.55 10.61% 33.616 0.25 0.58% 33.51 78.08% 0.20 0.47% 8.96 20.88% 184 Table 4.24(b) Concentrations of free inorganic and organically complexed Mn in HM solution 2+ Metal Free Mn MnSO4 Mn-phosphate Mn-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.011 21.57% 0.040 78.43% 0.0% 0.0% 0.00 0.0% 2.101 0.011 21.57% 0.039 76.47% 0.00 0.0% 0.001 1.96% 16.979 0.005 9.80% 0.018 35.29% 0.00 0.0% 0.028 54.90% 33.616 0.003 5.77% 0.011 21.15% 0.00 0.0% 0.038 73.08% Table 4.24(c) Concentrations of free inorganic and organically complexed Al in HM solution 3+ Metal Free Al Al2(SO4)3 Al-phosphate Al-citrate complex complex CA (g/L) percent (g/L) percent (g/L) percent (g/L) percent 0.00 0.00 0.0% 0.27 100.0% 0.00 0.0% 0.00 0.0% 2.101 0.00 0.0% 0.27 100.0% 0.00 0.0% 0.00 0.0% 16.979 0.00 0.0% 0.27 100.0% 0.00 0.0% 0.00 0.0% 33.616 0.00 0.0% 0.27 100.0% 0.00 0.0% 0.00 0.0% 4.4.4 Digestion of reeds propagated from wild rhizome cultured in solution Since the seedlings of reeds were propagated from contaminated rhizomes, Fe, Mn and Al were already contained in the biomass. In order to better assess the uptake of metals in reeds cultured in hydroponic solution, the concentrations of Mn, Fe and Al in reeds shown in Figure 4.99 to 4.107 did not include the background metals presented in Table 4.25. Table 4.25 Background values of metal accumulations in reeds propagated from wild rhizomes Metal Metal in Metal in Metal in Metal in (mg/g) roots rhizomes stems leaves Mn 0.02±0.00 0.02±0.01 0.01±0.01 0.01±0.00 Fe 0.99±0.18 1.36±0.32 0.44±0.08 0.29±0.03 Al 0.03±0.01 0.06±0.02 0.02±0.00 0.02±0.00 Results were reported as average±standard deviation, n=3 185 0.05 a a a a 0.04 0.03 a b a a a a a a rhizomes 0.02 roots b b b b b stems mg(Mn)/g(biomass) 0.01 leaves 0 Control solution NCNB LCNB MCNB HCNB 0.1 0.08 a a b 0.06 a b a b a a rhizomes 0.04 b a roots b b a a stems mg(Mn)/g(biomass) 0.02 a a a a leaves 0 Control solution NCWB LCWB MCWB HCWB 0.12 a a 0.1 a b a a a a a a b b b c c c a 0.08 a a a c b a b b a 0.06 c a b a b a a rhizomes c a c b a a a a a roots 0.04 a a b b b b b b b stems mg(Mn)/g(biomass) a a 0.02 b b leaves 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.99 Mn concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 186 0.06 a a a 0.05 0.04 a a a b b 0.03 a rhizomes b b b a a roots 0.02 b a a b c b stems mg(Mn)/g(biomass) b 0.01 leaves 0 Control solution NCNB LCNB MCNB HCNB 0.1 0.08 a b 0.06 b a a a b a a a rhizomes 0.04 roots a a b b b stems mg(Mn)/g(biomass) a 0.02 b b b b leaves 0 Control solution NCWB LCWB MCWB HCWB a 0.12 a b a a 0.1 b b a b c b a 0.08 b a c c a c a a b c b c a 0.06 c b b b rhizomes d c d d c c c c b b roots 0.04 b d d c c d c e d e stems mg(Mn)/g(biomass) c 0.02 c e leaves 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.100 Mn concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 187 0.09 a a 0.08 b 0.07 b a a 0.06 b a 0.05 c b c a a rhizomes 0.04 a roots 0.03 b b stems mg(Mn)/g(biomass) 0.02 b b b b leaves 0.01 0 Control solution NCNB LCNB MCNB HCNB a a 0.14 a 0.12 b 0.1 b 0.08 rhizomes c 0.06 b b a a roots c 0.04 a stems mg(Mn)/g(biomass) b b leaves 0.02 b c b c 0 Control solution NCWB LCWB MCWB HCWB a 0.2 a a b 0.18 a a a a 0.16 b b b c 0.14 c c b d b d c 0.12 0.1 a a rhizomes d b d a a a b 0.08 b b a b a b a roots b c b b b b c b 0.06 c c c c b c stems mg(Mn)/g(biomass) c c c 0.04 leaves 0.02 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.101: Mn concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 188 2 a a 1.5 a a a 1 b rhizomes b roots b b stems mg(Fe)/g(biomass) 0.5 leaves a b a b a a a a 0 Control solution NCNB LCNB MCNB HCNB 3 a 2.5 a 2 b a rhizomes 1.5 a b b b roots b b 1 stems mg(Fe)/g(biomass) leaves 0.5 a a a b b b b a 0 Control solution NCWB LCWB MCWB HCNB a 3 b a a a b b 2.5 c 2 a a b b a a a rhizomes 1.5 b b a a b roots b b b 1 stems mg(Fe)/g(biomass) a a a a a leaves 0.5 b b b b b b b b a a a 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.102 Fe concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 189 20 a a 15 b a a b a 10 b rhizomes b roots b stems mg(Fe)/g(biomass) 5 leaves c c c c b b a a 0 Control solution NCNB LCNB MCNB HCNB 20 a 15 b b b a 10 c rhizomes c b roots c c stems mg(Fe)/g(biomass) 5 leaves a a c c c c b b 0 Control solution NCWB LCWB MCWB HCWB 50 a 40 a a b b a c b b b 30 b c b c c rhizomes c c c d c d 20 e e roots f f stems mg(Fe)/g(biomass) 10 d a leaves d d e d d d e d b b c c a b b 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.103 Fe concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 190 40 a 35 a 30 a b a 25 b b 20 c c rhizomes roots 15 stems mg(Fe)/g(biomass) 10 leaves b a a 5 c c c c b 0 Control solution NCNB LCNB MCNB HCNB 60 a 50 b b 40 a rhizomes 30 b c c roots 20 c c stems mg(Fe)/g(biomass) leaves 10 b a c c c c b a 0 Control solution NCWB LCWB MCWB HCWB 70 a a a 60 b b c b a c c d 50 d b b c d e e 40 c c rhizomes d e d e 30 b roots e e 20 stems mg(Fe)/g(biomass) b a a leaves 10 d d e e d d e e b c c a b 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.104 Fe concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 191 0.15 a a a a 0.1 a rhizomes b b a a a a b b roots 0.05 b stems mg(Al)/g(biomass) b b b leaves 0 Control solution NCNB LCNB MCNB HCNB 0.25 a a a a 0.2 b b 0.15 b b a a b a a rhizomes b a b 0.1 b roots a b stems mg(Al)/g(biomass) b b a 0.05 b leaves 0 Control solution NCWB LCWB MCWB HCNB a b a 0.3 b a a c a b 0.25 b c a c a a c d c 0.2 d d d a a a a a rhizomes 0.15 a a b a a a a a roots a a b 0.1 a b a b b b stems mg(Al)/g(biomass) 0.05 leaves 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.105 Al concentration in the organs of reeds cultured in LM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 192 0.2 a a a a a 0.15 a a a a 0.1 rhizomes a a a roots a a a a stems mg(Al)/g(biomass) 0.05 a leaves 0 Control solution NCNB LCNB MCNB HCNB 0.3 a a a a 0.25 a a a a 0.2 b 0.15 rhizomes a a a a roots a a a 0.1 a stems mg(Al)/g(biomass) leaves 0.05 0 Control solution NCWB LCWB MCWB HCWB 0.4 a a a a 0.35 a a a a 0.3 a a a a a c 0.25 a a a rhizomes a 0.2 a a a a a a a a a a a a a a a roots 0.15 stems mg(Al)/g(biomass) 0.1 leaves 0.05 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.106 Al concentration in the organs of reeds cultured in MM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 193 0.4 0.35 a a a a 0.3 a a a a a 0.25 0.2 rhizomes roots 0.15 a a a a a a a a stems mg(Al)/g(biomass) 0.1 leaves 0.05 0 Control solution NCNB LCNB MCNB HCNB 0.6 a a a a 0.5 a a a a 0.4 b 0.3 rhizomes a roots a a a a a 0.2 a a stems mg(Al)/g(biomass) leaves 0.1 0 Control solution NCWB LCWB MCWB HCWB 0.8 0.7 a a a a a a a a a a a a a a a 0.6 a c 0.5 rhizomes 0.4 roots 0.3 a a a a a a a a a a a a a a a a stems mg(Al)/g(biomass) 0.2 leaves 0.1 0 Control NCNB NCWB LCNB LCWB MCNB MCWB HCNB HCWB solution Figure 4.107 Al concentration in the organs of reeds cultured in HM solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 194 Mn, Fe and Al were found in the biomass of reeds cultured in solution with different levels of metals. As expected, more metals were accumulated in reeds cultured in MM and HM solution than those in reeds grown in LM solution. For instance, the roots of reeds grown in LMNCNB solution for 12 weeks accumulated 0.04±0.01 mg/g Mn, 1.14±0.19 mg/g Fe and 0.09±0.01 mg/g Al while the roots in HMNCNB uptake 0.10±0.01 mg/g Mn, 31.75±2.37 mg/g Fe and 0.60±0.03 mg/g Al after 12 weeks. The stems of reeds grown in LMNCNB solution accumulated 0.01±0.01 mg/g Mn, 0.12±0.01 mg/g Fe and 0.05±0.00 mg/g Al while the stems in HMNCNB uptake 0.03±0.01 mg/g Mn, 2.50±0.02 mg/g Fe and 0.18±0.01 mg/g Al after 12 weeks. According to the results of Gramlich et al. (2013), increased supply of Zn can enhance the Zn uptake by wheat. Taylor et al. (1984) also indicated that accumulation of metals such as Fe and Mn in all plant tissues was correlated with concentrations in substrate. CA played a significant role in enhancing the Mn accumulation and translocation in reeds cultured in all the solutions (p<0.05). For example, the rhizomes cultured in LMMCNB for 4 weeks contained 0.03±0.01 mg/g Mn which was significantly higher (p<0.05) than that in the rhizomes grown in LMNCNB. The Mn concentrations in the stems cultured in LMHCNB for 12 weeks was 0.04±0.00 mg/g which was significantly higher (p<0.05) than that in the stems grown in LMLCNB for 12 weeks (0.01±0.01 mg/g). The Mn accumulated in rhizomes cultured in MMHCWB for 8 weeks (0.04±0.01 mg/g) was significantly higher (p<0.05) than that in the rhizomes grown in MMNCWB for the same time period (0.01±0.00 mg/g). The Mn 195 concentrations in the leaves cultured in HMHCNB for 12 weeks was 0.08±0.00 mg/g, which was significantly higher (p<0.05) than that in leaves grown in HMLCNB for 12 weeks (0.04±0.00 mg/g). According to the results of Najeeb et al. (2009), significant enhancement in Mn accumulation and translocation in J. effusus plants cultured in Mn hydroponic solution was recorded under the application of CA. The elevated Mn translocation caused by chelator may related to the activation of ATPase in the plasma membrane which can produce and then increase the translocation of both essential and non-essential metals (Najeeb et al., 2009; Williams et al., 2000). It was also reported that CA can enhance metal mobility by formation of metal-chelator complex or change of binding sites (Wang et al., 2009). Furthermore, CA may alleviate the toxic effects of Mn to plants which enable to increase the accumulation of Mn in plants (Najeeb et al., 2009). According to the calculation results in Table 4.22 to 4.24, more Mn-citrate complexes were formed in solution amended with more CA. Therefore it was not surprising that Mn accumulation were higher in reeds cultured in solutions added with middle and high level of CA than that in solutions with low or without CA. CA also played an important role in increasing the accumulation of Fe in both underground and above ground tissues of reeds during all the growth period (p<0.05). For example, the Fe levels in roots of reeds grown in solution LMHCWB for 8 weeks were 2.06±0.24 mg/g, which was significantly higher (p<0.05) than that in roots cultured in solution LMNCWB for the same period (0.90±0.11 mg/g). The stems of reeds grown in solution LMMCNB for 12 weeks contained 0.26±0.03 mg/g Fe. It was 196 significantly higher (p<0.05) than the Fe levels in stems cultured in solution LMNCNB for the same period, which was 0.12±0.01 mg/g. The Fe amounts in rhizomes of reeds grown in solution MMHCNB for 4 weeks were 9.49±0.80 mg/g, which was significantly higher (p<0.05) than that in rhizomes cultured in solution MMNCNB for the same period (4.40±0.44 mg/g). The leaves of reeds grown in solution HMMCWB for 12 weeks contained 2.41±0.04 mg/g Fe. It was significantly higher (p<0.05) than the Fe concentrations in leaves of reeds grown in solution HMNCWB for 12 weeks (1.46±0.08 mg/g). It was reported that metal may become more or less available to plants by interactions with organic matter, depending upon the molecular weight of the metal-chelate complexes, the surrounding pH, etc.(Macfie and Crowder, 1987). Extensive research demonstrated that CA can increase the bioavailability of metals and then enhance the metal accumulation in plants (Duarte et al., 2011; Kim and Lee, 2010). For example, the accumulation of Cd in the wheat roots was doubled by citrate compared to citrate-free treatments (Gramlich et al., 2013). Organic ligands such as citrate may enhance metal bioavailability by increasing the flux of metals to the root cell membranes or forming transient ternary complexes with the biotic ligands catalyzing transmembrane transfer (Gramlich et al., 2013). Citrate can maintain Fe in a soluble form by forming of stable plant-available organic-Fe3+ complexes at low pH value (<6.8) (Jones et al., 1996). CA also significantly elevated (p<0.05) the uptake of Al in different tissues of reeds grown in LM solution. For instance, the Al levels in rhizomes of reeds grown in 197 solution LMMCNB for 4 weeks was 0.09±0.02 mg/g, which was significantly higher (p<0.05) than that in rhizomes cultured in solution LMNCNB (0.05±0.01 mg/g). The leaves of reeds grown in solution LMHCNB for 12 weeks contained 0.15±0.02 mg/g Al, which was significantly higher (p<0.05) than the Al levels in leaves cultured in solution LMNCNB for the same period. According to the results in Table 4.22, citrate complexes were formed when middle and high level of CA were added into LM solution. Previous research found that Al-organic acid complexes which were less toxic than Al (Jones, 1998) may make the plants be tolerant to Al toxicity and enable more Al uptake into biomass. White et al. (2003) indicated that CA can increase the aqueous concentration of Fe, Al and Mn in soil. Boyle et al. (1967) also indicated that CA increased desorption of inorganic constituents such as Mn, Al in soil. It may elevate the mobility and bioavailability of metals to plants and then further promote the metal accumulation by plants. However, CA did not have important effect (p>0.05) in Al accumulation in different tissues of reeds cultured in MM and HM solution. For example, the Al levels in roots of reeds grown in solution MMHCWB for 8 weeks was 0.22±0.01 mg/g, which was similar to (p>0.05) to that in rhizomes cultured in solution MMNCWB (0.20±0.02 mg/g) for the same time period. The stems of reeds grown in solution HMHCNB for 12 weeks contained 0.19±0.01 mg/g Al. It was not significantly different (p>0.05) from that in stems cultured in solution HMNCNB for 12 weeks (0.18±0.01 mg/g). According to our calculation results in Table 4.22 and 4.24, most of CA reacted with Fe in MM and HM solution and then no citrate-Al complex were 198 formed. It was reported that the extent of complexation between metals and organic acid was related to the type of organic acid involved, the concentration of metals and the pH of solution, etc. (Jones, 1998). Since CA had higher affinity for Fe and Mn and the Fe concentrations were higher than Al, most CA reacted with Fe and Mn but not Al in MM and HM solution. Most Al still existed in the form of Al2(SO4)3. That may be the main reason why CA can no improve the Al uptake in reeds cultured in MM and HM solution. The rhizosphere bacteria did not have a significant effect on (p>0.05) the accumulation of Fe and Mn in reeds cultured in LM solution. However, the rhizosphere bacteria did have a significant role (p<0.05) in decreasing the accumulation of Fe and Mn in reeds cultured in MM and HM solution. For example, the roots grown in solution LMNCNB for 12 weeks uptake 1.14±0.19 mg/g Fe, which was not significantly different (p>0.05) from the Fe levels in roots cultured in solution LMNCWB for the same period (1.07±0.20 mg/g). The Fe concentrations in stems of reeds cultured in solution LMLCNB for 12 weeks were 0.11±0.01 mg/g, which was similar (p>0.05) to that in stems of reeds grown in solution LMLCWB. Similar trends were found for Mn in LM solution. The Mn concentrations in roots grown in solution LMHCNB for 12 weeks was 0.08±0.01 mg/g, which was not significantly different (p>0.05) from the Mn levels in roots cultured in solution LMHCWB for the same period (0.07±0.02 mg/g). The Mn concentrations in leaves of reeds cultured in solution LMMCNB for 12 weeks were 0.05±0.00 mg/g, which was similar (p>0.05) to that in leaves of reeds grown in solution LMMCWB (0.05±0.01 mg/g). However, 199 the Fe concentrations in rhizomes grown in solution MMMCNB for 12 weeks was 23.37±1.04 mg/g, which was significantly higher (p<0.05) from the Fe levels in rhizomes cultured in solution MMMCWB for the same period (18.27±1.38 mg/g). The Mn concentrations in rhizomes grown in solution HMNCNB for 12 weeks was (0.10±0.01 mg/g), which was significantly different (p<0.05) from the Mn levels in rhizomes cultured in solution HMNCWB for 12 weeks (0.05±0.01 mg/g). The rhizosphere bacteria mediated the formation of Fe plaque which may influence metal uptake in plants. Since plaque may sequester metals by adsorption or co-precipitation, and thus affect the availability of these elements and their accumulation and translocation in plants (Liu et al., 2010). Previous research indicated that Fe plaque may act as a ‘barrier’ for arsenate, leading to a lower influx into root cells, and then dramatically decrease arsenate accumulation in Oryza sativa (Chen et al., 2005). Greipsson and Crowder (1992) also found that the concentrations of Cu in the leaves were significantly lower in Oryza sativa with than that without Fe plaque. However, other researchers indicated that Fe plaque can increases the accumulation of essential and nonessential metals (Zhang et al., 1998). The overall effect of Fe plaque on the uptake of elements in plants may be related to the plant species, the amounts of plaque and the type and concentrations of cations and anions (Liu et al., 2004; Jiang et al., 2009). High amounts of metal plaque may inhibit the Fe and Mn accumulation in plants while low concentrations of plaque may not influence the elements uptake in plants. 200 However, the rhizosphere bacteria did not have important influence (p>0.05) on the Al uptake in reeds cultured in solution of different levels of metals. For example, the rhizomes grown in solution LMLCNB for 12 weeks accumulated 0.82±0.08 mg/g Al, which was not significantly different (p>0.05) from the Al levels in rhizomes cultured in solution LMLCWB for the same period (0.79±0.08 mg/g). The Al concentrations in stems of reeds cultured in solution LMMCNB for 12 weeks were 0.26±0.03 mg/g, which was similar to that in stems of reeds grown in solution LMMCWB. The roots grown in solution MMNCNB for 12 weeks accumulated 0.30±0.01 mg/g Al, which was not significantly different (p>0.05) from the Al levels in roots cultured in solution MMNCWB for the same period (0.30±0.01 mg/g). The Al concentration in leaves of reeds cultured in solution HMMCNB (0.18±0.00 mg/g) was similar to (p>0.05) that in leaves of reeds grown in solution HMMCWB for 12 weeks (0.19±0.02 mg/g). These findings were in agreement with the results of Batty et al. (2002) who found that no significant differences were found in Al concentrations in roots and shoots of reeds with or without Al, Fe and Mn plaque. Chen et al. (2006) also indicated that Al in the roots of Oryza sativa was not influenced by Fe plaque but Al in the shoots was reduced by Fe plaque. Many factors, such as age of root, concentrations of metal and pH of solution can influence the ability of metal plaque to inhibit or enhance elements uptake by plants (Ye et al., 1997a). Especially pH was one of the important factors for controlling uptake of metals (Chen et al., 2006). In our study, the pH of solution with different levels of metals was < 3.5 (secion 4.4.2) and 201 the solubility of Al was high under acidic conditions (Karathanasis and Johnson, 2003). That may inhibit or reduce influence of metal plaque on Al uptake in plants. The shoot concentrations and translocation factors of metals in reeds were calculated and shown in Table 4.26. The shoot concentrations of Fe were higher than Mn and Al. This may due to that the solution contained more Fe than Mn and Al. However, the translocation factors for Fe were less than Al and Mn. This may due to the different transfer ability of metals. For example, Cu, Mn, and Zn were classified as elements of intermediate mobility, Mg as phloemmobile, and Ca as phloem immobile (Taylor and Crowder, 1983b). Plants also possess different uptake and translocation mechanisms for different metals (Duman et al., 2007). In addition, the mobility and translocation of metals in plants depended on many environmental factors, such as pH, redox potential of soil or solution (Ye et al., 1998; Stoltz and Grege, 2005). Other factors, such as soil particle size, organic matter content, nutrients, and the presence of other ions may also affect metal accumulation and translocation in plants (Yang and Ye, 2009). The translocation factors of all these metals in reeds cultured in LM, MM and HM solution were all less than 1, which indicated that the below ground organs were the main site to store these metals. Baldantoni et al. (2009) found that Zn showed higher concentrations in leaves than roots of Phragmites australis, while the translocation of Mn and Fe were low which were mainly stored in roots and rhizomes. Ali et al. (2002) also indicated that the translocation of Cu and Fe to the shoots of reeds were low. 202 Table 4.26(a) Shoot concentration and translocation factor of metals in reeds cultured in LM solution for 4, 8 and 12 weeks Shoot Shoot Shoot Treat Week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.01±.00 0.03±.00 0.02±.00 0.28±.07 0.04±.01 0.45±.07 4 a a a a a a NCNB 0.01±.00 0.08±.01 0.05±.01 0.36±.08 0.08±.02 0.55±.15 12 c b a a a a 0.01±.00 0.04±.01 0.03±.00 0.13±.01 0.08±.02 0.25±.03 8 a a a a a a NCWB 0.02±.01 0.08±.01 0.06±.00 0.40±.14 0.08±.01 0.55±.04 12 bc b a a a a 0.01±.00 0.03±.00 0.02±.00 0.27±.06 0.05±.00 0.44±.04 4 a a a a a a LCNB 0.02±.00 0.09±.00 0.06±.00 0.42±.18 0.08±.02 0.53±.01 12 bc b a a a a 0.01±.00 0.05±.01 0.03±.00 0.21±.01 0.05±.01 0.46±.05 8 a a a a a a LCWB 0.02±.01 0.08±.01 0.05±.00 0.43±.16 0.08±.00 0.57±.02 12 bc b a a a a 0.02±.00 0.07±.00 0.05±.00 0.52±.08 0.05±.01 0.48±.03 4 b b b a a a MCNB 0.04±.00 0.20±.03 0.10±.00 0.52±.11 0.09±.00 0.57±.03 12 a a b a a a 0.02±.00 0.13±.00 0.08±.00 0.40±.01 0.06±.01 0.51±.04 8 b b b a a a MCWB 0.04±.00 0.21±.00 0.11±.02 0.59±.11 0.09±.00 0.58±.11 12 a a b a a a 0.02±.00 0.07±.00 0.06±.00 0.54±.00 0.05±.00 0.48±.06 4 b b b a a a HCNB 0.04±.00 0.20±.01 0.11±.00 0.57±.06 0.09±.01 0.62±.08 12 a a b a a a 0.02±.00 0.13±.01 0.09±.01 0.39±.03 0.06±.00 0.56±.09 8 b b b a a a HCWB 0.04±.00 0.22±.00 0.10±.01 0.57±.12 0.09±.01 0.58±.11 12 a a b a a a / indicated blow detection limits; results were average±standard deviation, n=3. 203 Table 4.26(b) Shoot concentration and translocation factor of metals in reeds cultured in MM solution for 4, 8 and 12 weeks Shoot Shoot Shoot Treat Week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.00±.00 0.21±.02 0.05±.00 0.12±.07 0.03±.00 0.31±.00 4 b c a a a a NCNB 0.02±.00 1.07±.02 0.13±.01 0.45±.07 0.05±.00 0.42±.03 12 cd d a a a a 0.00±.00 0.33±.00 0.08±.01 0.29±.04 0.04±.00 0.35±.04 8 b c a a a a NCWB 0.01±.00 0.80±.05 0.14±.02 0.51±.17 0.05±.01 0.42±.00 12 d e a a a a 0.01±.00 0.23±.01 0.05±.01 0.25±.02 0.03±.00 0.30±.00 4 b c a a a a LCNB 0.02±.01 1.12±.01 0.13±.00 0.47±.02 0.05±.00 0.42±.04 12 d d a a a a 0.01±.00 0.36±.02 0.07±.00 0.33±.08 0.04±.00 0.36±.05 8 b c a a a a LCWB 0.01±.00 0.95±.02 0.13±.01 0.42±.18 0.05±.01 0.43±.01 12 d d a a a a 0.02±.00 0.51±.02 0.05±.01 0.46±.09 0.04±.00 0.32±.04 4 a b a a a a MCNB 0.04±.01 2.05±.04 0.14±.01 0.49±.05 0.07±.01 0.43±.05 12 ab b a a a a 0.01±.01 0.63±.05 0.08±.00 0.41±.16 0.06±.00 0.35±.00 8 ab b a a a a MCWB 0.03±.01 1.61±.07 0.13±.01 0.46±.02 0.06±.00 0.41±.01 12 bc c a a a a 0.02±.00 0.79±.02 0.05±.01 0.42±.02 0.05±.00 0.33±.00 4 a a a a a a HCNB 0.05±.01 2.62±.06 0.13±.01 0.55±.01 0.07±.00 0.43±.03 12 a a a a a a 0.03±.01 0.94±.05 0.09±.00 0.59±.09 0.06±.00 0.39±.03 8 a a a a a a HCWB 0.03±.01 1.99±.03 0.13±.01 0.52±.00 0.06±.00 0.43±.01 12 b b a a a a / indicated blow detection limits; results were average±standard deviation, n=3. 204 Table 4.26(c) Shoot concentration and translocation factor of metals in reeds cultured in HM solution for 4, 8 and 12 weeks Shoot Shoot Shoot Treat Week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.01±.00 1.34±.05 0.09±.01 0.15±.01 0.07±.01 0.32±.01 4 b c a b a a NCNB 0.03±.01 2.50±.02 0.18±.01 0.32±.02 0.07±.01 0.30±.00 12 cd d a a a a 0.01±.00 0.77±.02 0.13±.02 0.17±.08 0.05±.00 0.27±.02 8 d c a a b a NCWB 0.02±.00 1.28±.02 0.18±.00 0.26±.01 0.06±.01 0.28±.00 12 e e e a a a 0.01±.00 1.38±.07 0.10±.00 0.27±.03 0.07±.00 0.31±.04 4 b c a ab a a LCNB 0.03±.01 2.52±.01 0.19±.02 0.32±.03 0.07±.01 0.30±.04 12 c d a a a a 0.01±.00 0.79±.01 0.13±.00 0.21±.02 0.05±.00 0.30±.02 8 c c a a b a LCWB 0.02±.00 1.33±.03 0.19±.01 0.31±.04 0.06±.01 0.31±.00 a 12 de e a a a 0.02±.00 2.88±.07 0.09±.02 0.28±.04 0.09±.01 0.32±.09 4 ab b a ab a a MCNB 0.05±.00 4.65±.10 0.18±.02 0.34±.00 0.08±.00 0.30±.02 12 b b a a a a 0.02±.00 3.36±.03 0.13±.04 0.21±.01 0.07±.00 0.31±.05 8 b b a a a a MCWB 0.03±.01 3.91±.05 0.19±.01 0.32±.00 0.08±.00 0.32±.04 12 bc c a a a a 0.03±.01 3.63±.01 0.10±.01 0.36±.04 0.09±.00 0.31±.02 4 a a a a a a HCNB 0.06±.01 6.38±.06 0.19±.01 0.38±.04 0.09±.01 0.30±.00 12 a a a a a a 0.03±.01 3.98±.17 0.13±.02 0.27±.03 0.07±.00 0.30±.03 8 a a a a a a HCWB 0.04±.01 6.00±.20 0.18±.00 0.34±.01 0.09±.00 0.30±.01 12 b a a a a a / indicated blow detection limits; results were average±standard deviation, n=3. 205 The shoot concentrations of Mn, Fe and Al in reeds cultured in LM solution elevated with the increase of CA (p<0.05). For example, the shoot concentrations of metals in reeds cultured in solution LMNCNB for 4 weeks were 0.01±0.00 mg/g for Mn, 0.03±0.00 mg/g for Fe and 0.02±0.00 mg/g for Al, while they increased to 0.02±0.00 mg/g for Mn, 0.07±0.00 mg/g for Fe and 0.06±0.00 mg/g for Al in reeds grown in solution LMHCNB for the same time period. These results were in accord with the findings discussed in above that CA can increase the uptake and translocation of Mn, Fe and Al in reeds cultured in solution with low level of metals. The shoot concentrations and translocation factors of Mn and Fe in reeds cultured in MM and HM solution also elevated with the increase of CA (p<0.05). The shoot concentrations of metals in reeds cultured in solution MMHCWB for 8 weeks were 0.63±0.05 mg/g for Fe, which were higher those in reeds cultured in solution MMNCWB for 8 weeks (0.33±0.00 mg/g for Fe). However, the shoot concentrations and translocation factors of Al in reeds cultured in MM and HM solution did not change too much with the addition of CA. For instance, the shoot concentrations of Al in reeds cultured in solution HMHCNB for 12 weeks were 0.10±0.01 mg/g, which were similar to that in reeds cultured in solution HMNCNB for 12 weeks (0.09±0.01 mg/g). As discussed above, most CA formed complex with Fe and Mn in MM and HM solution. That may be the main reason that CA did not increase the shoot concentrations and translocation factors of Al in MM and HM solutions. With the growth of reeds, the shoot concentrations of metals in reeds also increased. For example, the shoot concentrations of metals in reeds cultured in 206 solution LMLCNB for 4 weeks were 0.01±0.00 mg/g for Mn, 0.03±0.00 mg/g for Fe and 0.02±0.00 mg/g for Al. The shoot concentrations increased to 0.02±0.00 mg/g for Mn, 0.09±0.00 mg/g for Fe and 0.06±0.00 mg/g for Al in reeds grown in solution LMLCNB for 12 weeks. The shoot concentrations of Mn, Fe, Al in reeds cultured in solution MMMCNB for 4 weeks were 0.02±0.00 mg/g, 0.51±0.02 and 0.05±0.01 mg/g for Al, which increased to 0.04±0.01 mg/g, 2.05±0.04 and 0.14±0.00 in reeds grown in solution MMMCNB for 12 weeks. Similarly, the shoot concentrations of metals in reeds cultured in HM solution also increased with time. These phenomena indicated that accumulation and translocation of metals did occur in reeds. In addition, the synthetic hydroponic solution contained significantly higher levels of phosphate. The concentrations of phosphate will be lower in actual mine drainage (Batty et al., 2002), which may further decrease the complexation between phosphate and metals, especially Fe (Table 4.22 to 4.24). Thus the amounts of citrate-metals can be increased and the metal accumulation in reeds may be enhanced. Phragmites australis is known to widely distribute in the world and accumulate some heavy metals distinctly more than other wetlands plants (Duman et al. 2007). These advantages may make Phragmites australis a good selection for cleaning heavy metal contaminated water or soil. 4.4.5 Mass balance for metals in solution In order to the better understand the transport and transformation of metals in solution, the initial and eventual metal concentrations in solution were determined and 207 the mass balances of metals were calculated. In addition to plaque formed on the root/rhizome surface of plants and metals accumulated into plants, most metals still existed in solution. Table 4.27 showed the mass balance of metals in solution and plants sacrificed after 12 weeks. Table 4.27(a): Mass balance for metals in LM solution cultured with reeds for 12 weeks Treatment Metal Original Plaque In plants Remaining % amounts (mg) biomass in solution quantified in (mg) (mg) solution (mg) NCNB Mn 5.40 0.00 0.14 0.39 83.9% Fe 100.01 14.47 2.80 61.73 79.0% Al 6.74 0.00 0.38 5.32 84.5% NCWB Mn 5.40 0.00 0.11 0.42 83.9% Fe 100.00 20.17 2.64 61.36 84.2% Al 6.75 0.00 0.42 5.33 85.1% LCNB Mn 5.40 0.00 0.18 0.46 86.0% Fe 99.99 16.11 2.99 62.18 81.3% Al 6.74 0.00 0.49 5.37 86.9% LCWB Mn 5.40 0.00 0.13 0.40 83.9% Fe 100.03 20.34 2.87 62.41 85.6% Al 6.75 0.00 0.53 5.36 87.3% MCNB Mn 5.40 0.00 0.23 0.36 85.0% Fe 100.00 4.21 4.33 76.60 85.1% Al 6.75 0.00 0.69 5.21 87.5% MCWB Mn 5.40 0.00 0.23 0.31 84.1% Fe 100.00 4.81 4.12 78.70 88.8% Al 6.75 0.00 0.78 5.26 89.5% HCNB Mn 5.40 0.00 0.29 0.33 85.6% Fe 100.00 4.75 4.12 76.20 85.7% Al 6.75 0.00 0.74 5.29 89.4% HCWB Mn 5.40 0.00 0.25 0.34 87.8% Fe 100.02 4.32 4.77 77.14 86.7% Al 6.75 0.00 0.79 5.22 87.4% 208 Table 4.27(b): Mass balance for metals in MM solution cultured with reeds for 12 weeks Treatment Metal Original Plaque In plants Remaining % amounts (mg) biomass in solution quantified in (mg) (mg) solution (mg) NCNB Mn 10.00 0.32 0.14 8.38 88.4% Fe 5416.67 88.74 51.85 4722.50 89.8% Al 37.53 0.00 0.76 32.73 89.3% NCWB Mn 10.09 0.31 0.10 8.24 85.7% Fe 5416.83 124.14 36.27 4737.17 89.5% Al 37.52 0.00 0.77 32.71 89.2% LCNB Mn 10.08 0.30 0.16 8.39 87.8% Fe 5416.83 79.42 56.95 4737.17 90.0% Al 37.57 0.00 0.75 32.64 88.9% LCWB Mn 10.00 0.31 0.10 8.28 86.9% Fe 5417.00 124.01 38.08 4690.50 89.6% Al 37.46 0.00 0.80 32.71 89.5% MCNB Mn 10.04 0.16 0.26 8.50 88.8% Fe 5416.83 62.53 81.71 4756.67 90.5% Al 37.56 0.00 0.76 32.73 89.2% MCWB Mn 10.03 0.18 0.18 8.48 88.1% Fe 5417.50 92.67 66.00 4753.33 90.7% Al 37.59 0.00 0.77 32.74 89.1% HCNB Mn 10.05 0.08 0.34 8.54 89.2% Fe 5417.33 48.40 101.89 4749.83 90.5% Al 37.53 0.00 0.85 32.73 89.5% HCWB Mn 10.00 0.07 0.27 8.53 88.7% Fe 5416.50 65.55 85.79 4748.17 90.5% Al 37.58 0.00 0.84 32.74 89.3% 209 Table 4.27(c): Mass balance for metals in HM solution cultured with reeds for 12 weeks Treatment Metal Original Plaque In plants Remaining % amounts in (mg) biomass in solution quantified solution (mg) (mg) (mg) NCNB Mn 12.71 0.52 0.33 10.35 88.1% Fe 10737.33 169.40 80.01 9362.33 89.5% Al 136.17 0.00 1.86 120.00 89.5% NCWB Mn 12.73 0.50 0.18 10.03 84.1% Fe 10736.33 266.66 52.91 9072.33 87.5% Al 136.13 0.01 1.88 119.25 89.0% LCNB Mn 12.76 0.53 0.33 10.34 87.8% Fe 10734.67 153.25 84.65 9391.00 89.7% Al 135.58 0.00 2.00 119.63 89.7% LCWB Mn 12.76 0.53 0.19 10.13 85.0% Fe 10737.00 247.92 54.37 9065.00 87.2% Al 136.33 0.00 1.97 117.83 87.9% MCNB Mn 12.72 0.27 0.50 10.59 89.3% Fe 10736.00 108.63 124.57 9414.33 89.9% Al 136.13 0.00 1.93 118.83 88.7% MCWB Mn 12.66 0.29 0.40 10.32 87.0% Fe 10737.67 148.37 102.48 9175.00 87.8% Al 136.08 0.00 1.91 118.25 88.3% HCNB Mn 12.73 0.17 0.58 10.63 89.4% Fe 10738.33 73.45 154.45 9407.33 89.7% Al 136.04 0.00 1.88 119.96 89.6% HCWB Mn 12.73 0.19 0.51 10.33 86.7% Fe 10738.33 96.07 131.62 9248.67 88.2% Al 136.04 0.00 1.74 119.54 89.1% The quantified rates of metals in solution ranged from 70% to nearly 90% for Mn, Fe and Al. The main losses may due to that some metals were precipitated as pH changed and some were used by bacteria. In addition to the metal that was in the solution, plaque was the main existing form of metals on reeds cultured in solution without adding CA or added with low level of CA. According to the findings of Hansel and Fendorf (2001), the precipitation and adsorption of Fe, Mn, Pb and Zn on 210 the root surface accounted for great part of metals associated with aquatic plant roots. Ye et al. (1997b) also reported that, 89-90% of the total Fe of the seedlings of wetland plants presented as plaque. However, plaque which coats algae, plants, and sediments can cause the damage of the habitat for fish and other aquatic lives in streams (Jennings et al., 2008; Senko et al., 2008). With the increase of CA, less metal plaque formed on the root surface of reeds. As such, more metals were bioavailable and more metals were accumulated into the biomass of reeds. This indicated that CA may be effective to enhance the success of phytoremediation of AMD contaminated soil or water. 4.4.6 Summary of hydroponic experiments of reeds propagated from wild rhizomes Hydroponic experiments may yield better understanding of the fate of metals in the solution and plants due to the less mass transfer limitations. The amounts of Fe plaque on reeds cultured in HM and MM solution were higher than those on reeds grown in LM solution, since the numbers of Fe(II)OB were related to the presence of Fe. Although Mn plaque was not found on the root system of reeds cultured in LM solution, it was observed on the root system of reeds cultured in MM and HM solution. This may be related to the Mn concentrations. The amounts of Fe plaque and Mn plaque increased with time. However, most of Fe and Mn plaque were formed in the first 4 or 8 weeks due to the first order oxidation of Fe. Al plaque was not found on reeds cultured in LM, MM and HM solution, since most of phosphate complexed with Fe and prevented the formation of Al-phosphate plaque. 211 The metal plaque can inhibit the Fe and Mn accumulations in reeds, depending on the amounts of plaque. Fe plaque mediated by Fe(II)OB did not have a significant effect on (p>0.05) the accumulation of Fe and Mn in reeds cultured in LM solution. However, the metal plaque did have a significant role (p<0.05) in decreasing the accumulation of Fe and Mn in reeds cultured in MM and HM solution. High amounts of metal plaque may inhibit the Fe and Mn accumulation in plants while low concentrations of plaque may not influence the elements uptake in plants. However, the metal plaque did not have important influence (p>0.05) on the Al uptake in reeds cultured in either LM, MM or HM solution. This may be attributed to the high mobility of Al in acidic environments. CA decreased the amounts of Fe and Mn plaque, depending on the concentrations of metals and CA in solution. No Mn, Fe and Al citrate complex would be formed in LM solution added with low level of CA (0.021g CA/L). Thus the low level of CA did not have an effect to reduce the plaque formation in LM solution. However, with the increase of CA, more Mn and Fe citrate complex could be formed and less Mn and Fe plaque formed in LM, MM and MM solutions. CA also increased the metal accumulation in both belowground and above ground tissues of reeds, which were also related to the concentrations of metals and CA in solution. Low level of CA (0.021g CA/L) did not increase the metal accumulation in reeds, but middle and high level of CA significantly elevated (p<0.05) the uptake of Fe, Mn and Al in different tissues of reeds grown in LM solution. Similarly, CA elevated the Fe and Mn uptake in reeds cultured in MM and HM 212 solutions. The more CA added, the more Fe and Mn uptake in reeds. However, CA did not have important effect (p>0.05) in Al accumulation in reeds cultured in MM and HM solution, since most of CA reacted with Fe and Mn which and higher affinity with CA and then no citrate-Al complex were formed. 4.4.7 Comparison between HM hydroponic experiments and spiked soil experiments Comparing the results of spiked soil experiments and hydroponic experiments contained the same level of metals (HM), we can find that Fe(II)OB played an significant role in forming Fe plaque on reeds grown in both spiked soil and hydroponic solutions. CA also increased (p<0.05) the Fe and Mn uptake in reeds cultured in soil or solutions. However, there are some differences between the results of two sets of experiments. First, Al plaque was found on the root systems of reeds grown in spiked soil while no Al plaque was formed in the hydroponic solution. Second, CA increased the Al uptake in reeds grown in spiked soil while it had no significant influence on Al accumulations in reeds in hydroponic solutions. Third, wild reeds cultured in spiked soil formed more metal plaque and uptake more metals than reeds grown in solutions. For instance, wild reeds accumulated 0.63 mg Mn, 131.70 mg Fe and 7.11 mg Al from spiked soil NCWB while reeds accumulated 0.18 mg Mn, 52.91 mg Fe and 1.88 mg Al from solution NCWB. The differences may be attributed to the different environment in soil and solutions. Hydroponic experiments have less mass transfer limitations than soils (Asao, 2012). For example, the pH change may be dispersed throughout the solution. However, pH may change slowly in 213 soil and pH close to the root may be 1-2 units lower than that in bulk soil (Ye et al., 1997a). The pH may further influence the metal mobility and metal uptake in reeds. Besides, the wild reeds used in spiked soils were directly collected from the AMD site while reeds cultured in HM hydroponic solutions were propagated from wild rhizomes collected from AMD sites. The metal uptake may be different between adult plants in field and seedlings grown in metal solution condition (Ye et al., 1997a). In addition, plaque composition and structure might be different under soil and hydroponic conditions (Ye et al., 1997a). These may be the main reasons that cause the differences in results between soil and solution experiments. Further work under both soil and solution conditions may be required to elucidate the precise role of reeds in clean AMD contaminated field or water. 4.5 Hydroponic experiments for wild reeds collected from site E Hydroponic experiments were conducted for wild reeds collected from site E which were less exposed to the AMD contamination. The experoments were used to further assess the effect of different bacteria on the metal plaque formation and metal accumulation in “aged” reeds. 4.5.1 Rhizosphere bacteria in solution cultured with reeds collected from site E The control group, which only contained nutrient materials, was used to assess biomass growth of reeds. The treatment groups contained nutrient materials and middle level of metals (MM) (section 3.5) were separated into different conditions: 214 without inoculating bacteria and CA (NCNB); with 9.020 g/L CA without inoculating bacteria (WCNB); inoculated with only acidophilic heterotrophs (NCAB); inoculated with acidophilic heterotrophs and 9.020 g/L CA (WCAB); inoculated with only Fe (II) oxidizing bacteria (NCFB); inoculated with Fe(II)OB and 9.020 g/L CA (WCFB); inoculated with acidophilic heterotrophs and Fe(II)OB (NCAFB); inoculated with acidophilic heterotrophs, Fe(II)OB and 9.020 g/L CA (WCAFB). Reeds collected from site E were cultured in each of the hydroponic solutions for 4, 8 and 12 weeks, and then harvested for analysis. Heterotrophs, together with iron and sulfur oxidizing autotrophic microorganisms were important components of AMD microbial community (Willscher et al., 2007) Table 4.28 show the numbers of acidophilic heterotrophs which were enumerated on WAYE medium and Fe(II)OB (FETSB medium) from rhizosphere of reeds cultured in the different solutions of 4, 8 and 12 weeks. Acidophilic heterotrophs and Fe(II)OB were detected in all the solutions, including the control group. This may due to the fact that bacteria were already present in the rhizosphere of the reeds when collected from site E. According to the previous study (section 4.1.1), abundant acidophilic heterotrophs and Fe(II)OB existed in the root zone of reeds grown in the field. In addition, it was not surprising that the solution inoculated with bacteria contained more acidophilic heterotrophs or Fe(II)OB than the control group and treatment solution without adding with bacteria. The NCNB solution cultured reeds for 4 weeks had 2.15±0.09×104 CFU/ml acidophilic heterotrophs and 5.40±1.00×103 CFU/ml Fe(II)OB, while the solution 215 NCAB with reeds for 4 weeks possessed much higher (p<0.05) acidophilic heterotrophs (3.83±0.09×105 CFU/ml) and the solution NCFB possessed more (p<0.05) Fe(II)OB (4.70±0.50×104 CFU/ml). Table 4.28(a) Numbers of rhizosphere bacteria in solution cultured with reeds collected from site E for 4, 8 and 12 weeks Treatment Time WAYE (CFU/ml) FETSB (CFU/ml) Control 4 weeks 2.01 ±0.13×104 c 3.80 ±0.40×102 d 8 weeks 1.55 ±0.09×104 d 2.30 ±0.50×102d 12 weeks 1.11 ±0.09×104 c 1.80 ±0.04×102 d NCNB 4 weeks 2.15 ±0.09×104 c 5.40 ±1.00×103 c 8 weeks 1.85 ±0.11×104 d 4.20 ±0.60×103c 12 weeks 1.56 ±0.06×104 c 2.68 ±0.14×103 c WCNB 4 weeks 5.30 ±0.50×104 c 1.67 ±0.11×103 c 8 weeks 1.26 ±0.08×105 cd 1.77 ±0.07×103 c 12 weeks 2.02 ±0.08×105 c 1.45 ±0.07×103 c NCAB 4 weeks 3.83 ±0.09×105 b 8.30 ±1.1×103 c 8 weeks 4.60 ±0.80×105 bc 5.60 ±0.60×103 c 12 weeks 1.10 ±0.12×106 b 2.47 ±0.09×103 c WCAB 4 weeks 1.00 ±0.08×106 a 2.21 ±0.09×103 c 8 weeks 1.76 ±0.12×106 a 2.15 ±0.09×103 c 12 weeks 3.28±0.22×106 a 1.62 ±0.16×103 c NCFB 4 weeks 1.98 ±0.12×104 c 4.70 ±0.50×104 a 8 weeks 1.56 ±0.06×104 d 1.26 ±0.08×105 a 12 weeks 1.13 ±0.03×104 c 2.25 ±0.09×105 a WCFB 4 weeks 5.80 ±0.40×104 c 1.38±0.08×104 c 8 weeks 1.25±0.11×105 cd 1.95 ±0.11×104 bc 12 weeks 2.31 ±0.11×104 c 3.61 ±0.03×104 bc NCAFB 4 weeks 3.42±0.12×105 b 5.80 ±0.40×104 a 8 weeks 5.00 ±0.80×105 b 1.12 ±0.08×105 a 12 weeks 1.01 ±0.03×106 b 2.20 ±0.16×105 a WCAFB 4 weeks 8.20 ±0.40×105 a 3.03 ±0.07×104 b 8 weeks 1.82 ±0.04×106 a 4.03 ±0.07×104 b 12 weeks 3.36 ±0.14×106 a 5.00 ±0.06×104 b / indicated blow detection limits; results were average±standard deviation, n=10. 216 CA significantly increased the numbers of acidophilic heterotrophs in solution (p<0.05). The WCAB solution cultured with reeds for 4 weeks had 1.00±0.08×106 CFU/ml acidophilic heterotrophs, which was higher than that in NCAB solution (3.83±0.09×105 CFU/ml). The numbers of acidophilic heterotrophs in solution WCAFB, WCFB and WCNB cultured with reeds for 4 ,8 and 12 weeks were also higher than in solution NCAFB, NCFB, and NCNB cultured with reeds for the same time period, respectively. Unlike autotrophic species, heterotrophs need organic matter as carbon source (Schippers et al., 2010). So it was expected that CA, as an organic material, can increase the number of acidophilic heterotrophs. Jones (1998) also indicated that microorganisms can decompose organic acids such as citrate and malate acid in both soil and solution culture, which induce a growth of rhizosphere bacteria. However, CA significantly (p<0.05) inhibited the growth of the Fe(II)OB. The NCFB solution cultured reeds for 4 weeks had 4.70±0.50×104 CFU/ml Fe(II)OB, while the WCFB solution had much less (p<0.05) population of Fe(II)OB which were 1.38±0.08×104 CFU/ml after 4 weeks. Similarly, the WCAFB solution and WCNB solution also contained less Fe(II)OB than solution NCAFB and NCNB. This may be attributed to organic compounds being toxic to lithoautotroph Fe(II)OB (Hallberg, 2010), since Fe(II)OB obtain their carbon source by CO2 fixation instead of organic carbon. Further, it was worthwhile to note that acidophilic heterotrophs may improve the growth of Fe(II)OB. For the solution amended with CA, those inoculated with acidophilic heterotrophs and Fe(II)OB together contained more Fe(II)OB than the 217 solution only inoculated with Fe(II)OB. For instance, the WCAFB solution cultured reeds for 4 weeks contained 3.03±0.07×104 CFU/ml Fe(II)OB, which was significantly higher (p<0.05) than that of the WCFB system after 4 weeks (1.38±0.08×104 CFU/ml). The WCAFB solution cultured reeds for 8 and 12 weeks also contained slightly higher Fe(II)OB than that in WCFB solution over the same time period. This indicated that interactions between microorganisms may be critical to the overall AMD microbial community activity. Baker and Banfiel (2003) pointed out that an important relationship existed between heterotrophic and certain autotrophic species, since heterotrophs can consume organic compounds that maybe toxic to autotrophs. For example, the mutualistic relationship between Acidiphilium sp. was reported along with Fe(II)OB Leptospirillum in AMD environments, as Acidiphilium sp. consumed organic compounds toxic to Leptospirillum (Wan, et al., 2009). As mentioned above, CA can reduce the growth of Fe(II)OB. As the CA was consumed by acidophilic heterotrophs, the environment became more suitable for the growth of Fe(II)OB. As such, the numbers of Fe(II)OB were higher in solution added with CA and inoculated with acidophilic heterotrophs than that in solution added with CA but without adding with acidophilic heterotrophs. 4.5.2 pH, conductivity and DO change of solution cultured with reeds collected from site E Table 4.29 contained the change of pH, conductivity and DO in the solutions cultured with reeds for 4, 8 and 12 weeks. After adding CA, the pH decreased in the 218 solution, since organic acids can provide the H+ necessary to low the pH. Excluding the control group, the pH of all the treatment groups decreased after 4 weeks due to the oxidation of Fe2+ and precipitation of Fe3+. At pH values greater than 6, Fe2+ can be rapidly oxidized to Fe3+ by chemical process, but the abiotic oxidation of Fe2+ slows at pH below 4, thus the Fe(II)OB play an important role in Fe2+ oxidation under acidic conditions (Streten-Joyce et al., 2013). It was reported that the increased levels of Fe3+ and lower of pH was related to A. ferrooxidans, which was a typical Fe(II)OB (Melchiorre, 2011). As discussed in section 4.5.1, Fe(II)OB were found in all the treatment groups, which can oxidize Fe2+ rendering the solution more acidic. However, the pH of solution added with CA was higher than the pH of solution without CA after 4 weeks. For instance, the pH of solution WCFB was 2.12±0.02, which was higher than that in solution NCFB (2.02±0.01). This can be explained by two reasons. First, CA inhibited the growth of Fe(II)OB (as discussed in section 4.5.1) and then further affected the Fe(II) oxidation rate and pH of solution. Second, CA can form complex with Fe(II) (Jones, 1998) and then decrease the available Fe(II) for oxidation. The pH may decrease slightly or increase with the decrease of Fe2+ oxidation or reduction of Fe3+ ( Küsel et al., 2003). The pH of solution did not change dramatically after 8 and 12 weeks. As discussed before, the Fe(II) oxidation followed first-order equation which was fast in the first 4-5 weeks (Neubauer et al., 2007). As such, the pH also became stable after 4-5 weeks. 219 Table 4.29 (a) pH of solution under different treatment conditions Treatment Initial pH After 4 weeks 8 weeks 12 weeks adding CA Control 5.77±0.03 5.75±0.02 5.73±0.01 5.73±0.01 5.73±0.01 NCNB 3.50±0.01 3.50±0.02 2.15±0.01 2.13±0.01 2.11±0.00 WCNB 3.51±0.02 2.70±0.01 2.23±0.02 2.20±0.01 2.19±0.01 NCAB 3.50±0.02 3.50±0.02 2.14±0.02 2.12±0.02 2.09±0.01 WCAB 3.50±0.02 2.71±0.02 2.21±0.02 2.19±0.00 2.17±0.02 NCFB 3.50±0.01 3.49±0.01 2.02±0.01 1.99±0.01 1.97±0.02 WCFB 3.50±0.02 2.71±0.02 2.12±0.02 2.09±0.01 2.07±0.02 NCAFB 3.49±0.01 3.50±0.02 2.01±0.01 1.98±0.02 1.97±0.01 LWCAFB 3.51±0.01 2.70±0.02 2.10±0.01 2.07±0.01 2.06±0.02 Table 4.29 (b) Conductivity (ms/cm) of solution under different treatment conditions Treatment Initial After 4 weeks 8 weeks 12 weeks conductivity adding CA Control 2.08±0.01 2.07±0.01 1.87±0.02 1.76±0.02 1.61±0.01 NCNB 20.2±0.1 20.1±0.0 17.6±0.1 17.1±0.1 16.6±0.1 WCNB 20.1±0.1 22.5±0.3 17.2±0.2 16.3±0.0 15.4±0.2 NCAB 20.0±0.2 20.2±0.1 17.5±0.2 16.9±0.2 16.5±0.2 WCAB 20.1±0.2 22.3±0.3 17.1±0.1 16.4±0.1 15.6±0.1 NCFB 20.0±0.2 20.1±0.2 16.6±0.2 15.5±0.2 14.8±0.0 WCFB 20.0±0.1 22.4±0.4 16.3±0.1 15.3±0.2 14.7±0.1 NCAFB 20.2±0.2 20.1±0.2 16.5±0.2 15.4±0.0 14.7±0.2 WCAFB 20.1±0.1 22.2±0.4 16.4±0.1 15.3±0.1 14.6±0.0 Table 4.29 (c) DO (mg/L)of solution under different treatment conditions Treatment Initial DO After 4 weeks 8 weeks 12 weeks adding CA Control 5.26±0.27 5.38±0.27 5.13±0.20 5.28±0.13 5.16±0.22 NCNB 5.10±0.27 5.21±0.17 4.45±0.12 4.35±0.29 4.34±0.10 WCNB 5.18±0.22 5.14±0.16 4.37±0.16 4.47±0.41 4.30±0.20 NCAB 5.16±0.20 5.18±0.23 4.43±0.19 4.44±0.26 4.19±0.10 WCAB 5.20±0.18 5.09±0.23 4.38±0.27 4.40±0.14 4.23±0.24 NCFB 5.13±0.17 5.08±0.17 4.36±0.12 4.33±0.14 4.22±0.19 WCFB 5.10±0.16 5.16±0.21 4.24±0.20 4.31±0.11 4.29±0.29 NCAFB 5.10±0.24 5.23±0.04 4.39±0.10 4.55±0.20 4.28±0.17 WCAFB 5.03±0.14 5.10±0.20 4.37±0.07 4.40±0.09 4.20±0.15 220 The conductivity of the control solution was much lower than the treatment solution, since the metal ions added into treatment solution increased the conductivity. High conductivity is one of the typical characteristic of AMD (Herlihy et al., 1990) caused by the metals ions leaching from the mine waste. The conductivity of all the solution decreased after 4, 8 and 12 weeks, which indicated that the total metal ions in solution reduced (as well be corroborated in section 4.5.5). The Fe(II)OB can oxidize and cause the precipitate of Fe(II) and other metals, which may decrease the conductivity of solution. Kalin and Caetano Chaves (2003) also indicated that the precipitation and removal of some metals can reduce the conductivity of AMD. Hallberg and Johnson (2005) also found that the conductivity of AMD decreased after passing aerobic wetlands for iron oxidation and precipitation. In our study, the solution inoculated with Fe(II)OB also possessed lower conductivity than the solution without adding Fe(II)OB. For instance, the conductivity in solution NCFB was 16.6±0.2 ms/cm and was 17.6±0.1 ms/cm in solution NCNB after 4 weeks. As mentioned above, Fe(II) oxidation mainly occurred in the first several weeks. However, the conductivity of solution still decreased after 8 and 12 weeks. This indicated that plants also played an important role in reducing conductivity by uptake metals into biomass. The DO of solution was 4-5 mg/L, which was similar to the results of Ghosh et al. (2012) who found that the DO of an AMD contaminated stream to be 4-5 mg/L. After 4, 8 and 12 weeks, the DO of treatment solutions slightly decreased. This may due to the fact that the growth of microorganisms consumed oxygen in solution 221 (Streten-Joyce et al., 2013). There were not significant differences in different solutions added with different bacteria or CA. This may due to that other factors such as temperature, season and the growth activity of plants, also impact DO levels in solution. 4.5.3 DCB extraction of reeds collected from site E cultured in solution The metal plaque formed on reeds collected from site E and then cultured in different solutions for 4, 8 and 12 weeks were extracted and analyzed. In order to better assess the effect of CA and rhizosphere bacteria on the metal plaque formation, the amounts of metal plaque on reeds did not include the background metal plaque which were formed in the field. The background metal plaque amounts were show in Table 4.30. Mn plaque (Figure 4.108) and Fe plaque were found on reeds cultured under different treatment conditions. Table 4.30 Background values of metal plaque on reeds collected from site E cultured in solution Metal (mg/g) Plaque on roots Plaque on rhizomes Mn 0.01±0.00 0.00±0.00 Fe 9.12±0.82 8.75±1.84 Al 0.02±0.01 0.02±0.01 Results were reported as average±standard deviation, n=3 222 0.08 a a a 0.07 b a a b a 0.06 a b a a 0.05 a b rhizomes a a 0.04 b b b b b b roots 0.03 b mg(Mn)/g(biomass) 0.02 0.01 0.00 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB 0.10 a a b a a 0.08 b a b a a a a a b 0.06 b b b b a b b b rhizomes 0.04 b roots mg(Mn)/g(biomass) 0.02 0.00 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB a b a a 0.10 a b a a 0.08 b a a a c b a a 0.06 b b b b rhizomes b roots 0.04 b b mg(Mn)/g(biomass) 0.02 0.00 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB Figure 4-108 Mn plaque on the roots and rhizomes of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 223 CA significantly (p<0.05) decreased the formation of Mn and Fe plaque on the roots or rhizomes of reeds during all the growth period. The Mn plaque on rhizomes of reeds grown in solution NCNB for 4 weeks was 0.04±0.01 mg/g, which was significantly higher (p<0.05) than those on reeds cultured in solution WCNB for 4 weeks (0.02±0.01 mg/g). The Mn plaque on rhizomes of reeds grown in solution NCAB for 4 weeks was 0.04±0.00 mg/g, which was significantly higher (p<0.05) than those on reeds cultured in solution WCAB for 4 weeks (0.02±0.00 mg/g). The Mn plaque on roots of reeds grown in solution NCFB for 8 weeks was 0.04±0.01 mg/g. It was higher than those on reeds cultured in solution WCFB for 8 weeks which was 0.08±0.01 mg/g. Similarly, the Mn plaque on rhizomes of reeds grown in solution NCAFB for 12 weeks was 0.06±0.01 mg/g, which was significantly higher (p<0.05) than those on reeds cultured in solution WCAFB for 12 weeks (0.04±0.01 mg/g). Similar trends were found for Fe plaque (Figure 109). The Fe plaque on the rhizomes of reeds grown in solution NCNB for 4 weeks was 24.47±2.33 mg/g, which was significantly higher (p<0.05) than those on reeds cultured in solution WCNB for 4 weeks (15.77±1.95 mg/g). The Fe plaque on roots of reeds grown in solution NCFB for 8 weeks (49.60±0.29 mg/g) was significantly higher (p<0.05) than those on reeds cultured in solution WCFB for 8 weeks (33.69±1.88 mg/g). The Fe plaque on roots of reeds grown in solution NCAFB for 12 weeks was 52.66±2.42 mg/g. It was higher than those on reeds cultured in solution WCAFB for 12 weeks which was 40.92±0.72 mg/g. 224 As discussed in hydroponic experiments for reeds propagated from rhizome collected from the field, CA can increase Fe and Mn solubility by forming citrate-metal complex and then decrease the formation of metal plaque. (Weiss et al., 2004) also indicated that Fe plaque is a large reservoir of Fe(III) oxides which may include ferrihydrite lepidocrocite and/or goethite, and the organic acids can reduce Fe3+ and maintain Fe in soluble form. Organic acids such as malate and citrate can also release Mn from Mn oxides by oxidation and/or complexation (Jones, 1998). In this study, the rhizosphere bacteria did not have a significant influence on the formation of Mn plaque (p<0.05). For instance, the Mn plaque on roots of reeds cultured in solution NCNB for 4 weeks was 0.05±0.00 mg/g, which was similar to that on reeds grown in solution NCAB (0.05±0.01 mg/g) and NCFB (0.06±0.01 mg/g) for the same time period. It was not surprising since no biologically catalyzed Mn oxidation were significant at pH values of 4.0 or less (Wieder et al., 1990). The formation of Mn plaque was mainly caused by adsorbing or co-precipitation with Fe plaque. It was reported that Fe plaque may adsorb other metals such as Mn, Zn, Pb onto the root surface of plants (Hansel and Fendorf, 2001). Although the amounts of Mn plaque were always less than Fe plaque, the plaque element next in importance to Fe is generally Mn in nature and Fe and Mn plaque usually co-exist (Liu et al., 2010; Liu et al., 2005). 225 60 a a 50 a a b 40 b b b b c a c c c c c 30 d rhizomes d d d 20 d roots mg(Fe)/g(biomass) 10 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB 60 a a a 50 a b b c b c b 40 c c d c c d d b e 30 d e d rhizomes 20 roots mg(Fe)/g(biomass) 10 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB a 60 a a a 50 b b b b b b c 40 c c c b c c c d d 30 d d rhizomes 20 roots mg(Fe)/g(biomass) 10 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB Figure 4.109 Fe plaque on the roots and rhizomes of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 226 Nevertheless, rhizosphere bacteria, especially Fe(II)OB, had important influence (p<0.05) on the formation of Fe plaque in our study. More Fe plaque was formed on reeds cultured in solution inoculated with Fe(II)OB. For instance, the Fe plaque on roots of reeds grown in solution NCFB for 4 weeks was 46.65±3.19 mg/g, which was significantly higher (p<0.05) than those on reeds cultured in solution NCNB and NCAB for 4 weeks (23.93±0.36 mg/g and 27.37±0.45 mg/g, respectively). The Fe plaque on rhizomes of reeds grown in solution NCAFB for 12 weeks (46.16±1.39 mg/g) was significantly higher (p<0.05) than those on reeds cultured in solution NCAB and NCNB for 12 weeks (30.67±4.33 mg/g and 30.44±3.74 mg/g, respectively). It was in agreement with previous reports which indicated that Fe(II)OB played an important role in rhizosphere Fe plaque formation. The main reason for the formation of Fe plaque in the rhizosphere is that O2 release by plant roots creates microaerophilic zones suitable to the growth of Fe(II)OB, such as Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum (Weiss et al., 2004; Streten-Joyce et al., 2013). The Fe plaque formed on reeds inoculated with acidophilic heterotrophs were not significantly different from that on reeds cultured in solution without adding with rhizosphere bacteria. The Fe plaque on roots and rhizomes of reeds grown in solution NCNB for 4 weeks was 23.93±0.36 mg/g and 24.47±2.33 mg/g, which was not significantly different from (p>0.05) than those on reeds cultured in solution NCAB for 4 weeks (27.37±0.45 mg/g and 25.25±2.80 mg/g, respectively). This may indicate that acidophilic heterotrophs had no important or direct effect on Fe plaque formation. 227 The microbial diversity in AMD environment comprises aerobic and anaerobic species which are autotrophic heterotrophic as well as lithotrophic or organotrophic (Schippers et al., 2010). In general it is thought that the heterotrophs did not play important role in the geochemistry of AMD (Hallberg, 2010). However, it was worthwhile to note that the Fe plaque on reeds cultured in solution added with CA and inoculated with Fe(II)OB and acidophilic heterotrophs were higher than that on reeds grown in solution added with CA but only inoculated with Fe(II)OB. For instance, the Fe plaque on rhizomes of reeds grown in solution WCAFB for 8 weeks was 33.66±2.37 mg/g g, which was slightly higher than those on reeds cultured in solution WCFB for 8 weeks (29.91±2.01 mg/g). The Fe plaque on roots of reeds grown in solution WCAFB for 12 weeks (40.92±0.72 mg/g) was higher than those on reeds cultured in solution WCFB for the some time period (34.05±0.86 mg/g). As discussed in section 4.1.2, heterotrophs can consume organic compounds that can be toxic to Fe(II)OB and then create a less hostile environment for the growth of Fe(II)OB (Hallberg, 2010.) As such, more Fe plaque can be formed around the roots of reeds. The cooperative relationships between heterotrophic acidophiles and Fe(II)OB were also reported by other researchers. Autotrophs needed coexisting heterotrophs to remove organic compounds while heterotrophic acidophiles obtained nutrients from soil solution or got organic materials produced by acidophilic autotrophs to support their growth (Baker and Banfield, 2003). The abundance and diversity of microbial community in AMD may be relatively restricted due to the extreme conditions such as high level of toxic metals 228 and low pH (Ghosh et al., 2012). However, the interactions among microorganisms in AMD were also complicated. Further investigation may be needed to study the factors affect microbial community structure and the relationships between different microorganisms and other organisms such as plants. 4.5.4 Digestion of reeds collected from site E cultured in solution Reeds collected from site E cultured in solution for 4, 8 and 12 weeks were also harvested and digested. Since Fe, Mn and Al were already accumulated into the biomass of reeds when collected from field, the concentrations of Mn, Fe and Al in reeds shown in Figure 4.110 to 4.112 did not include the background metals to better assess the uptake of metals in reeds cultured in hydroponic solution. The background values were shown in Table 4.31, which were higher than the background values of metals in reeds propagated from wild rhizomes (Table 4.25). It was not surprising, since the reeds collected from site E have been exposed to the AMD longer than the reeds propagated from wild rhizomes. Table 4.31 Background values of metal accumulations in reeds collected from site E cultured in solution Metal Metal in Metal in Metal in Metal in (mg/g) roots rhizomes stems leaves Mn 0.05±0.01 0.04±0.01 0.02±0.01 0.02±0.00 Fe 2.88±0.12 1.99±0.59 0.72±0.23 0.38±0.03 Al 0.09±0.01 0.08±0.02 0.06±0.02 0.05±0.01 Results were reported as average±standard deviation, n=3 229 Fe, Al and Mn were detected in the biomass of reeds cultured under different conditions. As expected, CA significantly enhanced (p<0.05) the Mn and Fe accumulation in both aboveground and belowground tissues of reeds during all the growth periods. As shown in Figure 4.110 , the rhizomes cultured in WCNB solution for 4 weeks contained 0.06±0.01 mg/g Mn which was significantly higher (p<0.05) than that in the rhizomes grown in NCNB (0.04±0.01 mg/g Mn). The Mn concentrations in the stems and leaves cultured in WCAB for 8 weeks were 0.05±0.01 mg/g and 0.05±0.00 mg/g, respectively. They were significantly higher (p<0.05) than those in the stems and leaves of reeds grown in NCAB for 8 weeks (0.04±0.01 mg/g and 0.03±0.00 mg/g, respectively). Similarly, the roots and rhizomes cultured in WCNB solution for 4 weeks contained 34.44±0.81 mg/g and 24.74±2.00 mg/g Fe, which were significantly higher (p<0.05) than those in the roots and rhizomes grown in NCNB (26.04±1.88 mg/g and 15.09±1.93 mg/g). However, CA did not appear to have an important role in increasing Al accumulation in any tissues of reeds cultured in different solutions. The Al accumulated in the roots and rhizomes of reeds cultured in NCNB solution for 4 weeks were 0.32±0.03 mg/g and 0.22±0.03 mg/g Fe, which were similar to the Al levels in the roots and rhizomes of reeds cultured in WCNB for the same time. The Al concentrations in the stems and leaves cultured in WCAFB for 12 weeks were 0.26±0.02 mg/g and 0.20±0.02 mg/g. They were not significantly different (p>0.05) from those in the stems and leaves of reeds grown in NCAFB for 12 weeks (0.25±0.03 mg/g and 0.20±0.01 mg/g, respectively). 230 a rhizomes 0.1 a roots a b a stems 0.08 c a b a b b leaves b a c b a c c a c b 0.06 d b b a d c c c a a d b b c c 0.04 b d c d d b b b b b c c c c c c c mg(Mn)/g(biomass) 0.02 c c c c 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB b rhizomes 0.12 a roots a a b a 0.1 a a stems b a a b a leaves b b 0.08 b b a b b c 0.06 b a b c a c b c b b b c b 0.04 b c d c c d c c mg(Mn)/g(biomass) d c 0.02 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB c a rhizomes 0.16 a roots 0.14 a a a stems b a b 0.12 b a b a b c leaves c 0.1 d b a c b b b b d 0.08 b c c a a b c d d b b c c 0.06 b b d c b c c d c mg(Mn)/g(biomass) 0.04 c 0.02 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB Figure 4.110 Mn concentration in the organs of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 231 a rhizomes 50 roots a stems 40 a b leaves b b b a c b c 30 c b d b d c c c d d d d 20 e e mg(Fe)/g(biomass) 10 b a a c b c c d c b a e d c b e d d c 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB b rhizomes 50 a roots a b stems 40 b b b b c c a c leaves a c 30 c b b b b c 20 c mg(Fe)/g(biomass) 10 a a a b b a b b c b b b c c b c 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB c rhizomes 60 roots 50 a a stems b b c c d leaves d d e 40 a a e e f f f 30 b b b b c c 20 mg(Fe)/g(biomass) 10 b a a a b c c c b a d d b b d d c c 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB Figure 4.111 Fe concentration in the organs of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 232 a rhizomes 0.4 a a a a a a a a roots stems a a a a a a 0.3 a a leaves a a a a a a a a a a 0.2 a a a a a a mg(Al)/g(biomass) 0.1 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB b rhizomes 0.5 a a a a a a a a roots stems 0.4 a a a a a leaves a a a a a a a a a 0.3 a a a a a a a a a a 0.2 mg(Al)/g(biomass) 0.1 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB c rhizomes 0.6 roots a a stems 0.5 a a a a a a a a a a a a a a leaves 0.4 a a a a a a a a 0.3 a a a a a a a a 0.2 mg(Al)/g(biomass) 0.1 0 Control NCNB WCNB NCAB WCAB NCFB WCFB NCAFB WCAFB Figure 4.112 Al concentration in the organs of reeds collected from site E cultured in solution for (a) 4 (b) 8 (c) 12 weeks. Error bar represented the standard deviation of triplicate samples. Different letters on the same plant organ indicate a significant difference at p<0.05. 233 Organic acids such as CA, can form soluble complex with metals and may modify the fixation and mobility of soil, then enhance their phytoremediation (Chen et al., 2003). CA may also reduce the toxicity of some metals such as lead and cadmium and then increase their translocation from root to shoot (Chen et al., 2003). CA and other organic acids may reduce Mn(IV) to Mn(II) in the rhizosphere, increases the solubility of Mn in soil, and then consequently enhances its accumulation by the plants (Najeeb et al., 2009). The solubility of iron in soils is modified by the complexation and redox reactions of Fe3+ oxides,as the formation of Fe3+ and CA complex can avoid the precipitation of iron and increase its solubility (Escoda et al., 1999). It was also reported that the dominant metal ions complexed with CA in acid soil was Fe and CA may help to reduce the toxicity of Al (Jones and Brassington, 1998). This may also occur in aqueous environments. However, there were also researches indicated that CA may inhibit the uptake or translocation of metals. Duarte et al. (2007) found that Ni uptake in Halimione portulacoides decreased with an increase of CA, which may due to that CA caused an excess of nickel mobilization and turned this metal from a nutrient state to a phytotoxic form. In our study, Fe and Mn accumulation and translocation were enhanced by CA, whereas Al uptake was not influenced. According to our calculation (Table 4.23) based on the stability constants of complexes between metals and ligands and the methods provided by Morel and Hering (1994), when 9.02 g CA/L was added into MM solution, most of CA was complexed with Fe and Mn. It was in accordance with our results of plant digestion that CA can increase the uptake of Mn and Fe by forming 234 complex. The effectiveness of chelators may depend on the species of plants, the amounts of chelators, the types and concentration of metals and pH etc. (Kim and Lee, 2010; Jones and Brassington, 1998). The rhizosphere bacteria did have a significant role (p<0.05) in affecting the accumulation of Fe and Mn in reeds cultured in different solution. The Fe(II)OB appeared to inhibit the accumulation of Fe and Mn in reeds. The roots and rhizomes grown in solution NCNB for 4 weeks uptake 0.06±0.01mg/g and 0.04±0.01 mg/g Mn, which was significantly higher than (p<0.05) the Mn levels in roots and rhizomes cultured in solution NCFB for the same period (0.03±0.00 mg/g and 0.02±0.201 mg/g, respectively). The stems and leaves cultured in solution WCNB for 12 weeks accumulated 0.09±0.01 mg/g and 0.06±0.00 mg/g Mn, respectively. It was significantly higher than (p<0.05) that in stems and leaves of reeds cultured in solution WCFB for 12 weeks (0.05±0.00 mg/g and 0.04±0.00 mg/g, respectively). The roots and rhizomes cultured in solution NCNB for 8 weeks accumulated 33.24±0.52 mg/g and 20.72±2.01 mg/g Fe. It was significantly higher than (p<0.05) that in roots and rhizomes of reeds cultured in solution NCFB for the same time (26.42±0.96 mg/g and 15.25±1.72 mg/g, respectively). The stems and leaves of reeds grown in solution WCNB for 12 weeks uptake 3.11±0.07 mg/g and 2.72±0.01 mg/g Fe, which was significantly higher than (p<0.05) the Fe concentrations in stems and leaves cultured in solution WCFB (2.06±0.06 mg/g and 1.85±0.05 mg/g, respectively). 235 However, the Fe and Mn levels in reeds cultured in solution only amended with acidophilic heterotrophs were not significantly different (p>0.05) from that in reeds without inoculated with rhizosphere bacteria. The roots and rhizomes cultured in solution NCNB for 4 weeks accumulated 0.06±0.01 mg/g and 0.04±0.01 mg/g Mn. It was not significantly different from (p<0.05) that in roots and rhizomes of reeds cultured in solution NCAB for the same time (0.05±0.01 mg/g and 0.03±0.01 mg/g, respectively). The stems and leaves of reeds grown in solution WCNB for 12 weeks uptake 3.11±0.07 mg/g and 2.72±0.01 mg/g Fe, which was not significantly different from (p>0.05) the Fe concentrations in stems and leaves cultured in solution WCAB (3.00±0.02 mg/g and 2.68±0.01 mg/g, respectively). As discussed before, iron plaque mediated by Fe(II)OB may increase or inhibit the metal accumulation in plants. Uptake of arsenite by rice was enhanced by the iron plaque, while arsenate accumulation was dramatically inhibited by iron plaque (Chen et al., 2005). Cu concentrations were low in Lobelia dortmanna and Phragmites australis in the presence of plaque (Liu et al., 2010). In this study, Fe(II)OB may inhibit the metal accumulation of Fe and Mn in reeds. This may be attributed to the numbers of Fe(II)OB and amounts of Fe plaque. The Fe plaque may not inhibit metal ion uptake effectively, when the amounts of metal ions were higher than the adsorption capacity of the plaque (Ye et al., 1997b). As discussed in section 4.5.1, Fe(II)OB significantly enhanced the formation of Fe plaque, so it was not surprising that the uptake of Fe and Mn were lower in reeds cultured in solution inoculated with Fe(II)OB. Acidophilic heterotrophs can not oxidize Fe(II) into Fe plaque, thus they 236 did not affect the Fe and Mn accumulation in reeds. In addition, as discussed in section 4.5.1, the cooperative relationship existed between acidophilic heterotrophs and Fe(II)OB. That was why Fe and Mn uptake in reeds cultured in solution inoculated with both acidophilic heterotrophs and Fe(II)OB were also lower than that in reeds grown in solution without adding rhizosphere bacteria or only inoculated with acidophilic heterotrophs. For example, the roots and rhizomes of reeds grown in solution NCNB for 12 weeks uptake 0.10±0.01 mg/g and 0.07±0.01 mg/g Mn, which was significantly higher than (p<0.05) the Mn concentrations in stems and leaves cultured in solution NCAFB (0.06±0.00 mg/g and 0.05±0.01 mg/g, respectively). The Fe levels in stems and leaves of reeds grown in solution NCAB for 12 weeks (1.92±0.06 mg/g and 1.69±0.02 mg/g Fe), was also significantly higher than (p<0.05) than that in stems and leaves cultured in solution NCAFB (1.23±0.03 mg/g and 1.94±0.01 mg/g, respectively). However, the accumulation and translocation of Al was not affected by rhizosphere bacteria and metal plaque (p>0.05). This was contradictory with the findings of Chen et al. (2005) who indicated that Al translocation in rice were significantly decreased with iron plaque. The role of metal plaque may depend on various factors, such as the types and concentrations of metals and pH (Ye et al., 1997b). The low pH of solution in our study may reduce the adsorption of Al by Fe or coprecipitations of Al with Fe, thus maintain the bioavailability of Al to plants. In addition, Al was not known to participate in any biological functions (Singh et al., 237 2005). That may also be one reason that Al uptake was not influenced by rhizosphere bacteria. The shoot concentrations of metals in reeds were calculated. The translocation factors of metals in reeds were also presented in Table 4.32. As expected, CA significantly increased (p<0.05) the shoot concentrations and translocation factors of Mn and Fe. For instance, the shoot concentrations of Mn and Fe in reeds cultured in solution NCNB for 4 weeks were 0.02±0.01 mg/g and 1.63±0.05 mg/g, respectively. It was lower than that in reeds cultured in solution WCNB for the same time (0.05±0.01 mg/g and 2.69±0.12 mg/g). However, the shoot concentrations of Al in reeds were not enhanced by CA, since most CA were complexed with Fe and Mn. It was in accord with our above findings that the accumulation and uptake of Al was not influenced by CA. For example, the shoot concentrations of Al in reeds cultured in solution NCAB for 4 weeks were 0.16±0.01 mg/g, which was similar to that in reeds grown in solution WCAB (0.17±0.02 mg/g). The rhizosphere bacteria also had significant influence (p<0.05) on the shoot concentrations and translocation factors of Mn and Fe. Reeds in solution added with Fe(II)OB had lower Fe and Mn in shoots than reeds in solution without inoculating with Fe(II)OB or only inoculating with acidophilic heterotrophs. For instance, the shoot concentrations of Mn and Fe in reeds cultured in solution NCAB for 4 weeks were 0.02±0.00 mg/g and 1.56±0.09 mg/g, respectively, which was higher than that in reeds cultured in solution NCFB (0.01±0.00 mg/g and 0.95±0.07 mg/g). The translocation factors of Mn and Fe were lower in reeds grown in solution added with 238 Fe(II)OB than that in reeds cultured in solution without adding Fe(II)OB. For instance, the translocation factors of Mn and Fe in reeds cultured in solution NCAB for 4 weeks were 0.40±0.00 and 0.06±0.01, which were higher than that in reeds cultured in solution NCFB (0.22±0.08 and 0.04±0.00). Nevertheless, the shoot concentrations and translocation factors of Al in solution inoculated with different bacteria were similar (p>0.05) to that in solution without adding bacteria. These phenomena were in accord with our above results of plant digestion: CA increased the accumulation of Mn and Fe in stems and leaves of reeds while had no influence on Al uptake in reeds due to stability constants of complexes between metals and chelators; rhizosphere Fe(II)OB enhanced the formation of Fe plaque which inhibited the entering of Fe and Mn into biomass of reeds but had no influence on Al accumulation in different tissues of reeds. Table 4.32 Shoot concentration and translocation factor of metals in reeds collected from site E cultured in solution for 4, 8 and 12 weeks Shoot Shoot Shoot Treat Week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.02±.01 1.63±.05 0.16±.02 0.40±.00 0.06±.01 0.48±.07 4 b cd a abcd bc a 0.04±.01 1.86±.09 0.23±.02 0.43±.03 0.05±.00 0.52±.05 NCNB 8 b b a ab ab a 0.06±.00 1.90±.05 0.26±.02 0.48±.00 0.05±.00 0.61±.09 12 b bc a abc b a 0.05±.01 2.69±.12 0.16±.02 0.54±.02 0.08±.00 0.48±.01 4 a a a ab ab a 0.07±.01 2.88±.16 0.23±.03 0.58±.06 0.07±.00 0.52±.01 WCNB 8 a a a a a a 0.09±.01 3.11±.07 0.27±.01 0.64±.04 0.07±.00 0.63±.03 12 a a a a a a 239 Table 4.32 (cont.) Shoot concentration and translocation factor of metals in collected from site E cultured in solution for 4, 8 and 12 weeks Shoot Shoot Shoot Treatment Week Mn Fe Al TF (Mn) TF(Fe) TF(Al) (mg/g) (mg/g) (mg/g) 0.02±.00 1.56±.09 0.16±.01 0.41±.04 0.06±.00 0.48±.03 4 bc cd a abc cd a 0.04±.01 1.81±.05 0.24±.02 0.46±.07 0.05±.00 0.52±.01 NCAB 8 bc b a ab ab a 0.06±.01 1.92±.06 0.29±.01 0.48±.02 0.05±.00 0.60±.01 12 b c a abc b a 0.04±.01 2.43±.11 0.17±.02 0.55±.01 0.08±.00 0.48±.01 4 a b a a a a 0.05±.01 2.67±.05 0.22±.03 0.58±.02 0.07±.00 0.52±.04 WCAB 8 a a a a a a 0.08±.00 3.00±.02 0.26±.02 0.63±.04 0.07±.00 0.61±.11 12 a a a ab a a 0.01±.00 0.95±.07 0.17±.03 0.22±.08 0.04±.00 0.49±.07 4 d e a cd d a 0.02±.01 1.22±.17 0.22±.01 0.32±.02 0.04±.01 0.51±.05 NCFB 8 d c a b b a 0.03±.01 1.28±.04 0.25±.03 0.42±.03 0.04±.00 0.61±.01 12 c d a c b a 0.02±.00 1.63±.05 0.16±.03 0.35±.02 0.06±.00 0.50±.02 4 bcd c a bcd abc a 0.03±.00 2.01±.09 0.23±.02 0.43±.03 0.06±.00 0.52±.01 WCFB 8 bc b a ab ab a 0.05±.01 2.06±.06 0.26±.02 0.47±.05 0.06±.00 0.62±.04 12 b b a abc a a 0.01±.00 0.91±.05 0.16±.02 0.20±.00 0.04±.00 0.48±.03 4 cd e a d d a 0.02±.00 1.18±.09 0.23±.02 0.31±.03 0.04±.00 0.53±.06 NCAFB 8 d c a b b a 0.03±.00 1.23±.03 0.25±.03 0.42±.02 0.04±.00 0.60±.03 12 c d a c b a 0.02±.00 1.35±.08 0.17±.01 0.35±.02 0.06±.00 0.50±.02 4 bcd d a abcd cd a 0.03±.00 1.84±.17 0.23±.03 0.39±.04 0.06±.00 0.51±.07 WCAFB 8 c c a ab ab a 0.04±.00 1.92±.05 0.26±.02 0.45±.03 0.06±.00 0.60±.05 12 bc bc a bc a a / indicated blow detection limits; results were average±standard deviation, n=3. 240 4.5.5 Histological experiments of reeds collected from site E cultured in solution Histological experiments were conducted for reeds collected from site E cultured in solution. The following sections discussed the Fe and Al staining results. 4.5.5.1 Fe staining for reeds collected from site E cultured in solution Cross sections of roots and rhizomes of reeds grown in solution were cut and stained. Figure 4.113 shown stained roots and rhizomes cultured in control solution. 200µm 50µm a b Figure 4.113 Fe stained cross section of reeds grown in control solution (a) root (b) rhizome Although the control solution did not contain Fe, the cross sections of root and rhizomes of reeds grown in control solution turned blue after staining which indicated that Fe was accumulated into the tissues of reeds. It was due to the fact that reeds were collected from contaminated site E and Fe was already accumulated into the reeds. Fe was mainly sequestered in the exodermis, aerenchyma and endodermis of roots and the exodermis of rhizomes cultured in control solution. Figure 4.114 shown the stained roots cultured in treatment solution. Compared with roots of reeds cultured in control solution, the intensity of blue hue in roots of reeds grown in treatment solution was stronger. It was not surprising, since Fe was added into treatment solution and more Fe was accumulated into the roots of reeds. 241 200µm 200µm a b a b 200µm 200µm c d 200µm 200µm e f 200µm 200µm g h Figure 4.114 Fe stained cross section of roots of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e) NCFB (f) WCFB (g) NCAFB (h)WCAFB 242 According to Deng et al. (2004), elements in the underground tissues of wetland plants showed strong positive correlations with the sediment elements. Fe was also mainly stored in exodermis, aerenchyma and endodermis of roots grown in solution without adding CA (Figure 4.114 (a) (c) (e) (g)). According to the study of Vesk et al. (1999), Fe was present at high levels at the root surface and decreased centripetally across the root. Soukup et al. (2002) also reported that the exodermis of reeds can be an effective barrier to restrict the penetration of iron into root tissues. The existence of exodermis and endodermis may play an important role in the protection of the root tissues against of toxic metals (Siqueira-Silva et al., 2012). With the amendment of CA, the intensity of blue hue in exodermis, aerenchyma and endodermis became stronger and the stele of roots also turned blue (Figure 4.114 (b) (d) (f) (h)). This may be due to that citrate can form citrate-Fe complexes which is bioavailable and easier to be accumulated by plants (Jones et al., 1996). These phenomena further proved our results in section 4.5.4 that CA increased the accumulation and translocation of Fe in reeds. Besides, it was also worthwhile to note that the blue hue in roots cultured in solution NCFB and NCAFB were lighter than that in roots grown in solution NCNB and NCFB. Similarly, the blue color in stele of roots in solution WCFB and WCAFB were less than that in roots grown in solution WCNB and WCAB. It may be attributed to the inoculated Fe(II)OB. As discussed in section 4.5.1, Fe(II)OB enhanced the formation of Fe plaque and then inhibited the accumulation of Fe into reeds. 243 The rhizomes of reeds under different treatment conditions were also cut and stained. Figure 4.115 presented the stained rhizomes cultured in solution with or without CA and rhizosphere bacteria. As mentioned above, Fe depicted by a blue hue was mainly stored in the exodermis of rhizomes cultured in control solution without adding Fe. The blue hue around the vascular bundles of rhizomes in control solution was not very obvious. Fe was found in the epidermis, cortex and central cylinder of rhizomes cultured in treatment solution which contained Fe. Also, the intensity of blue in rhizomes cultured in treatment solution was stronger than that in control solution. With the amendment of CA, the area of blue color increased and the intensity of blue hue around the vascular bundles become stronger, since CA increased the accumulation of Fe in rhizomes. NCFB and NCAFB were lighter than that in roots grown in solution NCNB and NCAB. Similar to root cross section, the blue color in rhizome cultured in solution inoculated with Fe(II)OB (Figure 4.115 (e) (f)) was lighter than that in solution without adding Fe(II)OB (Figure 4.115 (a) (b)). These phenomena were in accord with our quantitative data that rhizomes treated with Fe(II)OB uptake less Fe than that without inoculated with Fe(II)OB, as Fe plaque mediated by Fe(II)OB may act as a “barrier” and decrease metal accumulation in plants (Chen et al., 2005). 244 50µm 50µm a b 50µm 50µm c d 50µm 50µm e f 50µm 50µm g h Figure 4.115 Fe stained cross section of rhizomes of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e) NCFB (f) WCFB (g) NCAFB (h)WCAFB 245 4.5.5.2 Al staining for reeds collected from site E cultured in solution Figure 4.116 shown the stained roots and rhizomes cultured in control solution. Since Al was already accumulated in reeds when collected from the contaminated field, magenta hue was found in the exodermis of roots in control solution. The vascular bundles of rhizomes grown in control solution also slightly turned magenta. 200µm 50µm a b Figure 4.116 Al stained cross section of reeds grown in control solution (a) root (b) rhizome Stained cross sections of roots and rhizomes cultured in treatment solution were shown in Figure 4.117 and Fig 4.118. The magenta hue was stronger in roots cultured in treatment solution added with Al than that in control solution without Al. Al was found in the epidermis, cortex and central cylinder of rhizomes in treatment solution (Figure 4.118). The magenta hue around the vascular bundles was also more obvious in rhizome cultured in treatment solutions contained Al than that in rhizome grown in control solution without adding with Al. 246 200µm 200µm a b 200µm 200µm c d 200µm 200µm e f 200µm 200µm g h Figure 4.117 Al stained cross section of roots of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e) NCFB (f) WCFB (g) NCAFB (h)WCAFB 247 50µm 50µm a b 50µm 50µm c c 50µm 50µm j l e f 50µm 50µm g h Figure 4.118 Al stained cross section of rhizomes of reeds grown in solution (a) NCNB (b) WCNB (c) NCAB (d) WCAB (e) NCFB (f) WCFB (g) NCAFB (h)WCAFB 248 Unlike Fe, the intensity of magenta hue in roots and rhizomes cultured in different treatment conditions were not significant different from each other. CA caused Fe enter the stele of roots or increased Fe around the vascular bundles of rhizomes, but did not affect Al. It was not surprising, since our quantitative data of plant digestion also indicated that CA and rhizosphere bacteria did not have significant effect in Al accumulation in reeds. In addition, different metals differ in their distribution in plants (Vesk et al., 1999). For instance, Fe decreased towards the root centre, but Cu, Zn and Pb were increased towards the root centre in water hyacinth (Vesk et al., 1999). 4.5.6 Mass balance for metals in solution cultured with reed collected from site E The mass balances of metals in solution were presented in Table 4.33. The main fates of metals included formation of metal plaque on the root system of reeds, uptake into plants and remained in the solution. The quantified rates of metals were around 80%, since there were some metals precipitated on the surface of bottles, participated into the metabolisms of bacteria (Francis, 1998), absorbed into the bacterial cells (Webster et al., 1998) which were hard to trace. Reeds cultured in solution added with CA accumulated more Mn and Fe into their biomass. For instance, the total Mn and Fe were 1.03 mg and 218.76 mg in plants grown in solution WCNB, while they were 0.73 mg and 162.52 mg in plants cultured in solution NCNB. The plants cultured in solution WCFB accumulated 0.70 mg Mn and 162.74 mg Fe into their biomass while the reeds grown in solution NCFB uptake 0.44 mg Mn and 117.27 mg Fe totally. 249 These results further proved that CA was effective to enhance the Mn and Fe accumulation in reeds. Besides, it was not surprising that reeds cultured in solution added with Fe(II)OB formed more Fe plaque on the roots system. The amounts of Fe plaque attached on reeds cultured in solution NCFB and NCAFB were 271.39 mg and 300.37 mg, which were higher than that on reeds grown in solution NCNB. Table 4.33: Mass balance for metals in solution cultured with reeds collected from site E for 12 weeks Treat Metal Original Plaque In plants Remaining % amounts (mg) biomass in solution Quantified (mg) (mg) (mg) NCNB Mn 10.09 0.38 0.73 7.57 86.0% Fe 5416.83 181.26 162.52 4300.38 85.7% Al 37.52 0.00 3.45 29.08 86.7% WCNB Mn 10.09 0.21 1.03 7.60 87.5% Fe 5416.83 132.44 218.76 4367.17 87.1% Al 37.52 0.00 3.54 29.32 87.6% NCAB Mn 10.08 0.41 0.69 7.54 85.7% Fe 5416.83 186.71 152.40 4301.17 85.7% Al 37.57 0.00 3.56 29.93 89.2% WCAB Mn 10.00 0.22 1.01 7.58 88.2% Fe 5417.00 160.23 218.90 4319.67 86.7% Al 37.46 0.00 3.45 29.80 88.8% NCFB Mn 10.04 0.45 0.44 7.52 83.7% Fe 5416.83 271.39 117.27 4288.00 86.3% Al 37.56 0.00 3.26 29.70 87.8% WCFB Mn 10.03 0.24 0.70 7.53 84.4% Fe 5417.50 190.47 162.74 4299.83 85.9% Al 37.59 0.00 3.42 29.43 87.4% NCAFB Mn 10.05 0.42 0.47 7.54 83.8% Fe 5417.33 300.37 115.01 4276.50 86.6% Al 37.53 0.00 3.43 29.45 87.6% WCAFB Mn 10.00 0.19 0.61 7.55 83.5% Fe 5416.50 174.81 158.25 4297.17 85.5% Al 37.58 0.00 3.40 29.88 88.5% 250 As a widely distributed plant, reeds grows equally well in both unpolluted sites and polluted field and can accumulate some heavy metals more than other aquatic plants (Duman et al., 2007). According to our results, chelator and rhizosphere microbial system can affect the metal uptake in reeds. More research may be needed to investigate the relationships among chelators, rhizosphere bacteria and metal accumulations in reeds grown in AMD contaminated field and to enhance the efficiency of phytoremediation of contaminated media by reeds. 4.5.7 Summary of hydroponic experements cultured with reeds collected from site E Hydroponic experiments for wild reeds collected from site E were used to further assess the effect different bacteria on the metal plaque formation and metal accumulation in “aged” reeds. Fe(II)OB oxidized Fe to form Fe plaque and decreased the pH of solution. CA inhibit the growth of the Fe(II)OB, inhibited the decrease of solution pH and the formation of Fe plaque. The Fe plaque formed on reeds inoculated with acidophilic heterotrophs were not significantly different from that on reeds cultured in solution without adding with rhizosphere bacteria which indicated that acidophilic heterotrophs had no direct influence on Fe plaque formation. However, it may improve the growth of Fe(II)OB by consuming CA which is toxic to Fe(II)OB. Thus the Fe plaque on reeds cultured in solution added with CA and inoculated with Fe(II)OB and acidophilic heterotrophs were higher than that on reeds grown in solution added with CA but only inoculated with Fe(II)OB. 251 Fe(II)OB did have a significant role (p<0.05) in decreasing the accumulation of Fe and Mn in reeds cultured in different solution. However, the Fe and Mn levels in reeds cultured in solution only added with acidophilic heterotrophs were not significantly different (p>0.05) from that in reeds without inoculated with rhizosphere bacteria. The accumulation and translocation of Al was not affected by rhizosphere bacteria and metal plaque (p>0.05). CA significantly enhanced (p<0.05) the Mn and Fe accumulation in both aboveground and belowground tissues of reeds, but had no important role in increasing Al accumulation. Since most of CA complexed with Mn and Fe which had higher concentrations in solution and higher affinity with CA. The results of histological experiments were in accord with the quantitative data. Fe was also mainly stored in exodermis, aerenchyma and endodermis of roots. Fe was found in the epidermis, cortex and central cylinder of rhizomes. The blue color in roots and rhizomes in solution added with Fe(II)OB were lighter than that in roots grown in solution without Fe(II)OB, since Fe(II)OB enhanced the formation of Fe plaque and then inhibited the accumulation of Fe into reeds .With the amendment of CA, the intensity of blue hue in exodermis, aerenchyma and endodermis of roots became stronger and the stele of roots also turned blue. Al depicted by magenta hue was found in exodermis, aerenchyma and endodermis of roots and in the epidermis, cortex and central cylinder of rhizomes. The color and area of magenta hue did not change with rhizosphere bacteria or CA, since they had no significant effect on Al accumulation, as shown in quantitative data. 252 4.5.8 Comparison between MM hydroponic experements cultured with reeds propagated from wild rhizomes and with wild reeds collected from site E Some similar results were found between MM hydroponic solutions cultured with reeds propagated from wild rhizomes and solutions wild reeds collected from site E. For example, no Al plaque was formed on root systems of reeds in both experiments. CA increased the Fe and Mn uptake in reeds while it had no significant effect on Al accumulations. The biggest difference between these two experiments was that more metal plaque were formed and more metals were accumulated into wild reeds collected from the site E than reeds developed from the wild rhizomes. The amounts of metal on wild reeds were 0.38 mg Mn and 181.26 mg Fe, while the Mn and Fe plaque were 0.32 mg and 88.74 mg on reeds propagated from wild rhizomes in solution NCNB. Wild reeds accumulated 0.73 mg Mn, 162.52 mg Fe and 3.45 mg Al, while reeds propagated from wild rhizomes uptake 0.14 mg Mn, 51.85 mg Fe and 0.76 mg Al from solution NCNB. This may due to the difference in plants. It was reported that plant structure may differ in adult plants and seedlings since adult plants may have better developed aerating system in roots than the seedlings (Ye et al., 1997a). The aerating system played an important role in oxygen transport and the formation of Fe plaque (Weis and Weis, 2004). That was why more metal plaque was formed on wild reeds than that on reeds seedling propagated from the wild rhizomes. As discussed in section 4.4.6, metal uptake may be different in adult plants and seedlings since adult plants had bigger biomass and may be more tolerant to toxic metals (Ernst, 2006). Thus more metals were accumulated in wild reeds that reeds 253 propagated from wild rhizomes cultured in hydroponic solution under similar conditions. 254 CHAPTER V CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions AMD is a serious environmental issue which affects many regions of the United States and other countries. This study conducted field and laboratory experiments to investigate the bioremediation potential of an AMD contaminated site via different types of Phragmites australis (reeds directly collected from AMD contaminated site or reeds propagated from purchased non-contaminated rhizomes or developed from rhizomes collected from AMD site), rhizosphere bacteria and CA. The relationships among CA, rhizosphere bacteria and metal accumulations in reeds were found and the overall objective of this study were achieved as outlined in conclusions followed. The results of field experiments indicated that Fe, Al and Mn plaque formed on the root systems of reeds and the uptake of Fe, Al and Mn by reeds did occur in a AMD contaminated site. The amounts of Fe plaque on roots/rhizomes were related to the abundance of Fe(II)OB in rhizosphere soil. The metal concentrations accumulated in the underground tissues of Phragmites australis depicted a strong and positive correlation with the concentrations in soil. The Fe concentrations in soil were 413.63±7.75 mg/g which were much higher than Mn (0.04±0.00 mg/g) and Al (1.39±0.03 mg/g). While the Fe amounts (13.43±5.98 mg/g) 255 in roots were also higher than Mn (0.09±0.03 mg/g) and Al (0.08±0.01 mg/g). The Fe levels in reeds were significantly greater (p<0.05) than Al and Mn, since the soil contained more Fe than Al and Mn. Most of metals were stored in the roots and rhizomes of reeds. According to the results of histological experiments, most of Fe was stored in the exodermis of roots grown in site that contained less Fe, while Fe had extended to the endodermis of roots grown in site with higher levels Fe. Al was found in the exodermis and endodermis of roots collected from site with lower Al concentrations, but the stele of roots grown in site with higher level of Al also contained Al. This may due to different mobility of different metals and translocation of metals also depends on various biological or chemical/physical factors. Besides, the metal uptake also related to the seasonal change. Higher levels of metals were stored in underground parts of reeds in autumn and winter while more metals transported to aboveground tissues of reeds during growth season. In order to study the effect of citric acid and rhizosphere bacteria on metal plaque formation metal uptake in different type of reeds without disrupting the site, wild reeds collected from the AMD contaminated site and reeds propagated from purchased uncontaminated rhizomes were cultured in spiked AMD soil added with different levels of CA and rhizosphere bacteria in greenhouse. Rhizosphere Fe(II)OB had significant effect (p<0.05) to enhance the formation of Fe plaque and decrease the pH of soil, but did not have significant impact (p>0.05) on the formation of Mn and Al plaque on the roots systems of reeds. Fe and Mn plaque did not prevent but restrain the accumulation of Fe and Mn into tissues of Phragmites australis; while the Al 256 uptake did not be influenced by the metal plaque. CA enhanced the numbers of acidophilic heterotrophs, inhibited the growth of Fe(II)OB, and then decreased the formation of metal plaque on the roots systems of reeds. For instance, the Fe plaque (42.77±5.18 mg/g) on roots of wild reeds cultured in soil added with 33.616 g/kg CA and inoculated with Fe(II)OB were lower than that on roots inoculated with Fe(II)OB but without adding CA (108.08±12.05 mg/g). Further, the mobility of metals in soil and the accumulation of Fe, Al and Mn in both underground and aboveground biomass of reeds were elevated by CA treatment. According to the results of histological experiments, low level of CA caused Fe enter the pith of roots, while middle and high level of CA made Fe enter the entire stele of roots. CA also enhanced Al entering stele of reeds and then further increased the accumulation and translocation of metals. The metal accumulations in reeds increased with time. Compared with purchased non-contaminated reeds cultured under the same conditions, wild reeds harvested from an AMD contaminated site accumulated more metals into plant tissues due to the adaptability to the hostile environments. Wild reeds cultured in soil added with 33.616 g/kg CA for 3 months accumulated 0.32±0.01 mg/g Mn, 96.99±5.75 mg/g Fe and 3.17±0.51 Al in roots, while purchased reeds uptake 0.20±0.00 mg/g Mn, 79.21±5.95 mg/g Fe and 0.74±0.02 Al mg/g. Phragmites australis can grow in both terrestrial and hydroponic habitats and hydroponic experiments have less mass transfer limitations than that associated with soil. So reeds propagated from wild rhizomes were cultured in hydroponic solutions added with different level of metals, CA and rhizosphere bacteria. Fe plaque was 257 observed on the roots/rhizomes of reeds cultured in LM, MM and HM solution. No Mn plaque was found around the root systems of reeds cultured in LM solution and no Al plaque were formed on reeds cultured in LM, MM and HM solutions. These phenomena may due to the low pH and low concentrations of Mn and Al in solutions. Fe plaque mediated by rhizosphere Fe(II)OB did not significantly influence (p>0.05) the uptake of metals into tissues of reeds cultured in LM solution, but significantly inhibit (p<0.05) the accumulation of metals in reed cultured in MM and HM solution. This may be related to the amounts of Fe plaque which were higher in solution added with high level of metals. CA significantly (p<0.05) inhibited the growth of Fe(II)OB and decreased the formation of Fe plaque. CA significantly improved (p<0.05) the accumulation of Fe and Mn in reeds grown in LM, MM and HM solutions, but had no important influence in Al accumulation in reeds cultured in MM and HM solution. For instance, reeds cultured in HM solution added with 16.979 g/L CA for 3 months accumulated 0.15±0.00 mg/g Mn, 47.72±1.57 mg/g Fe and 0.61±0.01 Al in roots while reeds cultured in solution without adding CA uptake 0.10±0.01 mg/g Mn, 31.75±2.37 mg/g Fe and 0.60±0.03 Al in roots. This may due to the fact that CA had higher affinity for Fe and Mn and the Fe concentrations were much higher than Al in MM and HM solution. In order to further assess the function of different rhizosphere bacteria on metal plaque formation and metal uptake, reeds collected from the AMD contaminated sites were cultured in MM solutions added with 9.020 g/L CA, acidophilic heterotrophs and/ or Fe(II)OB. As expected, CA inhibited the growth of Fe(II)OB and the 258 formation of Fe plaque. Further, CA increased the Mn and Fe accumulation and translocation in reeds. Reeds cultured in solution added with 9.020 g/L CA for 3 months accumulated 0.12±0.01 mg/g Mn, 44.58±0.60 mg/g Fe and 0.38±0.00 Al in roots while reeds cultured in solution without adding CA uptake 0.10±0.01 mg/g Mn, 37.81±0.94 mg/g Fe and 0.40±0.08 Al in roots. Acidophilic heterotrophs had no direct influence on the metal plaque formation and metal uptake in reeds. However, acidophilic heterotrophs can consume CA and make the environment became more suitable for the growth of Fe(II)OB. Thus, the numbers of Fe(II)OB and the amounts of Fe plaque were higher in rhizosphere of reeds cultured in solution inoculated with both Fe(II)OB and acidophilic heterotrophs than that in solution solely added with Fe(II)OB. Fe(II)OB enhanced the formation of Fe plaque and then inhibited the accumulation of Fe into reeds. The results of histological experiments also corroborated that roots and rhizomes treated with Fe(II)OB uptake less Fe than that without inoculated with Fe(II)OB. 5.2 Recommendations According to the results of this study, CA, rhizosphere bacteria and Phragmites australis had interconnected impacts on remediation of AMD sites. Phragmites australis which is known to be widely distributed in the world be able to survive under acidic conditions and accumulate some heavy metals distinctly more than other wetlands plants. Reed especially that initially grown in contaminated sites has strong adaptability which make it a good candidate for phytoremediation technique. 259 CA is the main factor to increase the metal mobility in soil and enhance metal uptake in reeds. Low level of CA may not have long term effect due to the consumption of bacteria. Also, low level of CA may not sufficient to form enough complex with metals and then increase the mobility of metals. Therefore, the use of 2.101 g/kg CA is not recommended for future application. The results indicated that the higher level of CA added into the AMD contaminated soil or solution, the more metal uptake into reeds. 33.616 g/kg CA was the best amount to increase metal accumulations in reeds in laboratory experiments. According to this study, Fe(II)OB and acidophilic heterotrophs should not be further inoculated into the AMD site, since the Fe plaque formed by Fe(II)OB can inhibit the metal uptake in plants while acidophilic heterotrophs can consume CA and enhance the growth of Fe(II)OB and formation of metal plaque. Based on the information at this time, it is recommend to use CA in conjunction with reeds to remediate other AMD contaminated sites. First, a thorough site characterization should be conducted to assess the type and concentrations of metals at the site. Second, the suitable amounts of CA needed should be determined depending on the levels of metals at sites. After application of CA to sites, reeds especially the adult reeds initially grown in AMD contaminated environments can be transferred to the sites needed to be cleaned to grow for certain time periods to uptake heavy metals. 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