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Bioremediation of Acid Mine Drainage Contaminated Soil By 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
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