phytoremediation of iron cyanide complexes in soil and groundwater

PHYTOREMEDIATION OF IRON CYANIDE COMPLEXES IN SOIL AND GROUNDWATER A Dissertation Submitted to the Faculty ofPurdue University byDong-Hee Kang In Partial Fulfillment of the Requirements

For the degree of

Final examining committee members

, Chair

Approved by Major Professor(s):

Approved by Head of Graduate Program:

Date of Graduate Program Head's Approval:

3239788 2007

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PHYTOREMEDIATION OF IRON CYANIDE COMPLEXES IN SOIL AND

GROUNDWATER

A Dissertation Submitted to the Faculty

ofPurdue University

byDong-Hee Kang

In Partial Fulfillment of the Requirements for the Degree

ofDoctor of Philosophy

August 2006 Purdue University West Lafayette, Indiana

ACKNOWLEDGMENTS

There are many people who I would like to thank for their contribution to this dissertation First and foremost, I would like to thank my advisor, Professor Katherine Banks of the School of Civil Engineering for her continuous support of my Ph.D program Professor Banks always gave me clarity when I had a question about my research or writing She consistently allowed this dissertation to be my own work and guided me in the right direction She showed me different ways to approach a research problem and the need to be persistent to accomplish any goal She is most responsible for assisting me with the writing of this dissertation as well as directing me towards a challenging research project Without her encouragement and constant guidance, I could not have finished this dissertation I sincerely want to thank Professor Paul Schwab of the Department of Agronomy for his assistance with the statistical analyses and analytical methods development He always made me comfortable and provided truthful advice about my future when I stopped by his office Thanks also to Professor Rao S Govindaraju of the School of Civil Engineering for asking insightful questions to help me think through my research direction I wish to thank Professor Cliff Johnston of the Department of Agronomy who offered guidance and research direction on the adsorption experimental design In addition, I would like to thank Professor James Alleman, Chair of

the Department of Civil, Construction, and Environmental Engineering at Iowa State University for instruction on the toxicity assays

I would like to acknowledge my colleagues from the Banks research group for their assistance and support during my doctoral work I would especially like to thank James Hunter, who gave me confidence when I doubted myself and helped me develop innovative research ideas (More importantly, he taught me to how to play hard and control stress!) I also would like to thank my fantastic coworker, Lee-Yen Hong, for her friendship, encouragement, and research discussions Also, many thanks to my other friends at Purdue University for their support: Yong Sang Kim, Sybil Sharvelle, Agnes Szlezak, Eric McLamore, and Jason Hickey I also appreciate the help provided by Dr Changhe Xiao with his patient explanations during instrument repair

Special recognition goes to Dr Mi-Youn Ahn, who provided insightful comments and reviewed my work on very short notice Also, thanks to Dr Andrew R Zimmerman (Assistant Professor of Geological Sciences at the University of Florida), who gave me useful information about oxidation and enzyme activity I want to thank Professor Won Chul Cho of the Department of Civil Engineering at Chung-Ang University (Seoul, Korea) who was my MS advisor for his continuous support Also, I appreciate the assistance of Dr David Tsao (BP Corporation, IL) and Dr Wang-Cahill Fan for guidance on the design of greenhouse study and financial support of the project

Finally, I would like to thank my lovely wife and best friend, Hye Jeong Lee, for her patience and for keeping my life in proper perspective and balance, and my parents for their endless encouragement and constant support

TABLE OF CONTENTS

Page

LIST OF TABLES vii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS xi

ABSTRACT xii

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 4

2.1 Manufactured Gas Plants Sites 4

2.2 Classification of Cyanide Compounds 6

2.3 Cyanide Toxicity 7

2.4 Solubility of Iron Cyanide Complexes 8

2.5 Fate and Transport of Cyanide 9

2.6 Microbial Degradation of Cyanide 11

2.7 Phytoremediation of Cyanide Contaminants 13

2.8 Modeling of Phytoremediation Processes 17

2.9 References 19

CHAPTER 3 DISSERTATION OBJECTIVES AND HYPOTHESES 33

CHAPTER 4 SELECTION OF PLANT VARIETIES FOR PHYTOREMEDIATION OF IRON CYANIDE COMPLEXES 35

4.1 Introduction 36

4.2 Materials and Methods 39

4.2.1 Soil Preparation 39

4.2.2 Plant Species 39

4.2.3 Germination Assay 40

4.2.4 Root Characteristics 41

4.2.5 Statistical Analysis 41

4.3 Results and Discussion 42

4.3.1 Germination 42

4.3.2 Root Characteristics 44

4.4 Conclusions 45

4.5 References 47

CHAPTER 5 PHYTOREMEDIATION OF IRON CYANIDE COMPLEXES USING CYANOGENIC AND NON-CYANOGENIC PLANT SPECIES 56

5.1 Introduction 57

5.2 Materials and Methods 59

5.2.1 Soil Preparation 59

5.2.2 Plant Selection 60

5.2.3 Greenhouse Methods 60

5.2.4 Analysis of Cyanide 61

5.2.5 Toxicity Assay 62

5.2.6 Statistical Methods 63

5.3 Results and Discussion 64

5.4 Conclusions 68

5.5 References 70

CHAPTER 6 SORPTION OF IRON CYANIDE COMPLEXES ONTO CLAY MINERALS, MANGANESE OXIDES, AND SOIL 80

6.1 Introduction 81

6.2 Materials and Methods 83

6.2.1 Soil Preparation 83

6.2.2 Clay Mineral Preparation 83

6.2.3 Manganese Oxide Synthesis 84

6.2.4 Adsorption 84

6.2.5 Cyanide Analysis 85

6.2.6 Acid Extraction 85

6.2.7 CEC and AEC 86

6.3 Results and Discussion 87

6.4 Conclusions 90

6.5 References 92

CHAPTER 7 THE ROLE OF TRAMETES VILLOSA LACCASE ON OXIDATION AND ADSORPTION OF FERROCYANIDE 100

7.1 Introduction 101

7.2 Materials and Methods 103

7.2.1 Materials 103

7.2.2 Enzyme Reactions 104

7.2.3 Adsorption Assessment 104

7.2.4 Cyanide Analysis 105

7.2.5 Laccase Analysis 106

7.3 Results and Discussion 106

7.4 Conclusions 110

7.5 References 111

CHAPTER 8 EFFECT OF PLANTS ON LANDFILL LEACHATE CONTAINING CYANIDE AND FLUORIDE 120

8.1 Introduction 121

8.2 Description of Field Site 125

8.3 Materials and Methods 128

8.3.1 Plant Selection 128

8.3.2 Soil and Leachate Analysis 128

8.3.3 Fluoride and Cyanide Adsorption 129

8.3.4 Greenhouse Study 130

8.3.5 Cyanide Analysis for Soil and Plant Biomass 130

8.3.6 Fluoride Analysis for Soil and Plant Biomass 131

8.3.7 Assessment of Root Characteristics 132

8.3.8 Statistical Analysis 133

8.4 Results and Discussion 133

8.4.1 Leachate Toxicity 133

8.4.2 Root Characteristic 137

8.4.3 Fluoride and Cyanide Adsorption 138

8.4.4 Soil pH 138

8.4.5 Fluoride Concentration 140

8.4.6 Cyanide Concentration 141

8.5 Conclusions 142

8.6 References 144

CHAPTER 9 CONCLUSIONS AND FUTURE RESEARCH 165

9.1 Conclusions 165

9.2 Future Research 167

APPENDIX 169

VITA 196

LIST OF TABLES

Table Page Table 2.1 Total Cyanide Concentrations in Contaminated

Soil and Groundwater 25

Table 2.2 Environmental Cyanide Compounds 26

Table 2.3 Potential Risks from Daily Exposure to Cyanide Compounds 27

Table 2.4 Equilibrium Reactions and Constants (log Ko) 28

Table 2.5 Adsorption of Iron Cyanide Complexes 29

Table 2.6 Plants Used in Phytoremediation Applications 30

Table 2.7 Phytoremediation of Cyanide 31

Table 4.1 Soil Characteristics 50

Table 4.2 Plant Varieties 51

Table 4.3 Germination Assays 52

Table 4.4 Root Characteristics of Surface Area, Average Diameter, and Tips 53

Table 5.1 EC50 (%) and Cyanide Concentration of Leachate 74

Table 5.2 EC50 (%) of Soil Samples 75

Table 5.3 Overall Mass Balance of Cyanide after 4 Months (%) 76

Table 6.1 Acid Extractable Aluminum, Calcium, Iron, Magnesium, and Manganese 95

Table 6.2 Freudlich Isotherm Parameters as a Function of Sorbents 96

Table 7.1 Freundlich Adsorption Isotherm Parameters 114

Table Page Table 8.1 Chemical Characteristics for Soils Surrounding the Landfill Site 151

Table 8.2 Sebree Landfill Leachate Composition 152

Table 8.3 Cyanide and Fluoride Concentrations in Plants 153

Table 8.4 Initial Cyanide and Fluoride Concentrations in Plant Samples

Before Initiation of Greenhouse Study 154

Table 8.5 Cyanide and Fluoride Concentrations in Leachate and Soil for

Greenhouse Study 155

Table 8.6 Plant Height as Affected by Landfill Leachate 156

LIST OF FIGURES

Figure Page Figure 2.1 Enzymatic Reactions Converting Free Cyanide into Asparagines 32

Figure 4.1 Germination of Sorghum, Switchgrass, and Flax after 7 Days

Exposure to Control, 500 mg/kg, and 1000 mg /kg Total Cyanide 54

Figure 4.2 Root Growth of Sorghum, Switchgrass, and Flax after 4 Weeks

Exposure to Control and 1000 mg /kg Total Cyanide 55

Figure 5.1 Cyanide Concentrations in Plant Biomass and Soil Samples

after 4 Months Exposure to 1000 mg /kg Total Cyanide A) Cyanide

Concentrations in Roots B) Cyanide Concentrations in Plants C)

Cyanide Concentrations in Soil Samples 77

Figure 5.2 Water Soluble Cyanide Concentrations in Soil after 4 Months

of Exposure to 1000 mg/kg Total Cyanide A) Water Soluble Cyanide

Concentrations in Soil Samples B) Average Fraction of Water Soluble Cyanide 78

Figure 5.3 Cyanide Concentrations in Soil after 4 Months of Exposure

to 1000 mg/kg A) Weak Acid Dissociable Cyanide Concentrations in Soil

B) Average Fraction of Weak Acid Dissociable Cyanide 79

Figure 6.1 Freundlich Isotherms for Clay Minerals and ACE and CEC Curve 97

Figure 6.2 Freundlich Isotherms for Prussian Blue on Manganese Oxides

as Affected by pH 98

Figure 6.3 Freundlich Isotherms for Prussian Blue on Drummer Soil

as Affected by pH 99

Figure 7.1 A) Disappearance of Ferrocyanide in the Presence of T Versicolor

Laccase as Influenced by Reaction Time B) pH Effect on Laccase Mediated

Disappearance of Ferrocyanide C) Effect of T Versicolor Laccase Activity

and Initial Concentration of Ferrocyanide on Oxidation 115

Figure Page Figure 7.2 Adsorption Isotherms of Iron Cyanide on Aluminum

Hydroxide at pH 3.7, 6.2 and 8.4 as Affected by Laccase 116

Figure 7.3 Adsorption Isotherms of Iron Cyanide on Montmorillonite at pH 3.7, 6.2 and 8.4 as Affected by Laccase 117

Figure 7.4 Adsorption Isotherms of Ferricyanide and Ferrocyanide on A) Aluminum Hydroxide or B) Montmorillonite at pH 3.7, 6.2 and 8.4 as Affected by Laccase 118

Figure 7.5 Laccase Adsorption on Aluminum Hydroxide or Montmorillonite at pH 3.7, 6.2 and 8.4 119

Figure 8.1 Sebree Landfill Site and Tree Establishment Areas 157

Figure 8.2 Cyanide Concentrations as a Function of Fluoride Concentration in Solution After Exposure to Soil at Two pH Levels 158

Figure 8.3 Fluoride Concentrations as a Function of Cyanide Concentration in Solution After Exposure to Soil at Two pH Levels 159

Figure 8.4 Daily Irrigation Rate per Species 160

Figure 8.5 Root Characteristics after Exposure to Landfill Leachate 161

Figure 8.6 Soil pH vs Leachate Concentration 162

Figure 8.7 Fluoride Concentrations in Soil and Plant Biomass A) Soluble Fluoride Concentration in Soil B) Fluoride Concentration in Roots C) Fluoride Concentration in Stems D) Fluoride Concentration in Leaves 163

Figure 8.8 Cyanide Concentrations in Soil and Plant Biomass A) Cyanide Concentration in Soil B) Cyanide Concentration in Roots C) Cyanide Concentration in Stems D) Cyanide Concentration in Leaves 164

LIST OF ABBREVIATIONS

ANOVA: analysis of variance

AOAC: Association of Official Analytical Chemists

APHA: American Public Health Association

ATP: adenosine triphosphate

AWWA: American Water Works Association

BET: surface analysis method (Brunauer, Emmett and Teller, 1938)

CDTA: cyclohexylene diamine tetraacetic acid

MGP: manufactured gas plant

NRT: normalized relative transpiration

PZC: Isoelectric point (pI) is the pH at which a molecule carries no net electrical charge

(Point Zero Charge)

RCRA: Resource Conservation Recovery Act

RSG: relative seed germination

SPL: spent potliner material

SPT: microtox solid phase test

STD: standard deviation

TISAB: total ionic strength adjustment buffer

WAD: weak acid dissociable cyanide

contaminated soil and groundwater Iron cyanides are often predominant in environmental samples and have low toxicity Unfortunately, free cyanides are the thermodynamically favorable species in solution, and degradation of iron cyanide compounds to the free cyanides can be accelerated by sunlight and microorganisms

There were two objectives of this research project The first objective was to investigate the potential for phytoremediation of cyanide contaminated soils using cyanogenic plants The second objective was to assess the fate and transport of cyanide compounds in vegetated soil The results indicate that germination and root growth for cyanogenic plants were higher than for the non-cyanogenic plant in the presence of cyanide In addition, root biomass had higher cyanide concentrations than plant shoots After 4 months of plant growth, soil cyanide concentration was reduced approximately 17~32% The mineral sorption capacity for cyanide was greatest for clay at low pH

Acid extractable elements also enhanced the adsorption capacity of the clays Manganese oxide and laccase enhanced oxidation of ferrocyanide to ferricyanide, resulting in a more mobile contaminant In addition, the use of phytoremediation to reduce landfill leachate volume, and cyanide and fluoride concentrations in groundwater was assessed Cyanide was degraded by the plants while fluoride accumulated in plant biomass The results reported in this dissertation can be used in the design of phytoremediation projects for cyanide impacted soil and groundwater

CHAPTER 1.

INTRODUCTION

Cyanide is a deadly poison that can result in human respiratory failure The

Cyanide generally refers to all compounds containing the –CN group which has a triple bond between carbon and nitrogen This compound tends to react readily with many chemical elements, subsequently producing a wide variety of cyanide complexes Iron cyanide complexes are common cyanide contaminants

Cyanide pollutants may be harmful to humans as a result of surface runoff and movement through soil into potable groundwater Soil contaminated with cyanide compounds may result from a variety of industrial and municipal activities Sodium

salts as anti-clumping agents Alkaline cyanide solutions are used in heap leaching of gold mining ores Soils contaminated with cyanide complexes are often located near former manufactured gas plant (MGP) sites

Manufactured gas was produced during the period between 1830 and 1950 The production facilities, or manufactured gas plants (MGPs), were used to provide gas for municipal lighting, heating, and residential use The last operational plant was closed in the 1950s At former gas works facilities, the process of degassing coal to produce

natural gas produced H2S and HCN at concentrations ranging from 500 to 1000 mL/m3(Kjeldsen, 1999) These contaminants were scrubbed from the gas using a purification box containing bog iron ore The spent ore with a pH of 2 to 5 and 1 to 2% cyanide (Young and Theis, 1991) was used as fill material in the surrounding area The form of cyanide found at these sites is predominantly iron cyanides, such as Prussian blue [FeIII4(FeII(CN)6)3].

Considerable attention recently has focused on cyanide near manufactured gas sites because of high concentrations in the soil and detection in groundwater (Kiikerich and Avin, 1993; Meeussen et al., 1994; Ghohs et al., 1999) Thermodynamic calculations indicate that free cyanide should predominate the chemical equilibrium in soil and that Prussian blue should govern solubility (Meeussen et al., 1995) However, complexed cyanide is often found in groundwater, indicating that the speciation of cyanide is determined not by chemical equilibrium but by decomposition kinetics (Meeussen et al., 1992) Iron cyanide complexes have low toxicity even at high levels of exposure The rate of conversation of ferrocyanide and ferricyanide complexes proceeds slowly in the dark but is greatly enhanced in the presence of low levels of light Temperature and, to

a lesser degree, pH also can impact degradation rates Therefore, iron cyanide complexes cannot be regarded as completely inert (Meeussen et al., 1994)

Phytoremediation is an innovative technology and a cost effective remediation method that utilizes plants to remove contaminants from soil and water The extraction and accumulation of contaminants in harvestable plant biomass, degradation of complex organic molecules to simple molecules and the incorporation of these molecules into

plant tissue, and stimulation of microbial and fungal degradation by release of exudates/enzymes into the root zone are all proven mechanisms of dissipation

Production of cyanide is not limited to anthropogenic activities; a number of plant species can produce this toxin as well This is of particular problem for food or forage crops that, under storage conditions, produce acutely toxic concentrations of free cyanide as a result of the degradation of cyanogenic glucosides These species of plants have the capability to produce cyanide, and they also possess the capacity to detoxify it; a detoxification capacity that is interesting from the perspective of phytoremediation Although phytoremediation is a popular remediation approach, there are limitations such

as extensive time compared to other remediation methods, difficulty in establishing vegetation, phytotoxicity due to hazardous wastes, and limited remediation depth A major concern about phytoremediation is the lack of understanding of the mechanisms of plant-chemical interactions

There are two objectives of this dissertation research project The first objective is to investigate the potential for phytoremediation of cyanide contaminated soils using cyanogenic plants Cyanogenic plants are those species that synthesize cyanogenic glucosides, compounds that readily decompose to cyanide when plant tissue

is injured Because cyanide is a natural component of these plants, they may possess enhanced capacities for degrading cyanide The second objective is to evaluate the fate and transport of cyanide compounds in planted soil

CHAPTER 2.

LITERATURE REVIEW

2.1 Manufactured Gas Plant Sites

Manufactured gas plants were constructed to provide cost-effective sources of energy for homes and industries from the mid-19th Century until the 1950s The manufacture of coal gas produced many toxic by-products Some of these wastes could

be sold after further processing, others were recycled on-site, and the remaining residual typically disposed of either on-site or at the local trash dump (ERL, 1987) Therefore, significant contamination of soils and groundwater has been reported at gasworks and disposal sites The production of gas from coal was based on the work of Robert Boyle (1691) and William Murdoch (1796) The first gasworks company, the Gas Light and Coke Company, was established in 1812, and supplied gas for light and heat for a large city (Turczynowicz, 1993) Gas usage was quickly adopted globally After 1812, over

1037 separate gas companies operated in the United Kingdom, each with at least one operational site resulting in approximately 3000 to 4000 sites within the UK alone (ERL, 1987; Turczynowicz, 1993)

The first use of manufactured gas in the United States was in Newport, Rhode Island in 1806 The city of Baltimore developed the first gas compamy in 1817 Across

the United States of America, from 1100 to 3000 gaswork sites operated between 1806 to the mid 1970s (Shifrin et al., 1996)

The coal gasification process generated a number of by-products Highly contaminated solid waste was produced from the final stage of gas purification, where the gas was filtered to remove cyanide and hydrogen sulfide Two possible purifier

Cyanides in the raw gas stream also may have been present as ammonium cyanide,

ferrocyanide compounds Ammonium ferrocyanide is soluble in water and readily

ferrocyanide ions can react to form thermodynamically stable ferric ferrocyanide,

Prussian blue is commonly used as a blue pigment in dyes The extensive underground infrastructure of a gas manufacturing plant, comprising of tanks, pipes and foundations for gasholders, also can be considered solid waste Elevated cyanide

concentrations near manufactured gas sites have been detected (Table 2.1) With a drinking water standard of 0.2 mg/L for free cyanide, nearly all of the reported values shown in Table 2.1 would violate the US EPA drinking water regulations if identified in the U.S Another important observation from the data in Table 2.1 is that there is very little relationship between soil and groundwater concentrations This strongly indicates that cyanide concentrations in water are controlled by dissolution/precipitation of solid phases.

2.2 Classification of Cyanide Compounds

dissociable and strong complexes, as shown in Table 2.2 This classification includes

released into solution by the dissolution and dissociation of cyanide compounds and complexes (Smith et al., 1991) In addition to WAD cyanide, other toxicologically important forms of cyanide are free cyanide, sodium cyanide (NaCN), potassium cyanide (KCN), and moderately and weakly complexed metal-cyanides

naturally into the environment by a number of living systems (Fuller, 1985) However,

the concentration of naturally occurring free cyanide is usually less than 0.01 mg/L, and

microorganisms and converted to carbonate and ammonia (Fuller, 1985) Iron-cyanide complexes are not produced by natural sources and their presence in the soil environment

is caused by anthropogenic input (Meeussen et al., 1992)

2.3 Cyanide Toxicity

Cyanide refers to all compounds containing the CN group in which there is a

HCN) is the result of its ability to bind heme iron in the oxygen-binding site of the mitochondrial enzyme, cytochrome oxidase, consequently blocking oxidative energy metabolism Brain cells and heart tissue are most vulnerable to serious damage from diminished oxygen utilization caused by cyanide poisoning (Shifrin et al., 1996) Cyanide does not accumulate in body tissue and therefore, any effects will be acute, chronic or

sub-chronic (US EPA, 1992; Shifrin et al., 1996) Acute cyanide poisoning in humans

may result in convulsions, vomiting, coma and death Chronic effects by exposure to lower concentrations of free cyanide include neuropathy, optical atrophy, and pernicious anemia (Raybuck, 1992) The toxicity of cyanide is dependent upon the species Cyanide may be inhaled, ingested, or adsorbed through dermal contact (Shifrin et al., 1992)

Combined forms of cyanide are quite common and have low toxicity Few studies are published on human and/or animal exposure to iron-complexed cyanides

Published literature strongly suggests that these compounds have low toxicity even at

high levels of exposure due to limited transformation to free cyanide (Shifrin et al., 1996)

The no-observed adverse effect level (NOAEL) of 3200 mg/kg-day was observed for rats exposed to ferric-ferrocyanide in drinking water for 12 weeks This is in contrast to fatal

ferric ferrocyanide for mice is projected to be more than 5000 mg/kg, which is greater than for the Fe (Shifrin et al., 1996) Table 2.3 highlights the potential risks from daily exposure to cyanide compounds The lower toxicity of some compounds appears to be the result of compound stability under acidic conditions and low absorption from the gut (Schmidt-Nielsen, 1990) However, iron cyanide complexes cannot be regarded as completely inert (Meeussen et al., 1994), because iron cyanide complexes are easily decomposed to HCN

2.4 Solubility of Iron Cyanide Complexes

Iron cyanide complexes are the predominant form of cyanide compounds at MGP sites, specifically Prussian blue These cyanide complexes are not stable and tend to decompose to free cyanide Thermodynamically, free cyanide should be the dominant species in soil and groundwater However, iron-cyanide complexes predominate in soil (Meeussen et al., 1992; Theis et al., 1994) due to the relatively slow decomposition rate

in absence of sunlight Table 2.4 shows equilibrium reactions and constants for cyanide species Iron cyanide complexes are slightly soluble under acidic conditions Acidic conditions prevail due to the generation of sulfuric acid at MGP sites The dissociation

constant (Ka) in water is 10-36.9 for ferrocyanide [Fe(CN)64-] and 10-43.9 for ferricyanide

2.5 Fate and Transport of Cyanide

Iron cyanide complexes have not been reported to be toxic Consequently, the

these compounds can be rapidly photodegraded to form toxic HCN (Rader et al, 1993; Meeussen, 1994) The transport of these compounds poses a serious risk of groundwater contamination Prediction of fate of these contaminants is often complicated by their physical characteristics and complex interactions in soil and groundwater The sorption and solubility characteristics of cyanide complexes play a major role in controlling contaminant fate and transport in the subsurface

The chemistry of cyanide in soils has been used to predict the equilibrium concentrations of free cyanide, cyanic acid, and complexed forms in both solution and solid phase (Meeussen et al., 1995; Kjeldsen, 1999) as well as the kinetics of the transformations (Meeussen et al., 1992) The solubility of Prussian blue is highest at moderate redox potential and basic pH Acidic pH favors precipitation of Prussian blue

as does high redox potential (pE>10) or low redox potential (pE<6) Conversion of cyanide complexes to free cyanide species is favored by acidic pH and high redox conditions

Fe-Meeussen et al (1992) stated the following regarding the stability of non-toxic iron-cyanide complexes in soil and groundwater “Iron cyanide complexes are

thermodynamically stable only under conditions which may be considered as rather extreme for soils and the environment, a relatively high pH combined with low redox potential and high total cyanide concentration Iron cyanide complexes present in groundwater will normally tend to dissociate to free cyanide The speciation of cyanide proceeds very slowly Hence speciation of cyanide will be governed by decomposition kinetics rather than by chemical equilibrium.” Thus, high concentrations of iron cyanides

in water will not pose significant risk unless associated with increased kinetics of degradation Decreased pH, exposure to light, and microbial degradation are all factors that could accelerate conversion of iron-cyanide solution complexes into free cyanide

Because cyanide complexes tend to be negatively charged, their movement in contaminated soils have few restrictions (Theis et al., 1994) Iron cyanide complexes have higher solubility and lower sorption capacity at neutral or alkaline pH, but may adsorb somewhat to aluminum oxides and kaolinite (Theis et al., 1986) Ghosh et al (1999a,b) found that the predominant iron cyanide species from MGP residuals are transported as non-reactive solutes in the sand gravel porous medium composing natural aquifers Transport of iron cyanide complexes in the subsurface will be increased under high pH conditions (Shifrin et al., 1996)

The concentration of iron cyanide complexes in soil solutions may be governed

by Prussian blue which has lower solubility and higher sorption capacity below pH 5 Adsorption experiments for iron cyanide complexes are summarized in Table 2.5 Sorption of iron-cyanide complexes onto soil surfaces is the main immobilizing process

at low concentrations (Meeussen, 1992) Although the soil has limited sorption of iron

conditions such as pH, redox potential, ionic strength, organic content, and organic ligands which could be exuded by microorganisms, plant roots, and soil type Of particular importance is pH, which controls sorption of iron cyanide complexes (Meeussen, 1992; Rennert, 2002) Ionic strength also significantly influences the sorption of iron cyanide complexes, with ferricyanide sorption decreasing with increasing ionic strength In contrast, ferrocyanide sorption is only slightly influenced by ionic strength Organic matter and the presence of clay minerals have been shown to promote the sorption of iron cyanide complexes (Fuller, 1985; Rennert, 2002)

2.6 Microbial Degradation of Cyanide

Microbial degradation of cyanide (Castric, 1981) has been shown for organisms that use cyanide as a source of both nitrogen (Castric and Strobel, 1969; Harris and Knowles, 1983a; Kunz and Nagappan, 1989) and carbon (Murphy and Nesbitt, 1964; Skowronsky and Strobel, 1969) Over the last 40 years, microbial systems have been successfully used to treat cyanide-contaminated wastewater from industrial and agricultural processes (Babu et al., 1996; Fallon et al., 1997; Siller and Winter, 1998; Dhillon and Shivaraman, 1999; Gijzen et al., 2000) Approximately 90% conversion of

A number of organisms have been identified that can degrade the simple forms

of cyanide, including Pseudomonas fluorescens (Harris and Knowles, 1983b),

Pseudomonas putida (Kunz et al., 1992), Escherichia coli (Figueira et al., 1996), Alcaligenes xylosoxidans (Ingvorsen et al., 1991), and Bacillus pumilus (Meyers et al.,

1991) Degradation mechanisms have been identified for these organisms under a

variety of conditions For example, the metabolism of cyanide by Escherichia coli,

involving a dioxygenase enzyme and the conversion of cyanide to ammonia, is performed without production of cyanate (Figueira et al., 1996)

Although research has been published indicating that microbial degradation of simple cyanide compounds is possible, few publications report degradation of cyano-metal complexes, particularly the Fe-cyanide complexes, present at MGP sites Two

(Barclay et al, 1998) White rot fungus, Phanerochate chrysosporium, is also capable of

ferrocyanide degradation (Shah and Aust, 1993) Finnegan et al (1991) reported that

Acinetobacter sp has the ability to degrade a wide range of cyano-metal complexes,

converting both ferri- and ferrocyanide to ammonia at a pH of 7.5 using cell-free extracts (Babu et al., 1996) The low rate of complexed cyanide degradation may be a direct function of limited bioavailability (Aronstein et al., 1994)

Microorganisms existing in the rhizosphere of cyanogenic plants possess the ability to degrade complexed cyanide Most fungal pathogens of cyanogenic plants are known to be tolerant to simple cyanide compounds (Fry and Meyers, 1981) A number

of fungal pathogens, including Rhizopus oryzae, Stemphylium loti, and Goleocercospora

sorghi, are capable of degrading the cyanogen glycosides produced by the cyanogenic

plants, Manihot esculenta (Padmaja and Balagopal, 1985), Lotus corniculatus, and

Sorghum vulgare, respectively (Fry and Myers, 1981) There is also evidence that

microbial degradation of cyanogenic roots can result in significant cyanide reduction (Siller and Winter, 1998) Published research is limited that assesses the potential

metabolic capability of microorganisms for cyano-metal complexes within the cyanogenic plant rhizosphere However, a cyanide degrading microorganism isolated from the root of a flax plant recently has been used in an industrial wastewater treatment system for removal of cyanide

2.7 Phytoremediation of Cyanide Contamimants

Phytoremediation is defined as the use of plants to remove contaminants from soil and water The most common type of phytoremediation includes absorption, accumulation, and precipitation by plant roots; reducing contaminant mobility and preventing migration to groundwater The extraction and accumulation of contaminants

in harvestable plant tissues including shoots and leaves (phytoextraction), degradation of complex organic molecules into simple molecules and the incorporation of these molecules into plant tissues (phytodegradation), and stimulation of microbial and fungal degradation by releasing exudates/enzymes into the root zone (rhizo-degradation) Plants used in phytoremediation applications are summarized in Table 2.6

Production of cyanide is not limited to anthropogenic activities; a number of plant species can produce the toxin This is a serious problem for forage crops that, upon storage, may produce acutely toxic concentrations of free cyanide as a result of the degradation of cyanogenic glucosides These species of plants not only have the capability to produce cyanide, they also possess the ability to detoxify it It is this detoxification capacity that is most intriguing from the perspective of phytoremediation Table 2.7 summarizes the published literature on phytoremediation of cyanide

Plant cyanogenic glucosides are largely distributed throughout the plant kingdom, occurring in over 3000 plant species (Conn, 1991; Conn, 1993; Halkier et al., 1988; Miller and Conn, 1980; Vetter, 2000) The majority of these compounds are formed from amino acids L-tyrosin, L-phenylalanine, L-valine, L-isoleucine, and L-leucine (Conn, 1980a,b; Conn, 1988; Du et al., 1995; Halkier et al., 1989a,b; Harborne and Baxter, 1993) These compounds also may be precursors for amino acid and protein synthesis during seedling development (Niedzwidez-Siegien, 1998) Linamarin and lotaustralin are the predominant cyanogenic glucosides found both in cassava and flax plants (Conn, 1980a; Cutler and Conn, 1982; Nahrstedt, 1985; Harborne and Baxter, 1993; O’Brien et al., 1994; Nambisan and Sundaresan, 1994; Du et al., 1995; White et al., 1998; Saka et al., 1998; Niedzwidez-Siegien, 1998; Vetter, 2000) Linamarin is produced by the decarboxylation of L-valine followed by the formation of a nitrile group and subsequent glycosylation A similar pathway produces lotaustralin from L-

isoleucine Dhurrin, present in Sorghum bicolor, is produced from a precursor of

L-tyrosine (Conn 1980a,b; Halkier et al., 1989a,b) In depth reviews of cyanogenic glucoside synthesis are available in Conn (1980a,b) and Halkier (1988, 1989a)

Figure 2.1 shows the enzymatic reactions in cyanogenic plants (Castric et al., 1972) L–cysteine combines with cyanide and produces E-cyanoalanine with the

incorporated into the normal metabolism of the plant (Castric et al., 1972, Conn, 1980a,b, Nambisan and Sundaresan, 1994)

As a result, these species of plants not only have the ability to produce cyanide, they can detoxify it, leading to the possibility of phytoremediation as a soil treatment

approach The role of cyanogenic glucosides in plants has been primarily attributed to plant defense against herbivores (Conn 1980a; Nahrstedt, 1985; Harborne and Baxter, 1993; Vetter, 2000) The mode of defense against herbivores involves evolution of HCN from the plant upon disruption of plant tissue Evidence also shows that these compounds play a role in various plant physiological processes Although HCN protects the plant from invading organisms, it also presents a risk to the plant Many cyanogenic plants have been shown to detoxify the HCN released upon injury through the use of the enzyme E-cyanoalanine synthase The HCN is converted to asparagine, which is then incorporated into the normal metabolism of the plant (Conn, 1980a; Miller and Conn, 1980; Manning, 1988; Nambisan and Sundaresan., 1994; Vennesland et al., 1982) The release of HCN can be used to quantitatively determine the amount of cyanogenic glucoside within a plant

Linum usitassimum (oil flax) is commonly grown for the production of linseed oil

(Thornton, 1917) The plant may reach a height of 0.75 meter, consisting of multiple stems branched from the base of the plant In addition, the presence of cyanogenic

glucosides in Linum usitatissimum is well documented The seeds of Linum

usitassimum predominately contain two cyanogenic diglucosides, linustatin and

neolinustatin, which disappear following germination and subsequent plant growth (Niedzwidez-Siegien, 1998) The cyanogenic monoglucosides, linamarin and lotaustralin, predominate in the leaves, stems, and roots of growing and mature plants (Conn, 1980a; Fan and Conn, 1985; Frehner et al., 1990; Nahrstedt, 1985; Harborne et al., 1993; Niedzwidez-Siegien, 1998; Vetter, 2000) Linamarin concentration exceeds that of lotaustralin in all tissues of flax by a factor of two

Sorghum bicolor (L.) (sorghum) exhibits a multi-branched fibrous root system

that extends throughout the soil occupying space approximately one meter away from the plant (Dogget, 1970; Bennet et al., 1990) Rooting depth can extend beyond two meters, although the roots are primarily bound in the first meter of soil This extensive fibrous root system may provide definite advantages relative to phytoremediation The extensive root system, low transpiration capacity, and physiological characteristics of its

leaves, reduce plant water loss, and provide sorghum with a high drought tolerance S.

bicolor has been shown to contain the cyanogenic monoglucoside dhurrin in its seeds,

shoots, leaves, fruit, and roots (Miller and Conn, 1980; Nahrstedt, 1985; Hosel et al.,

1987) The biosynthesis of dhurrin in S bicolor originates from the amino acid

L-tyrosine (Conn, 1980b; Halkier et al., 1989a,b)

The accumulation of cyanide-containing compounds in sorghum has been studied extensively for many years Cyanogenic glycosides tend to be present in high concentrations in the seedlings of most varieties of sorghum However, these compounds diminish as the plant matures When present at high concentrations, the cyanogenic glycosides are toxic to grazing ruminants, and the animals are generally removed from sorghum fields until the seedlings have somewhat matured The fact that cyano-compounds in the sorghum decrease with time is a strong indication of the plant's ability to degrade the toxins

Willows have been recently considered for use at phytoremediation projects with

high cyanide concentrations Willows (Salix spp.) are known for their high water uptake

potential and drought tolerance (Newsholme, 1992) The high water uptake of these plants is considered an advantage for phytoremediation in contaminated soil and

groundwater In particular, S aurita and S cinerea are capable of colonizing denuded areas highly impacted by human use S aruita L (eared willow) is a multi-branched

shrub that grows to a height of approximately 2 meters The plant is tolerant of acidic conditions, an advantage for reclamation of the commonly acidic soils found at

abandoned MGP sites S cinerea is a taller shrub, often reaching heights of 6-9 meters

2.8 Modeling of Phytoremediation Processes

Phytoremedation models have been developed to predict the fate and transport of contaminants in plants, air, soil, and groundwater (Burken and Schnoor, 1997: Trapp and Macfarlane, 1995; Bushey et al., 2004) A model to describe cyanide transport and metabolism in willows was developed by Bushey et al (2004) Transport of cyanide complexes is affected by plants since they reduce the water infiltration by blocking soil pores and extracting water from the soil for transpiration Root uptake is an important component in prediction of water movement and is linked to contaminant metabolism and accumulation of cyanide compounds in roots, stems, and leaves The transfer of iron cyanide complexes from the external zone to the interior of the root is generally considered to occur by active uptake since passive transport of cyanide compounds is restricted to the mitochondria (Beavis et al., 1992) The stem plays a major role as the channel, xylem and phloem for flow between the root and leaf (Burken and Schnoor,

compounds can volatilize from the leaves In addition, accumulation and transformation

of cyanide compounds can occur in the leaves

Modeling in the unsaturated zone requires a different approach than in a contaminated saturated groundwater model The water and solute transport in the unsaturated zone is complex due to both water content and plant activity (Sung et al., 2003) Inorganic chemicals behave differently than organic chemicals in this environment Inorganic compounds are often passively taken up by plants from the soil pore water (Reeves, 2000) It is assumed that solute transport is mainly achieved through advection and diffusion transfer processes, but sorption to soil and metabolism in the plant must be considered carefully to evaluate removal and reaction rates The shape of the uptake curve as a function of the electrochemical gradient can be described

by Michaelis-Menten kinetics, which was developed to represent enzyme reaction rates Model development can be a useful tool to evaluate the relative role of the different processes and to identify optimal conditions for phytoremediation (Corapcioglu et al., 1999)

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