.2411 human:

.11

 

“a . I
3.0%er .

33%.

”NW... ‘9me
. .. ﬂuid“;
3. u. .

.
.cl... urn» ._

“may

a - .
.5613... Quanta}. a"
‘3 $3.1“:le 4s!!! 1...».
If)... 1 .{ltsltnoﬂ

 

 

 

90’0”? LIBRARY
Michigan State
University

 

 

 

This is to certify that the
dissertation entitled

CLONING AND EXPRESSION OF A BACTERIAL CGTASE
AND IMPACTS OF TRANSGENIC PLANTS ON
PHYTOREMEDIATION OF ORGANIC POLLUTANTS

presented by

SARAH J. KINDER

has been accepted towards fulﬁllment
of the requirements for the

degree In prop and Soil Sciences

 

Wﬂe/A/

V Méjor Proféssor’s/Signature
444/ 3/, 2007-
/ 7

Date

 

MSU is an aﬁnnative-adion, equal-opportunity employer

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/07 p:/ClRCIDateDue.indd—p.1

 

CLONING AND EXPRESSION OF A BACTERIAL CGTASE
AND IMPACTS OF TRANSGENIC PLANTS
ON PHYTOREMEDIATION OF ORGANIC POLLUTANTS
By

Sarah J Kinder

A DISSERTATION

Submitted to
Michigan State University
In partial fulﬁllment Of the requirements
For the degree Of

DOCTOR OF PHILOSOPHY
Department of Crop and Soil Sciences

2007

ABSTRACT
CLONING AND EXPRESSION OF A BACTERIAL CGTASE
AND IMPACTS OF TRANSGENIC PLANTS
ON PHYTOREMEDIATION OF ORGANIC POLLUTANTS
By

SARAH J KINDER

One of the major limitations to biological remediation Of persistent organic pollutants is
water insolubility and the lack of bioavailability. Surfactants can be used to overcome the
limitations on contaminant water solubility for improved biological degradation. Addition
of surfactants to soil can be expensive and result in bacterial toxicity. Most surfactants,
require a minimum concentration for effectiveness, the critical micelle concentration.
Similar in properties to surfactant micelles, cyclodextrins are functional at any
concentration due to the toroidal shape of individual molecules, creating a hydrophobic
cavity and a hydrophilic exterior. Cyclodextrins can accommodate hydrophobic
compounds within the hydrophobic cavity, forming a complex that can improve the water
solubility of the “guest” molecule. Cyclodextrins are formed from the degradation of
starch by Cyclodextrin Glycosyl Transferase (CGTase, Cgt) secreted into the
environment by various Bacillus species. We have cloned a novel cgt gene from
Paenibacz'llus sp. strain C36, PI-cgt. Enzymatic studies showed Escherichia coli strains
harboring PI-cgt produced quantiﬁable amounts of BCD in solution. PI-cgt was
transformed into the plant species tobacco and Arabidopsis, resulting in transgenic plant
lines, which are also capable of degrading starch and producing quantiﬁable amount of

BCD. AS a test of transgenically produced CDS, Cgt plants were tested for

phytoremediation of contaminated soils containing polyaromatic hydrocarbons (PAHS) or
polychlorinated biphenyls (PCBS). Starch treated transgenic tobacco showed a signiﬁcant
reduction of the highest molecular weight PAH compound, Benzo[ghi]perylene (BGHP)
when compared to untreated, unplanted and wild type treatments. Results from PCB

- studies and total PAHs were inconclusive for Cgt-plants, with multiple treatments,
including unplanted, showing signiﬁcant reductions when compared to untreated soil.
Overall, the results showed that Cgt-plants are capable of producing CD3 and Cgt-plants

can have a positive effect on phytoremediation of some organic pollutants.

AKNOWLEDGEMENTS

Firstly I would like to thank my advisor Dr. Clayton Rugh, for his help, humor
and patience, through my course of study and in ﬁnishing this dissertation. I would also
like to thank the other members of my committee, Dr. Tiedje for helpful advice, use of
his laboratory facilities and emergency reagent borrowing. I would also like to thank Dr.
Smucker for his words of advice and encouragement and Dr. Gifford for advice,
suggestions and help with certain aspects of chapter 4 especially.

Outside of my committee I would like to thank Dr. Voice for allowing me to use
his laboratory facility for PCB analysis. Dr. John Davis, Dr. Quensen, John Davis, Chris
Saffron and Theresa Cox for helping me learn and troubleshoot the HPLC and CC for
contaminant analysis. I would like to thank all of my lab mates co—workers and friends
for help in media preparation, sample analysis, and friendship: Endang Susilawati,
Rachada Settavongsin, Michael Roberts, Andrew Bender, Danielle Abshagen, and Kevin
Christ.

My research was supported by a variety of grants and institutions. For the ﬁrst
three years I was supported by a USDA-National Needs Fellowship for the training of
scientists in communication with the public concerning the risks and beneﬁts of plant
biotechnology. The Ford Motor Company, US EPA STAR —HSRC, MAES, MSU Center
for Microbial Ecology, MSU Ofﬁce of Diversity and Pluralism and the Graduate
School’s Dissertation Completion Fellowship, also supported me for various lengths of
time during my studies.

I would also like to thank my family members, especially my husband Douglas

MacDonald who aside from emotional and ﬁnancial support, was willing to come into the

iv

laboratory and help me ﬁnish my last samples. I would also like to thank my parents and
grandparents for their advice, support and visits to the lab for general encouragement and
questions. I also thank the Lord for giving me the opportunity to learn so much from
those around me and enjoy the company of so many wonderful people during my Ph.D.
program, and the providence of my various forms of ﬁnancial support. I thoroughly
enjoyed my studies at MSU and hope that they will enable me to continue learning in

new and exciting ways.

TABLE OF CONTENTS

PAGE
LIST OF TABLES ................................................................................. viii
LIST OF FIGURES ................................................................................. ix
INTRODUCTION ................................................................................... 1
REVIEW OF LITERATURE ....................................................................... 5
Environmental Contamination ............................................................. 5
Organic Chemical Pollutants ............................................................... 6
Soil and Pollutant Interaction ............................................................. 10
Soil Remediation ........................................................................... 12
Biotic Soil Pollutant Interactions ......................................................... 13
Phytoremediation ........................................................................... 17
Bioengineered Rhizosphere Phytoremediation ......................................... 18
Limitations on Phytoremediation ......................................................... 20
Summary and Conclusion ................................................................. 25
Literature Cited ............................................................................. 27
CHAPTER 1. CLONING AND CHARACTERIZATION OF
THE CYCLODEXTRIN GLYCOSYLTRANSFERASE FROM
PAENIBA CILL US SP. STRAIN C36 .......................................... 38
Introduction ................................................................................. 38
Materials and Methods ..................................................................... 40
Results ....................................................................................... 47
Discussion ................................................................................... 60
Conclusion .......................................................................... ' ........ 63
Literature Cited ............................................................................. 65
CHAPTER 2. PLANT EXPRESSION OF BACTERIAL
CYCLODEXTRIN GLYCOSYLTRANSFERASE FOR
CYCLODEXTRIN PRODUCTION ............................................ 69
Introduction .................................................................................. 69
Materials and Methods ..................................................................... 72
Results ....................................................................................... 82
Discussion ................................................................................... 91
Conclusion .................................................................................. 93
Literature Cited ............................................................................. 95

vi

CHAPTER 3. EFFECT OF CYCLODEXTRIN GLYCOSYL TRANSFERASE
EXPRESSING PLANTS ON THE REMEDIATION OF PAHS

AND PCBS ........................................................................ 98
Introduction ................................................................................. 98
Materials and Methods ................................................................... 104
Results and Discussion .................................................................. 109
Conclusion ................................................................................. 120
Literature Cited ........................................................................... 123

CHAPTER 4. IS PHYTOREMEDIATION SAFE? A COMPARISON OF RISKS AND
MANAGEMENT STRATEGIES OF PLANT-BASED
ENVIRONMENTAL REMEDIATION TECHNOLOGIES AND THEIR

ENGINEERING-BASED COUNTERPARTS .............................. 128
Introduction ................................................................................ 128
Risk Assessment and Risk management ............................................... 129
Risks and Beneﬁts from Standard Remediation Technologies ..................... 130
Risks and Beneﬁts from Phytoremediation ........................................... 131
Is Phytoremediation Safe? ............................................................... 143
Conclusion ................................................................................. 146
Literature Cited ........................................................................... 148

vii

LIST OF TABLES

PAGE

Table 1.1. Source organisms and references for

CGTases included in Figure 1.1, and 1.2 ....................................... 52-53
Table 2.1a. Selective marker segregation analysis for

T2 generation tobacco seeds .......................................................... 83
Table 2.1b. Selective marker segregation analysis for

T2 generation Arabidopsis seeds ..................................................... 83
Table 2.2a. Gene integration, expression, function

summary of Tobacco lines ............................................................ 89
Table 2.2b. Gene integration, expression, function

summary of Arabidopsis .............................................................. 89
Table 3.1. Percentage of relative concentration of each

of the individual PAH compounds

in reference to the total PAH content of the soil ................................. 106
Table 4.1. Summary of Accepted Remediation Technologies

and Associated Potential Risks ..................................................... 132
Table 4.2. Summary of Phytoremediation Technologies

and Associated Potential Risks ...................................................... 142
Table 4.3. Direct comparison of exposure based risks

from remediation technologies ................................................ 144-145

viii

LIST OF FIGURES

PAGE

Figure 1.1. Evolutionary phylogram comparison of Cgts to PI-Cgt ........................... I
Figure 1.2. Alignment of CGTases ............................................................... 52
Figure 1.3. The predicted PI-cgt signal sequence ............................................... 54
Figure 1.4. Bacterial clear-zone formation ....................................................... 55
Figure 1.5. Typical standard curve for colorimetric analysis of [3CD using

phenolphthalein ........................................................................ 57
Figure 1.6. Temperature optimum determination for P. Sp. C36 .............................. 57
Figure 1.7. CGTase Kinetic reaction Showing [3CD production over time .................. 59
Figure 1.8. Thin layer chromatography analysis ofCDs

by CGTase producing bacteria ....................................................... 59
Figure 2.1. Diagram of the pAPC9K and pCAMBIA 1300 cloning vectors ................ 73
Figure 2.2. Clear zone formation by cgt-arabidopsis ........................................... 85
Figure 2.3a. RT-PCR ofArabidopsis lines ...................................................... 86
Figure 2.3b. RT—PCR of tobacco lines ............................................................ 86
Figure 2.4. Production of BCD by Arabidopsis seedlings

as shown by colorimetric assay ....................................................... 88
Figure 2.5. TLC analysis of plant-produced cyclodextrins .................................... 90
Figure 3.1. tPAH content of soil from PAH Soil

Cgt-Phytoremediation treatments .................................................. 1 10

Figure 3.2. Benzo[ghi]perylene (BGHP) content of soil PAH Soil Cgt-Phytoremediation
treatments .............................................................................. 111

Figure 3.3. Napthalene content of soil PAH Soil
C gt-Phytoremediation treatments .................................................. 1 12

ix

Figure 3.4. Plant dry biomass from soil PAH Soil
C gt-Phytoremediation treatments .................................................. 1 13

Figure 3.5. PCB content of soil from PCB Soil
Cgt-Phytoremediation treatments .................................................. 1 16

Figure 3.6. Biomass of Arabidopsis shoot tissues in PCB Soil
Cgt-Phytoremediation treatments .................................................. 1 19

Figure 3.7. Percentage change in soil PCB content from spiked PCB Soil
C gt-Phytoremediation treatments as compared to untreated control .......... 119

Figure 3.8. Tobacco fresh and dry biomass in spiked PCB Soil Cgt-Phytoremediation
treatments .............................................................................. 121

INTRODUCTION

Environmental contamination by organic pollutants is a pervasive and important
threat to the biosphere. Organic pollutants typically originate due to spillage and
intentional release of industrial, agricultural or waste products. Once in the environment
organic compounds can cause environmental damage, more directly through
mutagenicity or indirectly though the disruption of physiological processes.

The problem of organic contaminants in soil has been addressed in the past
through a wide variety of methods based around engineering technologies, such as
physical soil removal, chemical reactions and stabilization. Physical and chemical
technological solutions to polluted land, known as remediation, have been joined by
biologically based technologies such as bioremediation, which is using bacteria to
decompose or stabilize toxic soil contaminants. Phytoremediation, or the use of plants to
degrade or render contaminants harmless, has recently gained favor as a viable
technological alternative to engineering and bioremediation installations.

One major property of many organic contaminants that acts as a powerful barrier
to remediation technologies that attempt to remove or destroy organic contaminants
within soil, is the very low water solubility and bioavailability of many organic
contaminants. Organic pollutants are chemically similar to soil organic matter and tend to
partition to, essentially dissolving in the organic fraction of soil. Bound organic
contaminants can be nearly inert to biological methods of degradation and removal but
may still cause environmental harm via slow loss from soil or direct soil consumption by
biota. One proposal to help solve the difﬁculty in removal of organic contaminants from

soil, is the use of surfactant or surface active agents on soil to help solubilize organic

contaminants. Surfactants are molecules with a hydrophilic “head” and a hydrophobic
“tail” portion, enabling the hydrophobic portion of the molecule to associate with
contaminant molecules, bringing them into the aqueous phase. However, surfactant
molecules are not without problems, they are capable of enhancing biological degradation
of organic pollutants. However, surfactants can also cause toxic effects on bacteria
capable of biodegradation. Biologically based surfactants or biosurfactants are thought to
be a more environmentally friendly, less toxic alternative to synthetic surfactants.
Although not a true biosurfactant, one compound stands out as a potential enhancer of
biological soil remediation, cyclodextrin. Cyclodextrins (CDs) have similar properties to
surfactant micelles and are formed by bacteria. The enzyme CGTase is secreted into the
soil by microbes, which acts on available starch molecules forming CD5. CDs are cyclic
molecules usually composed of 6-8 glucose units. The hydroxyl groups of cyclodextrins
face towards the exterior of the molecule giving cyclodextrins a hydrophilic exterior
while the interior remains largely hydrophobic. This property, in common with surfactant
micelles, allows the inclusion of hydrophobic compounds inside of the doughnut-shaped
molecule, forming a complex. CD complexes can make hydrophobic pollutants more
available for both bacterial and plant degradation, speeding up the process of contaminant
removal.

The objectives of this project are four-fold:
I. Isolate, clone and characterize a bacterial cyclodextrin glycosyl transferase
II. To create transgenic plants which are capable of secreting CGTase and forming

cyclodextrin

III. Testing transgenic plants for improvements in biological degradation of persistent
organic pollutants.

IV. Examination of the safety of phytoremediation as a whole, in comparison to standard
engineering based remediation practices.

In this project we isolated and cloned a novel CGTase from Paem'bacillus sp.
strain C3 6. This CGTase was sequenced and compared to known CGTases through
different software programs, direct comparison, analysis of signal peptides. The bacterial
cgt gene was minimally modiﬁed for bacterial and plant expression using PCR based
techniques. Escherichia coli DHSOL was used as the bacterial expression system. Assays
were performed using the E. coli cgt, and P. sp. C36 to determine optimal reaction
temperature, quantitative BCD production and qualitative CD production.

PI-cgt was placed into a plant expression cassette, containing a plant functional
promoter and terminator sequence. Plant constructs were used to generate transgenic
plants, both tobacco and Arabidopsis, which were screened for gene integration, cgt
expression, starch clearing and CD production using similar methods to those used in
testing bacterial expression. These plants were then used in experiments with the
hydrophobic contaminants, polychlorinated biphenyls (PCBS) and polyaromatic
hydrocarbons (PAHS). Chronically contaminated soil as well as spiked soils, were used to
test the effectiveness Of cgt-expressing plants. Some of the soils were starch treated to aid
in the in-situ production of CD5. Soils were tested for contaminant content after treatment
and plants were weight for biomass production. Promising results were seen in the

degradation of some contaminants.

AS an additional aspect of phytoremediation technology, the safety of
phytoremediation technology as a whole was also examined in comparison to standard
engineering remediation technologies. The varying and unique risks posed by each were
compared and contrasted. Attention was given to transgenic phytoremediation and special
risks that are presented by the use of transgenic plants in phytoremediation. A
comparison of standard engineering based technologies to their individual
phytoremediation counterparts was performed. The ultimate determination of “safety” of
phytoremediation technologies was made based on comparison to the existing, accepted

engineering technologies.

REVIEW OF LITERATURE

Sarah Kinder
Department of Crop and Soil Sciences
Michigan State University

Environmental Contamination

Anthropogenic environmental pollution is as old as civilization; human and
livestock nutrient waste has long polluted rivers and streams, rudimentary metalworking
by post-agrarian cultures released toxic metals, and more recently, the industrial
revolution rapidly disbursed a vast array of mined and manufactured pollutants. Fossil
fuel processing and combustion has caused widespread, persistent impacts to land surface
and air quality. Synthetic organic chemistry advances of the 20‘h century resulted in
versatile new chemical products, though also a variety of novel ecotoxicological
pollutants. The discovery of unintended effects of manmade organic compounds like
polychlorinated biphenyls (PCBS) was largely accidental (Jensen, 1972) and in the United
States, led to various regulations such as the Comprehensive Environmental Response,
Compensation, and Liability Act (CERCLA) (EPA, 2006). These policies are typically
called the Superfund and the Clean Water and Air Act and were enacted to reduce
ongoing anthropogenic pollution of the environment and force cleanup of existing
polluted sites.

Despite laws enacted for the prevention and cleanup, soil and sediment
contamination continues to be a common and often persistent problem in many areas of
the world, with the United States alone harboring 1,303 contaminated Sites on the
National Priorities List (N PL) and an estimated 450,000 low level or potentially

contaminated sites called brown ﬁelds (EPA, 2006). Around the world, contaminated

sites persist and continue to propagate as the rate of industrialization and modernization
rapidly accelerates in the world’s developing nations.

Due to the human and wildlife health hazards posed by environmental
contamination, considerable research has been performed to develop effective methods of
cleaning polluted soils, sediments and waterways. This review will discuss the nature and
interaction of various pollutants with soils, challenges to their remediation, methods of
engineering- and biologically-based remediation, and various approaches to improve

biological remediation.

Organic Chemical Pollutants

The ecotoxicity of organic chemicals is dependent on contaminant level, rate of
organismal uptake and retention, mechanism of dispersal, and environmental persistence.
Some environmental contaminants such as low molecular weight hydrocarbons can be
easily degraded or dissipated with little opportunity for biological exposure.
Alternatively, chlorinated organic compounds like polychlorinated biphenyls (PCBS)
persist in the environment for long periods of time and, even though not acutely toxic,
pose widespread biological risk. Due to variation in contaminant biochemical properties,
toxicant presence is not synonymous with biological risk: there must be a route of
exposure. Contaminants that are tightly bound to the soil matrix may be relatively stable
and inaccessible to potential receptor organisms. Hydrophobic organic contaminants and
relatively immobile metals such as lead are unlikely to leach to groundwater. More
mobile pollutants, such as water-soluble organic compounds and less stably complexed

metals, may increase potential risk if conditions favor contaminant leaching. Even if

pollutants are tightly bound to soil, the particles themselves may be moved by wind or
water, thus dispersing the pollutants. If humans or animals ingest soil, direct absorption
of contaminant molecules from the soil matrix can occur. Other routes of exposure
include, direct contact, inhalation, and through contaminated food stocks.

Environmental contaminants are generally grouped into two major classes,
organic and inorganic contaminants, with organic compounds being broken into many
distinct categories based on their chemical properties. Since the focus of this project is
on organic chemicals, these compounds will be the primary focus of this review.
Persistent organic pollutants (POPS) include DDT, aldrin, dieldrin, endrin, chlordane,
heptachlor, toxaphene, hexachlorobenzene, mirex, PCBS, dioxin, and furans according to
the United Nations Environmental Programme (Rodan et al., 1999). POPS can cause
considerable environmental problems even at very low concentrations due to a
combination of environmental persistence and lipophilicity. POP compounds can be
incorporated into the fatty tissues of an organism, where they are typically too
hydrophobic to be excreted except at very low levels (Connolly and Glaser, 2002). At
only a few parts per million, strongly lipophilic contaminants that incorporate into small
organisms will be magniﬁed through each trophic transfer. Eventually, at high trophic
levels in apex predators, such as eagles, the toxicological effects become detrimental
(Kumar et al., 2002). POPS in oceanic and riverine food chains are thought to be partially
responsible for population declines in many marine mammals via immune and
reproductive system impairment (Barron et al., 2003; Bitman and Cecil, 1970; Colbom et

aL,1993)

Aromatic and aliphatic pollutants include petroleum-based compounds often
grouped as total petroleum hydrocarbons (TPHs). TPHs typically possess a range of
hydrophobicity, toxicity, and biodegradability characteristics in parallel with increasing
molecular weight. TPH compounds are classiﬁed by nomenclature and distillation
fractions as the lighter weight gasoline-type fractions of benzene, toluene, ethylbenzene,
and xylene (BTEX), heavier oils, tar and polyaromatic hydrocarbon compounds (PAH).
PAHS are created naturally from incomplete burning of organic materials as well as
anthropogenically from industrial processes, such as hydrocarbon burning and coal
processing (Wilson and Jones, 1993). High molecular weight PAHS, such as
benzo[a]pyrene, are considered carcinogenic and genotoxic (Alexander et al., 2002;
Brown et al., 1999). Photomodiﬁcation may produce oxygenated radicals from PAH
molecules, which can react with biological molecules such as DNA (Mallakin et al.,
2002)

POPS also include synthetically created pesticides, explosives and assorted
compounds used as dielectric ﬂuids, ﬁre retardants, and solvents. One group of POP
synthetic compounds is halogenated organic compounds, which contain chlorine,
bromine or ﬂuorine bonded to carbon atoms in place of hydrogen. Halogenated aromatic
compounds include polychlorinated biphenyls (PCBS), polybrominated biphenyls
(PBBS), dichlorodiphenyltrichloroethane (DDT), polychlorinated dibenzofurans (PCDF S)
and polychlorinated dibenzodioxins (PCDDS). PCBS were used primarily in electrical
transformers and capacitors, carbonless copy paper, paint, plastics and ﬂame retardant
materials. PCBS have been shown to possess carcinogenic and estrogenic properties

(Safe, 1989). DDT was among the most wide used synthetic pesticides seeing resulting in

unforeseen environmental impacts, such as thinning of bird eggshells (Blus, 1984). POPS,
such as PCBS and DDT, persist for many decades in soils and sediments, long after the
manufacture and sale of the substances were banned (Erickson, 1993). PCBS and other
chlorinated organics are ubiquitous in global distribution, being detected in pristine
environments due to volatilization and atmospheric transport (Atlas and Giam, 1981;
Risebrough et al., 1968). PCBS, PBBS, PCDDS and PCDFS are mixtures of aromatics
compounds with varying chlorination, however only PCBS and PBBS were intentionally
synthesized. PCDDS and PCDFS are unintended by products of combustion, pesticide
manufacture and are contaminants of concern at 130 sites on the USEPA National
Priorities List as of 2006 (EPA, 2006). One PCDD congener, 2,3,7,8-
tetrachlorodibenzene-para-dioxin, is thought to be one of the most toxic organic
compounds known (Steenland et al., 2004).

Chlorinated aliphatic compounds, like trichloroethylene (TCE) and
tetrachlorothylene (PCE), are common groundwater contaminants. Chlorinated aliphatics,
like most chlorinated organic compounds, are chemically stable and persistent in the
environment, more so as the degree of chlorination increases. TCE and PCB are
biological harmful compounds, though may be converted to more toxic vinyl chloride by
bacterial processes (Nelson, 1988).

Nitroaromatic compounds (NACs), e. g. trinitrotoluene (TNT),
cyclotetramethylene-tetranitramine (HMX) and cyclotrimethylenetrinitramine (RDX), are
primarily used as explosives, though also as dyes and pesticide intermediates.

Environmental contamination from NACS results from manufacture or distribution of

unexploded residuals during detonation. Nitroaromatics are directly toxic, though are also

harmful at trace levels via oxidative DNA damage (Homma-Takeda et al., 2002).

Soil and Pollutant Interaction

Soil is a complex physicochemical medium with highly dynamic inﬂuences on
contaminant fate. Soils are composed of highly varied mineral and organic fractions with
large proportions of water and air. Mineral fractions are typically size-classed from
largest to smallest as sand, silt and clay, respectively. Chemical composition of the
mineral portion of soil includes silica and aluminum oxides arranged in ordered
crystalline forms (Dragun, 1998). Soil texture consists of many larger scale structural
elements, such as aggregates, cracks and old root tunnels, each of which inﬂuences water
and air ﬂow. The ratio of soil air space to soil hydration inﬂuences mechanical aspects of
the soil, such as plasticity. Soil organic matter (SOM) has complex chemical composition
and generally serves as the major sorption/partitioning matrix for organic pollutants.
Under the International Humic Substances Society Standard, soil organic matter is
composed of several fractions, including fulvic acid, humic acid and humin. SOM
fractions are largely deﬁned by their extractability. Humin is the acidic, neutral and
alkaline insoluble fraction. Fulvic acid is readily soluble in water at neutral pH. Humic
acid is only soluble in high pH conditions. Fulvic and humic acid are more labile than
humin and turn over more rapidly in the soil (Mobed et al., 1996). Organic compounds,
such as PAHS and PCBS, partition to soil organic fractions due to their chemical afﬁnity,
becoming largely unavailable for microbial biodegradation. Very hydrophobic organic

compounds partition to humin more strongly than the other SOM fractions due to the

10

Similarity in chemical composition (Petruzzelli et al., 2002). Once sorbed to soil organic
matter, pollutants may be retained almost indeﬁnitely with extremely slow transfer to the
aqueous phase. Permanent binding to soil organic matter, often called irreversible
sorption, occurs possibly by covalent linking to SOM molecules. However, irreversibly
bound contaminant molecules may be released during decomposition of organic matter
by SOM-degrading microbes (Reemtsma et al., 2003).

Water-soluble contaminants may be held within soil pore water or temporarily
bound by charged particles such as clays. Some nitroaromatic explosives interact very
speciﬁcally with certain types of clay (Haderlein et al., 1996). Nearly neutral
nitroaromatic compounds (e.g. dinitrotoluene) can be held between smectite clay
interlayers in spacing geometries created by the hydration spheres of associated ions such
as cesium or potassium. The larger hydration spheres of ions such as calcium and
magnesium cause clay layer spacing to increase such that inclusion of nitroaromatics is
no longer favorable and the compounds are released into the aqueous phase (Li et al.,
2004). Other charged compounds and ions may also be held onto the surface or in-
between negatively charged clay layers (Colbom et al., 1993). Nanopores in the soil
mineral fraction also sequester hydrophobic pollutants, where they may be inaccessible
for microbial bioremediation due to lack of space for microbial entry or growth (Sun et
aL,2003)

In addition to the solid soil phase, soil vapor is important for contaminant fate in
that it supplies oxygen and other gases for biotic and abiotic chemical reactions. The soil

vapor phase is typically in equilibrium with the liquid phase in porespaces between solid

11

 

soil particles. Volatile contaminants may escape from soil to react with other compounds

while in the gaseous state.

Soil Remediation
Conventional Remediation Technologies

In most contaminated sites, engineering based approaches are used to remediate
hazardous contaminants. Engineering based remediation largely focuses only on either
removing or destroying either the contaminants or the soil that contains them. The most
common of these practices is excavation or removal of contaminated soil for off Site
burial or disposal. Excavation and re-burial of contaminated soil can be extremely
expensive and labor intensive with costs usually in the range Of $270 to $460 per ton
(Deuren et al., 2002; EPA, 2001). Disturbed soils are subject to wind or rain erosion, at
least temporarily increasing exposure risk during excavation activities. Excavation may
be coupled to a secondary treatment such as soil washing, chemical or physical
stabilization, biological treatment or soil incineration followed by offsite disposal or
onsite reburial, each step with additional costs.

An alternative to excavation and removal of contaminated soils is in-situ soil
treatment, which is contaminant stabilization or decomposition on site. There are a wide
variety of in-situ soil treatment technologies including chemical solidiﬁcation,
vitriﬁcation, air sparging, electrokinetic migration, soil ﬂushing, bioremediation as well
as other emerging technologies. Chemical solidiﬁcation immobilizes contaminants by
addition of various types of chemicals such as asphalt, concrete and silicate based

additives. However, sequestered contaminants may be released as they weather over time

12

so long-term assessments of stability must be performed (Sellers, 1999). In-situ
vitriﬁcation is similar to chemical stabilization except that electrical energy is applied to
melt soil into glass-like blocks, which may retain contaminants for longer periods of time
with reduced leaching risk relative to chemical solidiﬁcation. Air sparging utilizes forced
air to remove volatile soil contaminants from the soil for atmospheric dispersal. Many
solvents such as TCE and light petroleum compounds may be treated by air sparging,
though this practice may be restricted by local or national regulations. Electrokinetics
uses electrical energy to mobilize contaminant compounds through the soil matrix for
concentration and removal. Electrokinetic effectiveness is inﬂuenced by soil moisture

and the presence of other conductive soil constituents (Saichek and Reddy, 2005).

Biotic Soil Pollutant Interactions

In addition to abiotic interactions, biological processes are important determinants
of environmental persistence of organic contaminants. Bioremediation, the use of living
organisms to remove, destroy or detoxify contaminants, is occasionally used alongside
standard engineering practices or as a site treatment. The implementation of biological
process for pollutant remediation could technically be called bioremediation, but the term
is most often used speciﬁcally to describe the action of microorganisms. In some cases,
bacteria or fungi may utilize organic pollutants as metabolites and completely mineralize
these contaminants to C02, water, chloride, or nitrate, depending on the compound.
Organic molecules may also be transformed to other compounds, some of which may still

be environmentally damaging, such as bacterial transformation of trichloroethylene to

13

vinyl chloride by anaerobic dechlorination (Enzien et al., 1994; Freedman and Gossett,

1989)

Organic pollutant biometabolism

Biotic transformation inﬂuences incorporation of organic contaminants or
metabolic byproducts into humic materials. Bacterial biotransfonnation of organic
compounds occurs either by direct utilization of the pollutant as a growth substrate or via
non-speciﬁc degradation due to broad speciﬁcity of some bacterial enzymes. This latter
mechanism is known as co-metabolism, which is the primary process for degradation of
many chlorinated compounds, e.g. PCBS. Bacterial cells synthesize dioxygenase and
other enzymes directed towards carbon substrates capable of serving as a carbon source.
For most PCB degraders PCBS cannot serve as a primary carbon source, and biphenyl or
other phenolic compounds ﬁll the role instead. In PCB co-metabolism, the same
enzymatic pathway induced by biphenyl also transforms moderately chlorinated PCBS to
utilizable products (Brenner et al., 1994).

Polyaromatic hydrocarbons (PAHS) are composed of two or more fused benzene
or furan rings. PAHS with less than four rings are termed light PAHS and are potential
targets for direct bacterial degradation and assimilation. Light PAHS are typically
degraded by dioxygenase enzymes, which catalyze cleavage of the aromatic rings leading
to production of ATP and carbon assimilation. Simpler hydrocarbons such as benzene,
toluene, ethylbenzene and xylene (BTEX) are common soil and groundwater

contaminants and more easily degraded than PAHS. Aerobic or anaerobic BTEX

14

biodegradation allows rapid contaminant removal under optimal conditions (Tsao et al.,
1998)

Highly chlorinated PCBS and tetrachloroethylene (PCE) are not efﬁciently
dehalogenated under aerobic conditions (Enzien et al., 1994). Chlorinated organic
compounds may be transformed under anaerobic conditions by bacteria that are capable
of reductive dechlorination. In this process, PCBS or trichloroethylene are selectively
dechlorinated with microbial use of chlorine as a terminal electron acceptor in place of
oxygen, nitrate or other more typical electron acceptors leading to release of the chlorine
atom from the molecule (Freedman and Gossett, 1989; Quensen et al., 1988). Reductively
dechlorinated organics are more amenable to aerobic degradation due to removal of
halogen-caused steric hindrances to dioxygenase enzyme attack.

Nitroaromatic compounds such as 2,4,6-trinitrotoluene and the heterocyclic
compounds HMX and RDX can also be biologically degraded, though more typically
serve as nitrogen sources rather than as carbon substrates (Esteve-Nunez et al., 2001).
Bacteria transform TNT to 2,4- and 2,6-monoamino,dinitrotoluene via nitroreductase
activity which converts the nitro groups to amino substituents (French et al., 1998; Labidi
etaL,2001)

Fungi that are capable of biodegrading naturally recalcitrant compounds such as
lignin have been applied to the degradation of anthropogenic compounds. The lignolytic
white rot fungus, Phanerochaete chrysosporium, is capable of degrading a wide variety
of organic compounds such as PAHS, PCBS and NACS (Sheremata and Hawari, 2000;
Yadav and Reddy, 1993; Zheng and Obbard, 2002). Fungal attack, unlike bacterial

degradation, is almost always via non-speciﬁc enzyme activities, such as by peroxidases

15

or laccases, which are involved in lignin metabolism degradation and usually released

only under lignolytic degrading conditions (Reddy, 1993).

Limitations of Bioremediation

Though it is possible for bacteria and fungi to degrade organic compounds under
laboratory conditions, most persistent organic pollutants are very slowly transformed and
degraded under ﬁeld conditions. Slow bacterial degradation rates are frequently linked to
low water solubility of the contaminant compounds and slow desorption from soil
organic matter. Contaminant desorption from the soil matrix and transfer to the aqueous
phase is thought to be the primary limiting factor for biological degradation of
hydrophobic contaminants (Bosma et al., 1997). Microbes utilize alternative strategies to
overcome mass transfer limitations, including direct colonization on sorbed substrates or
production of solubilizing compounds such as biosurfactants (Johnsen and Karlson,
2004). Direct microbial contact with contaminant molecules may be limited due to soil
tortuosity, low surface to volume ratios of contaminant globules, and protozoan predation
(Bouchez-Naitali et al., 1999). Microbially enhanced solubilization may be limited due to
low biosurfactant substrate availability or insufﬁcient bacterial cell density for effective
levels of biosurfactant production. Consequently, a large proportion of the bacterial
community may access only aqueous phase contaminant prior to degradation. Applied
bioremediation is limited by these same factors, as well as poor persistence of introduced
microbes, lack of sustained induction of desired biodegradative pathways, or concerns
over containment of improved genetically engineered organisms (GEMS) (Giddings,

1998).

16

Phytoremediation

Phytoremediation is similar to bioremediation, though is focused on the use of
plants to remove or detoxify environmental contaminants. Phytoremediation processes
include volatilization of contaminants from leaves (phytovolatilization), direct plant
decomposition of organic contaminants (phytodegradation) and plant sequestration and
concentration of contaminants in the above ground parts (phytoaccumulation). Organic
contaminants may be directly metabolized by plant processes, though plant-enhanced
biodegradation typically occurs via enhanced bacterial enzymatic activity in the root
zone, subsequently termed phytostimulation or rhizodegradation. Plant species that
naturally accumulate higher quantities of metals, such as arsenic, nickel, zinc, cadmium
and cobalt, in their above ground parts are termed hyperaccumulators (Baker and Brooks,
1989). Plants like the zinc hyperaccumulator T hlaspi caerulescens are able to concentrate
toxic metals in their tissues above 1% of dry tissue mass, which is many times higher
than that found in bulk soil (Brown et al., 1994; Nedelkoska and Doran, 2001; Salido et
al., 2003). Hyperaccumulators have the potential to remediate metal contaminated sites
by concentrating the disbursed metals in a small quantity of tissue, allowing for easy
harvest and removal rather than excavation of the entire volume of contaminated soil
(Baker et al., 1994). For economically valuable elements such as nickel,
phytoaccumulation also has the potential to provide revenue to defer cleanup costs (Li et
al., 2003).

Phytovolatilization is a process in which volatile metals and organic compounds

are removed from soil by water uptake followed by evapotranspiration from shoot

17

tissues. Hybrid poplar tree phytoremediation of hydrocarbon contaminated groundwater
is a combination of phytostimulation, phytovolatilization, and phytodegradation
processes (Widdowson etal., 2005). Like bioremediation, plant-based cleanup
technologies are largely experimental and are limited by environmental and
physicochemical factors, though they possess certain ancillary advantages over microbial
treatments, including self-sustenance through photosynthesis and containment of

contaminated media by stabilization against erosion.

Bioengineered Rhizosphere Phytoremediation

Crop bioengineering has become a standard technique in crop improvement for
agricultural purposes with 56% percent of soybean and 28% of cotton global crop
acreage genetically modiﬁed (James, 2004). However the application of biotechnological
approaches to phytoremediation is still largely in the experimental stages. The most
advanced of these approaches is mercury phytovolatilization, which is undergoing ﬁeld
testing in several states (APHIS release #05-045-01). These plants express mer bacterial
genes for detoxiﬁcation of bioaccumulative methylmercury and phytovolatilization of
less toxic elemental mercury (Rugh et al., 1998; Rugh et al., 1998). Recent advancements
in selenium phytoremediation have allowed the genetic enhancement of the innate ability
of Indian mustard to accumulate selenium (LeDuc et al., 2004). In another study, plant
arsenic accumulation was improved via RNAi silencing of the plant arsenic reductase
gene, which normally converts arsenic to an insoluble, immobile form. Without the
function of this gene in roots, the plants transported and sequestered soluble arsenic in

shoot tissues (Dhankher et al., 2006).

18

Transgenic phytoremediation of organic pollutants has also been achieved in
numerous laboratories. Engineered plants have effective expressed genes for degradation
and detoxiﬁcation of TNT and HMX nitroaromatics (French et al., 1998; Rylott et al.,
2006). Mammalian cytochrome P450 monooxygenases has been utilized to enhance
transgenic plant degradation of a wide range of organic contaminants (Kawahigashi et al.,
2002)

Despite recognized successes in genetically engineered phytoremediation, this
approach may present difﬁculties. Foreign genes may fail to be expressed in transgenic
plants due to GC bias or atypical codon usage (Slimko and Lester, 2003), resulting in
silenced or low expressing genes (Haseloff et al., 1997; Rugh et al., 1996). Bacillus gene
sequences are typically A/T rich, which may contain pseudo mRNA splice sites that are
recognized by the host transcriptional systems leading to transcript instability and
potential silencing. Monocot plants have a considerably higher GC codon bias than
dicotyledous species (Kawabe and Miyashita, 2003). In one study, no expression was
detected of an unmodiﬁed bacterial B-glucanase expressed in barley, but a higher GC,
codon-optimized gene was expressed successfully (Jensen et al., 1996). High GC coding
regions often contain an abundance of CpG motifs, which are targets for eukaryotic DNA
methylases in some hosts (Ingelbrecht et al., 1994; Vanyushin and Kimos, 1988). Early
attempts to express an unmodiﬁed bacterial Tn21 transposon merA gene in Arabidopis
were unsuccessful, though gene sequence modiﬁcation to reduce GC abundance utilizing
more common dicot codons allowed transgene expression (Rugh et al., 1996). A highly
GC-biased chlorocatechol degradation gene from Ralstonia eutropha NH9 was inserted

into tobacco BY2 cells without detectable expression, though the GC-rich rice genome

19

supported expression of the transgene (Shimizu et al., 2002). Chromosomal insertion
position may also strongly inﬂuence transgene expression, with genes inserted into

heterochromatic regions resulting in low expression (Matzke and Matzke, 1998).

Limitations of phytoremediation

Basic limitations on phytoremediation include long treatment time and
requirement for agronomically suitable site conditions. Most phytoremediation research
on organic compounds has focused on uptake and sequestration or degradation of water-
soluble compounds. However, for some strongly non-polar contaminants such as PCBS
and PAHS, very small quantities of these compounds will be available to plant tissues for
direct degradation (Shrout et al., 2006). One approach to dealing with extremely
hydrOphobic pollutants using plants is indirect, using root-produced molecules to
stimulate available microbes which can more readily ac'cess hydrophobic contaminants
due to higher surface to volume ratios and potential for direct contact with pollutant
globules. For plants to be capable of direct degradation, more creative methods are
necessary to increase the bioavailability of hydrophobic compounds. One method of
increasing apparent solubility of strongly hydrophobic contaminants is the addition of
surfactants.

Surfactants are amphiphilic compounds, which possess both hydrophilic and
hydrophobic domains within a single molecule. At low concentrations, surfactants
congregate at the aqueous, non-aqueous interfaces, reducing surface tension in
immiscible liquid systems. As the concentration of surfactant in an aqueous system is

increased, the surface tension will continue to decrease up to a certain point when it will

20

no longer decrease, which is known as the critical micelle concentration (CMC). Once
the CMC is reached, surfactant molecules spontaneously form spherical vesicles called
micelles, which are capable of assimilating hydrophobic compounds and increasing their
apparent water solubility and bioavailability. Surfactants have been widely used in
bioremediation treatment strategies to enhance in situ degradation rates of organic
compounds or in ex situ operations for soil washing procedures. However, due to their
solubilizing effect, surfactants have the potential to cause contaminant leaching when
used in situ. Chemical surfactants may result in bacterial or plant toxicity when used at
chemically effective concentrations or become unintended contaminants due to their
environmental persistence in soils (Rouse et al., 1995). In some situations, synthetic
surfactants may inhibit microbial biodegradation even while enhancing contaminant
desorption (Laha and Luthy, 1991) by toxicity to bacterial cells (V olkering et al., 1997)
or inaccessibility of entrapped contaminants (Makkar and Rockne, 2003).

Biologically-synthesized surfactants or biosurfactants are considered more
environmentally benign than synthetic surfactants due to shorter half-life, lower toxicity,
and higher biodegradability. Biosurfactants are grouped in several major classes,
glycolipids, lipoproteins, phospholipids, and polymeric biosurfactants. Bacterial
biosurfactant production has been proposed as a biological mechanism for transport of
less water-soluble compounds, to promote enhanced cell-matrix adhesion, or to function
as defense compounds (Maier, 2003; Neu, 1996). Glycolipids are sugar-lipid containing
molecules with a well-studied example being rhamnolipids. Rhamnolipids were Shown to
enhance removal of PAHS in soil washing treatments (Noordman et al., 1998).

Rhamnolipids were observed to be similar in desorption effectiveness to the chemical

21

surfactant Triton-X across a wide range of compounds in chronically contaminated soil
(Berselli et al., 2004).

Cyclodextrins (CDS) are unique biosurfactant-like molecules unlike most
surfactant chemicals. Cyclodextrins are not surface active (do not reduce surface tension)
and therefore are not true surfactants. Due to their mono-molecular activity, CDS have no
CMC requirement and are capable of contaminant solubilization activity at any
concentration. CDs are composed of a—l,4 linked glucose units with the primary
hydroxyl groups directed towards the interior of the torus-shaped molecule and secondary
hydroxyls directed towards the exterior (Szejtli, 1988). The exterior hydroxyls form the
hydrophilic portion of the molecule while the interior portion remains relatively
hydrophobic. Hydrophobic compounds can become included into the center cavity of the
CD and are called “guest” molecules. CD is typically formed from 6, 7, or 8 glucose
units; comprising or, B and yCD respectively. The differing number of glucose units
results in a different cavity size, giving each CD a Slightly different range of
contaminants to solubilize. Cyclodextrin-producing bacteria synthesize mixtures of CD3
usually with one or two different cyclodextrin types predominating depending on the
speciﬁcity of the bacterial cyclodextrin glycosyltransferase (CGTase) (Qi and
Zimmermann, 2005). CD5 generally cannot solubilize hydrophobic compounds larger
than the interior CD cavity but portions of larger molecules may become encapsulated
with complex ratios of 1:1, 2:1 or occasionally 3:1 of cyclodextrin to guest molecule.
CDS are thought to function in one of two ways, one in which the CD functions as an
inert agent for simple solubilization, with another being the speciﬁc cellular import and

cytoplasmic degradation of intact CD-complexes (Pajatsch et al., 1998). Some microbes

22

such as Klebsiella oxytoca are capable of both producing and utilizing CD as a sole
carbon source (Fiedler et al., 1996).

Cyclodextrins have been used for enhancement of environmental remediation, but
chemically modiﬁed cyclodextrins are frequently utilized, especially modiﬁed BCD. This
is due to the fact that [3CD has relatively low water solubility, which is due to the fact that
the hydroxyl groups of [3CD tend to hydrogen bond with one another rather than with the
surrounding solvent (Szejtli, 1988). To increase the water solubility of BCD, hydrophobic
or hydrophilic groups are chemically added to the surface of the molecule to break up the
intrarnolecular hydrogen bonding. Given the nearly unlimited water solubility of most
CD derivatives, they are often preferred for exogenous applications. Modiﬁed CDs may
actually have lower solubilization power than their natural counterparts at lower
concentrations, likely due to steric hindrance from functional groups attached to the CD
ring (Gao et al., 1998). Since chemical modiﬁcation of cyclodextrins is unfeasible for in-
vivo CD production, works concerning modiﬁed cyclodextrins must be viewed with
recognition that chemically modiﬁed CDS could only be added exogenously rather than
solely biologically produced. However the solubilization properties of modiﬁed CDs may
still be indicative of the potential of the parent natural CDS. Modiﬁed CDS are
considerably more persistent and resistant to degradation than natural CDs; randomly
methylated BCD (RAMEB) was found to be fully resistant to biodegradation (Fenyvesi et
al., 2005). Low degradation rates may lead to potential problems with persistence of the
modiﬁed CD compounds similar to those caused by synthetic surfactants, including

contaminant leaching or increased toxicity to receptor organisms.

23

Cyclodextrins are commonly used to increase desorption of various soil
contaminants in biodegradation or soil washing treatments. Modiﬁed hydroxypropyl BCD
(HPCD) was used to enhance the transport of: anthracene, pyrene and a PCB congener
(Brusseau et al., 1994). HPCD was found to be very effective in part due to lack of
sorption to soil, which has been observed to occur with most true surfactants. HPCD and
[3CD were both demonstrated to signiﬁcantly enhance the apparent aqueous solubility of
the low molecular weight PAHS naphthalene and phenanthrene (Badr et al., 2004). [3CD
enhanced biodegradation in liquid cultures spiked with PAHS (naphthalene and
anthracene) and linear hydrocarbons (tetracosane and dodecane) (Bardi et al., 2000). CD5
were also observed to reduce Pseudomonas putida growth inhibition and toxicity by
toluene and toluic acid, while increasing biodegradation rates (Schwartz and Bar, 1995).
HPCD has been Shown to enhance phenanthrene degradation in liquid cultures with 97%
of the compound removed in 48 hours in comparison to 54.8% removal in treatments
without HPCD (Wang et al., 1998). yCD and HPCD were shown to signiﬁcantly enhance
biodegradation of individual PCB congeners when compared to control treatments
lacking cyclodextrin under both slurry and ﬁxed phase column soil bioreactor conditions
(Fava et al., 1998). Under greenhouse conditions, several planted systems amended with
[3CD showed a signiﬁcant reduction of initial soil PAH levels, ostensibly via enhanced
rhizodegradation (Settavongsin, 2005). HPCD enhanced removal of a wide range of
compounds, including BTEX and TCE, in ﬁeld aquifer conditions (McCray and

Brusseau, 1998).

24

S_um_n;ary & Conclusion

Environmental contamination of soils is a continuing and pervasive problem in many
areas of the world. Organic pollutant compounds cause environmental harm even at low
levels due to their persistence and bioaccumulation through food webs. These compounds
may be mineralized, transformed into their basic constituents of carbon dioxide and
water. However transformation of organic compounds is limited by their low solubility in
water and resulting unavailability for rapid degradation. Engineering-based approaches to
site decontamination, while often quick and effective, may not represent the most cost
efﬁcient and environmentally compatible treatment for organic pollutants.
Phytoremediation has been proposed as an environmentally friendly alternative to
engineering based approaches. Plants are considered to be more environmentally friendly
and capable of enhancing the removal of a wide range of compounds. Genetic
engineering has also been used to generate plants with improved capabilities for
environmental restoration, including genes for biodegradation and contaminant
concentration. Despite genetic improvements phytoremediation is still constrained by the
physical properties of organic contaminants and soil. Organic compounds tend to
partition to the humic materials within soil, protecting them from biological
decomposition.

Surfactants are compounds, which are capable of solubilizing organic
contaminants, bringing them into the aqueous phase. Surfactants have been used to
improve biological degradation of organic compounds, but may be expensive and labor
intensive to add to a Site. Surfactants may also cause bacterial toxicity and contaminant

leaching. Cyclodextrins are biologically synthesized compounds capable of overcoming

25

the limitations of low water solubility and bioavailability in a similar fashion to
surfactants. Instead of relying either on exogenously added cyclodextrins or bacterial
synthesis, we propose a more manageable and consistent solution is the plant production
of extracellular CGTase coupled with exogenous addition of starch.

In this research project we cloned a novel cgt gene was cloned from a soil isolated
bacterium. This gene was then expressed in 3 different species, Esherichia coli,
Arabidopsis thaliana and Nicotiana tabacum. Functional CGTase was detected in at least
one line or clone of all three species. Several plant lines were tested for improvements to
degradation of PAHS and PCBS. The transgenic lines examined in this work are a ﬁrst
step towards ﬁeld implementation of in-situ plant-based production of CDs.
Contaminated soils containing CDS may exhibit enhanced biological degradation due to
both plants and bacteria. Plant produced CD may be more controlled than either direct
CD addition or bacterial production due to location within the rhizosphere and direct
observation of plant growth. Cgt-plants provide potential for acceleration of in-situ

biological degradation of organic contaminants.

26

REFERENCES

Alexander, R. R., Tang, J. X., and Alexander, M. (2002). Genotoxicity is unrelated to
total concentration of priority carcinogenic polycyclic aromatic hydrocarbons in
soils undergoing biological treatment. Journal of Environmental Quality 31, 150-

154.

Atlas, E., and Giam, C. S. (1981). Global transport of organic pollutants - ambient
concentrations in the remote marine atmosphere. Science 211, 163-165.

Badr, T., Hanna, K., and de Brauer, C. (2004). Enhanced solubilization and removal of
naphthalene and phenanthrene by cyclodextrins from two contaminated soils.
Journal of Hazardous Materials 112, 215-223.

Baker, A. J. M., and Brooks, R. R. (1989). Terrestrial higher plants which hyper-
accumulate metallic elements: A review of their distribution, ecology and
phytochemistry. Biorecovery 1, 8 1 - l 26.

Baker, A. J. M., McGrath, S. P., Sidoli, C. M. D., and Reeves, R. D. (1994). The
possibility of in situ heavy metal decontamination of polluted soils using crops of
metal-accurnulating plants. Resources Conservation and Recycling [1, 41-49.

Bardi, L., Mattei, A., Steffan, S., and Marzona, M. (2000). Hydrocarbon degradation by a
soil microbial population with beta-cyclodextrin as surfactant to enhance
bioavailability. Enzyme and Microbial Technology 27, 709-713.

Barron, M. G., Heintz, R., and Krahn, M. M. (2003). Contaminant exposure and effects
in pinnipeds: implications for Steller sea lion declines in Alaska. Science of the
Total Environment 311, 111-133.

Berselli, S., Milone, G., Canepa, P., Di Gioia, D., and F ava, F. (2004). Effects of
cyclodextrins, humic substances, and rhamnolipids on the washing of a
historically contaminated soil and on the aerobic bioremediation of the resulting
efﬂuents. Biotechnology and Bioengineering 88, 111-120.

Bitman, J., and Cecil, H. C. (1970). Estrogenic activity of DDT analogs and
polychlorinated biphenyls. Journal of Agricultural and Food Chemistry 18, 1108-
1112.

27

Blus, L. J. (1984). DDE in birds' eggs: Comparison of two methods for estimating critical
levels. The Wilson Bulletin 96, 268-276.

Bosma, T. N. P., Middeldorp, P. J. M., Schraa, G., and Zehnder, A. J. B. (1997). Mass
transfer limitation of biotransforrnation: Quantifying bioavailability.
Environmental Science & Technology 31, 248-252.

Bouchez-Naitali, M., Rakatozafy, H., Marchal, R., Leveau, J. Y., and Vandecasteele, J. P.
(1999). Diversity of bacterial strains degrading hexadecane in relation to the mode
of substrate uptake. Journal of Applied Microbiology 86, 421-428.

Brenner, V., Arensdorf, J. J ., and F ocht, D. D. (1994). Genetic construction of PCB
degraders. Biodegradation 5, 359-377.

Brown, D. G., Knightes, C. D., and Peters, C. A. (1999). Risk assessment for polycyclic
aromatic hydrocarbon NAPLS using component fractions. Environmental Science
& Technology 33, 4357-4363.

Brown, S. L., Chaney, R. L., Angle, J. S., and Baker, A. J. M. (1994). Phytoremediation
potential of Thlaspi caerulescens and bladder campion for zinc-contaminated and
cadmium-contaminated Soil. Journal of Environmental Quality 23, 1151-1157.

Brusseau, M. L., Wang, X. J ., and Hu, Q. H. (1994). Enhanced transport of low polarity
organic compounds through soil by cyclodextrin. Environmental Science &
Technology 28, 952-956.

Colbom, T., Saal, F. S. V., and Soto, A. M. (1993). Developmental effects of endocrine-
disrupting chemicals in wildlife and humans. Environmental Health Perspectives
10], 378-384.

Connolly, J. P., and Glaser, D. (2002). p,p '-DDE bioaccumulation in female sea lions of
the California Channel Islands. Continental Shelf Research 22, 1059-1078.

28

Deuren, J. V., Lloyd, T., Chhetry, S., Liou, R., and Peck, J. (2002). Remediation
Technologies Screening Matrix and Reference Guide, 4th Edition: Federal
Remediation Technologies Roundtable).

Dhankher, O. P., Rosen, B. P., McKinney, E. C., and Meagher, R. B. (2006).
Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate
reductase (ACR2). Proceedings of the National Academy of Sciences of the
United States of America 103, 5413-5418.

Dragun, J. (1998). The Soil Chemistry of Hazardous Materials, Second Edition (Amherst,
MA: Amherst Scientiﬁc Publishers).

Enzien, M. V., Picardal, F., Hazen, T. C., Arnold, R. G., and Fliermans, C. B. (1994).
Reductive dechlorination of trichloroethylene and tetrachloroethylene under
aerobic conditions in a sediment column (V 01 60, Pg 2202, 1994). Applied and
Environmental Microbiology 60, 4646-4646.

EPA. (2006). Laws and Regulations: Major Environmental Laws: United States
Environmental Protection Agency).

EPA. (2006). National Priorities List: United States Environmental Protection Agency).

EPA. (2001). US Environmental Protection Agency - Remediation Technology Cost
Compendium - Year 2000. In EPA 542-R-01-009, U. E. P. Agency, ed.

Erickson, M. D. (1993). Remediation of PCB Spills (Boca Raton, FL: Lewis Publishers).

Esteve-Nunez, A., Caballero, A., and Ramos, J. L. (2001). Biological degradation of
2,4,6-trinitrotoluene. Microbiology and Molecular Biology Reviews 65, 335-+.

F ava, F., Di Gioia, D., and Marchetti, L. (1998). Cyclodextrin effects on the ex-situ
bioremediation of a chronically polychlorobiphenyl-contaminated soil.
Biotechnology and Bioengineering 58, 345-355.

29

Fenyvesi, E., Gruiz, K., Verstichel, S., De Wilde, B., Leitgib, L., Csabai, K., and
Szaniszlo, N. (2005). Biodegradation of cyclodextrins in soil. Chemosphere 60,
1001-1008.

Fiedler, G., Pajatsch, M., and Bock, A. (1996). Genetics of a novel starch utilisation
pathway present in Klebsiella oxytoca. Journal of Molecular Biology 256, 279-
291.

Freedman, D. L., and Gossett, J. M. (1989). Biological reductive dechlorination of
tetrachloroethylene and trichloroethylene to ethylene under methanogenic
conditions. Applied and Environmental Microbiology 55, 2144-2151.

French, C. E., Nicklin, S., and Bruce, N. C. (1998). Aerobic degradation of 2,4,6-
trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate
reductase. Applied and Environmental Microbiology 64, 2864-2868.

Gao, S. X., Wang, L. S., Huang, Q. G., and Han, S. K. (1998). Solubilization of
polycyclic aromatic hydrocarbons by beta-cyclodextrin and carboxymethyl-beta-
cyclodextrin. Chemosphere 3 7, 1299-1305.

Haderlein, S. B., Weissmahr, K. W., and Schwarzenbach, R. P. (1996). Speciﬁc
adsorption of nitroaromatic: Explosives and pesticides to clay minerals.

Environmental Science & Technology 30, 612-622.

Haseloff, J ., Siemering, K. R., Prasher, D. C., and Hodge, S. (1997). Removal of a cryptic
intron and subcellular localization of green ﬂuorescent protein are required to
mark transgenic Arabidopsis plants brightly. Proceedings of the National
Academy of Sciences USA 94, 2122-2127.

Homma-Takeda, S., Hiraku, Y., Ohkuma, Y., Oikawa, S., Murata, M., Ogawa, K.,
Iwamuro, T., Li, 8., Sun, G. F., Kumagai, Y., Shimojo, N., and Kawanishi, S.
(2002). 2,4,6-trinitrotoluene-induced reproductive toxicity via oxidative DNA
damage by its metabolite. Free Radical Research 36, 555-566.

Ingelbrecht, I., Vanhoudt, H., Vanmontagu, M., and Depicker, A. (1994).
Posttranscriptional silencing of reporter transgenes in tobacco correlates with
DNA methylation. Proceedings of the National Academy of Sciences of the
United States of America 91, 10502-10506.

30

James, C. (2004). Preview: Global Status of Commercialized Biotech/GM Crops (Ithaca,
NY: ISAA Briefs), pp. 60.

Jensen, L. G., Olsen, 0., Kops, 0., Wolf, N., Thomsen, K. K., and Von Wettstein, D.
(1996). Transgenic barley expressing a protein-engineered, thermostable (1,3-

l,4)-B-glucanase during germination. Proceedings of the National Academy of
Sciences of the United States of America 93, 3487-3491.

Jensen, S. (1972). The PCB story. AMBIO 1, 123-131.

J ohnsen, A. R., and Karlson, U. (2004). Evaluation of bacterial strategies to promote the
bioavailability of polycyclic aromatic hydrocarbons. Applied Microbiology and
Biotechnology 63, 452-459.

Kawabe, A., and Miyashita, N. T. (2003). Patterns of codon usage bias in three dicot and
four monocot plant species. Genes & Genetic Systems 78, 343-352.

Kawahigashi, H., Hirose, S., Hayashi, E., Ohkawa, H., and Ohkawa, Y. (2002).
Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing
the human C YPZB6 gene. Pesticide Biochemistry and Physiology 74, 139-147.

Kumar, K. S., Kannan, K, Giesy, J. P., and Masunaga, S. (2002). Distribution and
elimination of polychlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls, and
p,p '-DDE in tissues of bald eagles from the Upper Peninsula of Michigan.
Environmental Science & Technology 36, 2789-2796.

Labidi, M., Ahmad, D., Halasz, A., and Hawari, J. (2001). Biotransforrnation and partial
mineralization of the explosive 2,4,6-trinitrotoluene (TNT) by rhizobia. Canadian
Journal of Microbiology 47, 559-566.

Laha, S., and Luthy, R. G. (1991). Inhibition of phenanthrene mineralization by nonionic
surfactants in soil-water systems. Environmental Science & Technology 25, 1920-
1930.

LeDuc, D. L., Tarun, A. S., MonteS-Bayon, M., Meija, J., Malit, M. F., Wu, C. P.,
AbdelSamie, M., Chiang, C. Y., Tagmount, A., DeSouza, M., Neuhierl, B., Bock,
A., Caruso, J ., and Terry, N. (2004). Overexpression of selenocysteine

31

methyltransferase in Arabidopsis and Indian mustard increases selenium tolerance
and accumulation. Plant Physiology 135, 377-383.

Li, H., Teppen, B. J ., Laird, D. A., Johnston, C. T., and Boyd, S. A. (2004). Geochemical
modulation of pesticide sorption on smectite clay. Environmental Science &
Technology 38, 5393-5399.

Li, Y. M., Chaney, R., Brewer, E., Roseberg, R., Angle, J. S., Baker, A., Reeves, R., and
Nelkin, J. (2003). Development of a technology for commercial phytoextraction
of nickel: economic and technical considerations. Plant and Soil 249, 107-115.

Maier, R. M. (2003). Biosurfactants: Evolution and diversity in bacteria. Advances in
Applied Microbiology 52, 101-121.

Makkar, R. S., and Rockne, K. J. (2003). Comparison of synthetic surfactants and
biosurfactants in enhancing biodegradation of polycyclic aromatic hydrocarbons.
Environmental Toxicology and Chemistry 22, 2280-2292.

Mallakin, A., Babu, T. 8., Dixon, D. G., and Greenberg, B. M. (2002). Sites of toxicity of
speciﬁc photooxidation products of anthracene to higher plants: Inhibition of
photosynthetic activity and electron transport in Lemna gibba L. G-3 (duckweed).
Environmental Toxicology 1 7, 462-471.

Matzke, A. J. M., and Matzke, M. A. (1998). Position effects and epigenetic silencing of
plant transgenes. Current Opinion in Plant Biology 1, 142-148.

McCray, J. E., and Brusseau, M. L. (1998). Cyclodextrin-enhanced in situ ﬂushing of
multiple-component immiscible organic liquid contamination at the ﬁeld scale:
Mass removal effectiveness. Environmental Science & Technology 32, 1285-
1293.

Mobed, J. J ., Hemmingsen, S. L., Autry, J. L., and McGown, L. B. (1996). Fluorescence
characterization of IHSS humic substances: Total luminescence spectra with
absorbance correction. Environmental Science & Technology 30, 3061-3065.

32

Nedelkoska, T. V., and Doran, P. M. (2001). Hyperaccumulation of nickel by hairy roots
ofAlyssum species: Comparison with whole regenerated plants. Biotechnology
Progress 17, 752-759.

Nelson, M. J. K., Montgomery, 8.0., and Pritchard, PH. (1988). Trichloroethylene
metabolism by microoganisms that degrade aromatic compounds. Applied and

Environmental Microbiology 54, 604-606.

Neu, T. R. (1996). Signiﬁcance of bacterial surface-active compounds in interaction of
bacteria with interfaces. Microbiological Reviews 60, 151-&.

Noordman, W. H., Ji, W., Brusseau, M. L., and Janssen, D. B. (1998). Effects of
rhamnolipid biosurfactants on removal of phenanthrene from soil. Environmental
Science & Technology 32, 1806-1812.

Pajatsch, M., Gerhart, M., Peist, R., Horlacher, R., Boos, W., and Bock, A. (1998). The
periplasmic cyclodextrin binding protein CymE from Klebsiella oxytoca and Its

role in maltodextrin and cyclodextrin transport. Journal of Bacteriology 180,
2630-2635.

Petruzzelli, L., Celi, L., Cignetti, A., and Marsan, F. A. (2002). Inﬂuence of soil organic
matter on the leaching of polycyclic aromatic hydrocarbons in soil. Journal of

Environmental Science and Health Part B-Pesticides Food Contaminants and
Agricultural Wastes 3 7, 187-199.

Pilon-Smits, E. (2005). Phytoremediation. Annual Review of Plant Biology 56, 15-39.

Qi, Q. S., and Zimmermann, W. (2005). Cyclodextrin glucanotransferase: from gene to
applications. Applied Microbiology and Biotechnology 66, 475-485.

Quensen, J. F ., Tiedje, J. M., and Boyd, S. A. (1988). Reductive dechlorination of
polychlorinated biphenyls by amerobic microorganisms from sediments. Science
242, 752-754.

Reddy, C. A. (1993). An overview of the recent advances on the physiology and
molecular-biology of lignin peroxidases ofPhanerochaete chrysosporium.
Journal of Biotechnology 30, 91-107.

33

Reemtsma, T., Savric, I., and Jekel, M. (2003). A potential link between the turnover of
soil organic matter and the release of aged organic contaminants. Environmental
Toxicology and Chemistry 22, 760-766.

Risebrough, R. W., Peakall, D. B., Herman, S. G., and Kirven, M. N. (1968).
Polychlorinated biphenyls in the global ecosystem. Nature 220, 1098-1102.

Rodan, B. D., Pennington, D. W., Eckley, N., and Boethling, R. S. (1999). Screening for
persistent organic pollutants: Techniques to provide a scientiﬁc basis for POPS

criteria in international negotiations. Environmental Science & Technology 33,
3482-3488.

Rouse, J. D., Sabatini, D. A., and Harwell, J. H. (1995). Inﬂuence of anionic surfactants
on bioremediation of hydrocarbons. In Surfactant-Enhanced Subsurface
Remediation -- Emerging Technologies, D. A. Sabatini, R. C. Knox and J. H.
Harwell, eds. (Washington, D. C.: American Chemical Society), pp. 124-141.

Rugh, C. L., Gragson, G. M., Meagher, R. B., and Merkle, S. A. (1998). Toxic mercury
reduction and remediation using transgenic plants with a modiﬁed bacterial gene.
Hortscience 33, 618-621.

Rugh, C. L., Senecoff, J. F ., Meagher, R. B., and Merkle, S. A. (1998). Development of
transgenic yellow poplar for mercury phytoremediation. Nature Biotechnology
1 6, 925-928.

Rugh, C. L., Wilde, H. D., Stack, N. M., Thompson, D. M., Summers, A. 0., and
Meagher, R. B. (1996). Mercuric ion reduction and resistance in transgenic
Arabidopsis thaliana plants expressing a modiﬁed bacterial merA gene.
Proceedings of the National Academy of Sciences USA 93 , 3182-7.

Rylott, E. L., Jackson, R. G., Edwards, J ., Womack, G. L., Seth-Smith, H. M. B.,
Rathbone, D. A., Strand, S. E., and Bruce, N. C. (2006). An explosive-degrading
cytochrome P450 activity and its targeted application for the phytoremediation of
RDX. Nature Biotechnology 24, 216-219.

Safe, S. (1989). Polychlorinated biphenyls (PCBS): Mutagenicity and carcinogenicity.
Mutation Research 220, 31-47.

34

Saichek, R. E., and Reddy, K. R. (2005). Electrokinetically enhanced remediation of
hydrophobic organic compounds in soils: A review. Critical Reviews in
Environmental Science and Technology 35, 115-192.

Salido, A. L., Hasty, K. L., Lim, J. M., and Butcher, D. J. (2003). Phytoremediation of
arsenic and lead in contaminated soil using Chinese Brake Ferns (Pteris vittata)
and Indian mustard (Brassicajuncea). International Journal of Phytoremediation
5, 89-103.

Schwartz, A., and Bar, R. (1995). Cyclodextrin-enhanced degradation of toluene and p-
toluic acid by Pseudomonas putida. Applied and Environmental Microbiology 61,
2727-2731.

Sellers, K. (1999). Fundamentals of Hazardous Waste Site Remediation (Boca Raton, FL:
CRC Press).

Settavongsin, R. (2005). Application and Transgenic Production of the Biosurfactant
Cyclodextrin for Enhanced Polyaromatic Hydrocarbon Phytoremediation. In Crop
and Soil Sciences (East Lansing: Michigan State University), pp. 175.

Sheremata, T. W., and Hawari, J. (2000). Mineralization of RDX by the white rot fungus
Phanerochaete chrysosporium to carbon dioxide and nitrous oxide. Environmental
Science & Technology 34, 3384-3388.

Shimizu, M., Kimura, T., Koyama, T., Suzuki, K., Ogawa, N., Miyashita, K., Sakka, K.,
and Ohmiya, K. (2002). Molecular breeding of transgenic rice plants expressing a

bacterial chlorocatechol dioxygenase gene. Applied and Environmental
Microbiology 68, 4061 -4066.

Shrout, J. D., Struckhoff, G. C., Parkin, G. F., and Schnoor, J. L. (2006). Stimulation and
molecular characterization of bacterial perchlorate degradation by plant-produced
electron donors. Environmental Science & Technology 40, 310-317.

Slimko, E. M., and Lester, H. A. (2003). Codon optimization of Cenorhabditis elegans
GluCl ion channel genes for mammalian cells dramatically improves expression
levels. Journal of Neuroscience Methods 00, 1-7.

35

Steenland, K., Bertazzi, P., Baccarelli, A., and Kogevinas, M. (2004). Dioxin revisited:
Developments since the 1997 IARC classiﬁcation of dioxin as a human
carcinogen. Environmental Health Perspectives 112, 1265-1268.

Sun, H., Tateda, M., Ike, M., and Fujita, M. (2003). Short- and long-term
sorption/desorption of polycyclic aromatic hydrocarbons onto artiﬁcial solids:
effects of particle and pore sizes and organic matters. Water Research 3 7, 2960-
2968.

Szejtli, J. (1988). Cyclodextrin Technology (Dordrecht ; Boston: Kluwar Academic
Publishers).

Tsao, C. W., Song, H. G., and Bartha, R. (1998). Metabolism of benzene, toluene, and
xylene hydrocarbons in soil. Applied and Environmental Microbiology 64, 4924-
4929.

Vanyushin, B. F., and Kimos, M. D. (1988). DNA methylation in plants. Gene 74, 117-
121.

Volkering, F., Breure, A. M., and Rulkens, W. H. (1997). Microbiological aspects of
surfactant use for biological soil remediation. Biodegradation 8, 401-417.

Wang, J. M., Marlowe, E. M., Miller-Maier, R. M., and Brusseau, M. L. (1998).
Cyclodextrin-enhanced biodegradation of phenanthrene. Environmental Science
& Technology 32, 1907-1912.

Widdowson, M. A., Shearer, 8., Andersen, R. G., and Novak, J. T. (2005). Remediation
of polycyclic aromatic hydrocarbon compounds in groundwater using poplar

trees. Environmental Science & Technology 39, 1598-1605.

Wilson, S. C., and Jones, K. C. (1993). Bioremediation of soil contaminated with
polynuclear aromatic hydrocarbons (PAHS): A review. Environmental Pollution
81, 229-249.

Yadav, J. S., and Reddy, C. A. (1993). Degradation of Benzene, Toluene, Ethylbenzene,
and Xylenes (BTEX) by the lignin-degrading basidiomycete Phanerochaete
chrysosporium. Applied and Environmental Microbiology 59, 756-762.

36

Zheng, Z. M., and Obbard, J. P. (2002). Oxidation of polycyclic aromatic hydrocarbons
(PAH) by the white rot fungus, Phanerochaete chrysosporium. Enzyme and
Microbial Technology 31, 3-9.

37

CHAPTER I

Cloning and Characterization of the Cyclodextrin Glycosyltransferase
from Paem'bacillus sp. strain C36

Sarah Kinder
Department of Crop and Soil Sciences
Michigan State University

INTRODUCTION

Cyclodextrins

Cyclodextrins (CDS) are versatile surfactant-like molecules used for commercial
and analytical purposes including pharmaceutical and pesticide chemical production,
plant growth regulator enhancers, chiral chromatographic separation, and many other
applications (Apostolo et al., 2001; Gines et al., 1996; Greenberg—Ofrath et al., 1993;
Kamiya and Nakamura, 1995; Kilsdonk et al., 1995; Nunez-Delicado et al., 1997;
Uekama et al., 1998). CD3 are cyclic sugars composed of six, seven, or eight 0t-1,4 linked
glucose units (orCD, BCD and yCD, respectively) produced by bacterial starch
degradation. CDS are doughnut-shaped with a hydrophilic exterior and a hydrophobic
cavity, which can accommodate appropriately sized hydrophobic “guest” molecules in
ratios of 1:1, 2:1, or more rarely, 3:1 Cnguest molecule (Shen et al., 1998).
Complexation with CDS has the effect of solubilization, stabilization, or sometimes
precipitation of the included compound (Szejtli, 1988). CD3 have been found to
effectively in enhance dissolution and biodegradation of soil-sorbed organic
contaminants (Badr et al., 2004; Bardi et al., 2000; Molnar et al., 2005; Sheremata and

Hawari, 2000; Wang et al., 2005).

38

CGTases
Cyclodextrins are produced from starch by the action of a bacterial enzyme,
cyclodextrin glycosyltransferase (CGTase), which is thought to be functionally and

phylogenetically linked to or-amylase. Both enzymes act on starch, with CGTase forming

cyclic products and a-amylases forming linear products (delRio et al., 1997). The typical
reaction of CGTase, cyclization, is the covalent linkage of the non-reducing end of the
sugar and another glucose unit of the same oligosaccharide, forming cyclic products
(Uitdehaag et al., 1999). CGTases perform other reactions such as disproportionation,
coupling, and hydrolysis. The coupling reaction is the reverse of the cyclization reaction.
In the hydrolysis reaction a linear oligosaccharide is broken down into smaller linear
fragments.

CGTases are used industrially for the production of cyclodextrins and for
glycosylation of various sugars and other compounds (Stames, 1990). CGTases are 60-75
kDa extracellular enzymes secreted into the environment via the action of a diverse array
of transit peptides (Schmid, 1989). CGTases have been found in a wide variety of
microbes, primarily Bacillus and related species but also, Klebsiella, Brevibacillus, and
Thermoanaerobacter (Binder et al., 1986; Wind et al., 1995). Full genome sequencing of
several microbes has revealed putative cgt genes in Xanthomonas and Streptococcus
strains (da Silva et al., 2002; Ferretti et al., 2001).

CGTases contain ﬁve recognized domains, most of which are shared with or-
amylases. Domain A contains a calcium-binding domain, which comprises the active site
of the enzyme. The B and C domains provide stability for the active site during substrate

binding, while the function of the D domain remains unknown and is generally limited to

39

CGTases and not shared with cr-amylases (Qi and Zimmerrnann, 2005). Domain E is
found in Ot-amylases from diverse sources and is thought to function in binding raw
starch (Janecek et al., 2003). CGTases are relatively similar across bacterial species with
at least 51 completely conserved amino acid residues located primarily within the active
site, which contains a ([3/0t)g barrel tertiary structure (Qi and Zimmerrnann, 2005).
Individual CGTases tend to exhibit differing chD, BCD or yCD biosynthetic
speciﬁcities, which may also be inﬂuenced by reaction conditions (Szejtli, 1988). Amino
acid sequences are generally conserved among BCGTase and ctCGTase proteins, both of
which are more divergent from the less conserved yCGTases. For example, yCGTaseS
usually contain a deletion of six amino acids at the beginning of the B region, which may
serve as a hinge, to Open the active site allowing formation of the larger (8 glucose unit)
cyclic product (Qi and Zimmerrnann, 2005).

The objective of this study is to examine the sequence and enzymatic function of
a novel CGTase, PI-Cgt, in the original host strain, Paenibacillus sp. C36, and as

expressed in Escherichia coli strain DHSOL.

MATERIALS AND METHODS
Gene cloning and modiﬁcation
Cyclodextrin producing bacteria were isolated from ﬁeld soil and the cgt gene
cloned via PCR from genomic DNA isolated from Paenibacillus sp. (formerly classiﬁed
within Bacillus) strain C36 (Settavongsin, 2005). To make the cgt gene (PI-cgt) more

amenable to in vitro manipulation and allow cloning into the vector pBluescript SK' the

40

recognition sites for NotI and EcoRI restriction enzymes (Stratagene, La Jolla, CA) were
introduced into the 5’ UTR of the PI-cgt using the forward primer:

PIcgt-F 1: 5’GAA TTC GGC GGC CCG "ITA AAG AGG ATT AAC AAT GTT AAT
GG. The recognition sites of the restriction enzymes Sac] and BamHI were introduced
into the 3’ UTR of P1-cgt using the reverse primer: PIcgt-Rl: 5’CTG TAC GGA TCC
GAG CTC ATT AAG GCT GCC AGT T. These changes allow for the expression of
pBS-PI-cgt in E. coli DHSOL. Primer sections containing engineered restriction sites are
underlined, with remaining DNA sequence of primer PIcgt-Rl complementary to PI-cgt.
Primer F 1 contains other modiﬁcations including an in-frame stop codon in reference to
the pBS LacZ fragment and a new ribosomal binding sequence to replace the original
which was removed by restriction site addition. The in—frame stop codon causes
translation of the LacZ fragment to terminate while the ribosomal binding site attracts the
ribosome to the cgt initiation codon, resulting in the translation of PI-cgt.

PCR for cloning and modiﬁcation were performed in 40ul volume reactions
containing, 0.1 pl template, 4ul 10XPCR Buffer, 4p] 25mM MgClz, 2p] of 10pm forward
and reverse primers 0.8ul of lOOmM dNTPs, 1.6ul BSA (10mg/ml) and 0.4ul of
Amplitaq (Invitrogen, Carlsbad, CA). The PCR conditions were as follows: A primary
denaturation step, 96°C for 3 minutes followed by 40 cycles of 96°C 3OSeconds, 50°C
annealing temperature for 30 seconds and a 72°C extension temperature for 45 seconds.
The PCR products were ﬁrst checked for correct size and ampliﬁcation by agarose gel
electrophoresis. DNA preparations displaying positive PCR reactions were then cloned
directly into pCR2.l using the TOPO TA cloning kit (Invitrogen, Carlsbad, California)

used according to manufacturer speciﬁcations. TA-cloned cgt was transformed into E.

41

coli DHSOL and were plated on selective LB plates (lOg/L NaCl, Sg/L yeast extract, lOg/L
Bacto tryptone, 15g/L Bacto agar; Beckton Dickinson, Sparks, MD) containing 100mg/L
kanamycin. Positive colonies were picked, grown overnight (16hrs) in 5mls LB with
lOOmg/L kanamycin or 50mg/L ampicillin. DNA was extracted using a truncated
standard alkaline lysis procedure (Maniatis et al., 1982). Cells were Spun down at 10,000
X g for 1 minute using 1.5m] micro tubes and 1.5ml of culture. Supernatant was removed
by pouring and the tubes were blotted dry using a paper towel. Cells were then
resuspended by vortexing in lOOul of (SOmM glucose, 25mM Tris-HCI, lOmM EDTA)
(pH 8.0). Cells were lysed by 200141 of (0.2% sodium dodecyl sulfate and 0.2M NaOH)
and repeated inversions of the tubes. The resulting mixture was neutralized by 150ml
(60mL 5M potassium acetate, 11.5mL acetic acid, 28.5mL H20), mixed well and spun
down for 2 min at 10,000 X g. To precipitate DNA, 200p] of the supernatant was added
to lml of 98% ethanol and mixed well. The solution was spun down at 10,000 x g for 5
minutes. Ethanol was carefully decanted, the tubes blotted dry, and tubes were allowed to
stand to air-dry. Once pellets were mostly dry they were resuspended in 27ml of Tris-
EDTA containing approximately 150ug/ml RNase. The resulting plasmid DNA solutions
were screened via restriction enzyme digestion using EcoRI (Invitrogen, Carlsbad, CA)
performed according to manufacturer’s instructions followed by agarose gel
electrophoresis on a 1% gel in Tris-acetate EDTA at 100 V for 1 hour. Agarose gels were
stained aﬁer running with 0.8mM ethidium bromide and visualized on a Bio-Rad
Quantity One Gel Documentation system (Bio-Rad, Hercules, CA).

Positive clones were grown up for 16hrs in LB with selection at 37°C and

extracted using the Wizard DNA minprep kit. DNA solutions were subjected to micro-

42

dialysis prior to sequence submission, which consisted of placement of the DNA solution
on a13mm, 0.025um pore size nitrocellulose ﬁlter (Millipore, Billerica, MA) ﬂoating on
sterile water for 5 minutes. Afterwards the DNA solution was placed in a new Eppendorf
tube, and sequenced using Applied Biosystems (Foster City, CA) sequencing technology
in one direction as a primary screen for mutations and was performed by the Michigan
State Research Technology Support Facility (MSU, East Lansing, MI).

A single mutation free, completely sequenced clone was chosen for subsequent
subcloning into pBS using EcoRI and BamHI. pBS and pCR2.1 containing PI-cgt were
digested using EcoRI and BamHI enzymes. Both reactions were run on 1% Agarose gels.
The 2100bp PI-cgt gene fragment was excised from the gel using a scalpel and puriﬁed
using the QIAquick gel extraction kit according to manufacturer’s instructions (Quiagen,
La Jolla, CA). Only a portion of the pBS reaction was run to check for complete
digestion. The remainder of the reaction was heat inactivated at 65°C for 20 minutes. The
puriﬁed PI-cgt fragment was combined with the digested pBS in a 3 to 1 molecular ratio
along with DNAligase and fresh ligation reaction buffer as per manufacturers
instructions. Ligations were allowed to proceed overnight and were heat inactivated the
following day at 65°C for 20 minutes. Ligations were digested by an enzyme found
within the polylinker segment of pBS, but not within PI-cgt, to cut self-ligated plasmids.
Linearized plasmids will not be replicated in bacterial cells — biasing the transformation
recovery to cells containing the desired insert. Bacterial transformation was carried out
using E. coli DHSor cells prepared to be chemically competent utilizing the following
method. A single colony was used to inoculate an overnight 5mL culture of LB, which

was used to inoculate 1L of LB medium split between 4 1L ﬂasks incubated at 37°C at

43

200rpm. After the optical density of the cultures reached approximately 0.3 they were
spun down for 8 minutes, 10,000 x g at 4°C and resuspended in 0.1M CaClz twice. After
the second resuspension the cells were held overnight on ice. The next day cells were
diluted with 80% glycerol, aliquoted into 1.5m] micro-tubes and ﬂash frozen using liquid
nitrogen.

For transformation, DHSOL competent cells were thawed and held on ice. The

entire ligation was added to 200ul of cells. Cells were heat shocked by placement in a
42°C water bath for 2 minutes followed by 2 minutes on ice. After heat shock 500ml of
SOC medium (20g Bacto tryptone, 5g Bacto yeast extract, 2ml of 5M NaCl, 2.5m] of 1M
KCl, 10ml of 1M MgClz, 10ml of 1M MgS04, 20ml of 1M glucose in IL) was added and
the transformation reaction was incubated at 37°C, 200rprn for 30 minutes.
Transformation reactions, one plate with 10 III the other with lOOul were plated
on solid LB ampicillin plates spread with 50ul X-gal (20mg/ml in dimethyl forrnarnide)
to allow for blue-white screening of potential clones. Those clones that were white in
color due were more likely to contain an insert due to disruption of the B—galactosidase
fragment. Positive clones were screened via restriction analysis using EcoRI and BamHI

as described above. A single positive clone was selected for further experiments.

Sequence Analyses
Alignments, phylograms and bootstrap values were analyzed with Clustal W
version 1.83 (Thompson et al., 1994). Trees were drawn by Phylodraw version 0.8

(Graphics Application lab, Pusan National University, South Korea) with the neighbor-

44

joining method using the Clustal W output. Detection of signal peptide and cleavage site

was performed using SignalP analysis software (Bendtsen et al., 2004).

Starch Clearing Analysis

For screening of CGTase producing strains for starch degradation, visual starch
clearing was utilized as a diagnostic screen. Bacterial colonies were grown on solid Basic
medium containing 1% soluble starch, 0.5%yeast extract, 0.5% tryptone, 0.1%K2HP04,
0.02%MgSO4 * 7H20, 0.02%CaC12 * 2H20, 1%(NH4)ZSO4, pH to 7.0 and 15g of Bacto
Agar per liter. Media was sterilized prior to use, poured into 100mm X 15mm disposable
plates (Fisher Scientiﬁc, Hampton, NH) and allowed to solidify overnight. Bacterial
colonies were touched to plates and allowed to incubate at their respective temperature
optima, 28-30°C for P. sp. C36, 37°C for E. coli for 16 hr and P. sp C36 for 32 hr. After
growth was completed, plates were stained with a 1:30 aqueous dilution of an iodine
solution consisting of 10% potassium iodide 1% iodine and 50% ethanol with water.
Starch forms a deep blue colored complex with iodine. Cyclodextrin fails to form a
colored complex with the iodine dye causing areas of starch degradation and CD

production appear as colorless, clear zones on agar plates.

Colorimetric Assay of B-CD

A colorimetric dye assay was used to quantify [3CD production (Kaneko et al.,
1987), modiﬁed for use in a microplate. Crude enzyme extract was obtained from
overnight cultures of E. coli and P. sp. C36 respectively, via centrifugation for 1 minute

at 10,000xG and ﬁlter sterilized to exclude any remaining cells. 50p] of enzyme extract

45

was added to 200ul of 1.25% starch in lmM phosphate buffer in 1.5ml microtubes. After
incubation of up to 24 hrs for P. sp C36 and E. coli-PI-cgt, 50ul of the enzymatic reaction
was removed and placed in a 96 well plate. 25p.l of 0.4mM phenolphthalein and 20ul of
1M NaC04 were added to the enzyme/starch mixture. CD forms a complex with the
colored phenolphthalein dye causing color reduction. The amount of color reduction is
proportional to the quantity of CD in solution, with the relationship being a logarithmic
reduction in absorbance. Absorbance was measured at 550nm with a Spectra Max 190
(Molecular Devices, Sunnyvale, CA) microplate reader after 2 minutes of incubation
accompanied by light shaking. Samples were tested alongside a standard curve done in
triplicate, measuring from 15ug/ml to 1000ug/ml.

The Speed and rate of BCD production by both C36 and pBS PI-cgt were
measured. An enzymatic digest was setup as previously described with subsaniples being
analyzed for BCD content via the colorimetric method. Three replicates of each enzyme
source were included, with incubation performed at 50°C. Subsarnples were analyzed at

zero, one, two, and three hours after incubation start.

Thin Layer Chromatography

Thin Layer Chromatography (TLC) was performed to separate and identify the
different CDS. The same in-vitro enzymatic reactions as those used for the colorimetric
assays, were spotted in 2ul aliquots, 3 times onto the base of a 10cm X 20cm silica gel
60/Kieselguhr p254 aluminum TLC sheet (EM Science, Gibbstown, NJ). on, B, and yCDs
(Sigma, St. Lois, M0) were used as standards in 1% aqueous solutions and spotted to the

same sheet as the enzymatic reactions. The mobile phase was acetonitrile-water-

46

ammonium hydroxide (6:321) with plates being run in a sealed glass TLC tank.
Completed TLC plates were sprayed with Vaugh’s solution (1 g Ce(SO4)2, 24g

(N H4)2M004, 50ml concentrated H2804 and 450ml H20) using a TLC Sprayer and
developed by heating on a hot plate until blue spots appeared. Blue Spots were compared
to spots generated in lanes containing the standards, if the migration distances were

similar to that of the CD standards, it was deemed a positive result.

RESULTS

Sequence comparisons

Since PI-cgt was not shown to be identical in amino acid sequence to any other
CGTase sequence in the Genbank database, sequence comparisons to known CGTases
were undertaken. A Blast search performed utilizing the entire amino acid sequence of
PI-cgt found the most similar protein to be a CGTase from Bacillus lichenformis
(Genbank accession # CAA33763) at 86% identity and 89% similarity using the Blosum
62 amino acid substitution matrix (Henikoff, 1992). Using the same methods, PI-cgt was
73% identical and 78% similar to the well-characterized CGTase of Paenibacillus
illinoisensis strain 251. A phylogenetic tree of several bacterial CGTases (archaeal
CGTases were excluded) was created using Clustal W and Phylodraw using the neighbor
joining method (Figure 1.1, Figure 1.2). This method uses the assumption that in any set
of data the quantity of evolution between the various enzymes should be minimized, it is

generally assumed to frequently produce a tree that is very close to the true tree.

47

 

 

 

100

100

CAM840

CAA33763

PLCgt
MR32682
ABC-02281
CAAssozs

 

c AAA22298

 

c E05456

 

1c BAI121 7
o CAH61550

 

 

. NP_269428

 

100

a CAL2 5733

 

o YP_242734

Figure 1. Evolutionary phylogram comparison of Cgts to PI-Cgt. Branch lengths indicate

evolutionary distance. Bootstrap values are indicated on the branches.

CAA48401
CAA33763
PI-Cgt

AAR32682
Aecozzai
CAA55023
AAA22298
E05456

8A891217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI-Cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
£05456

8A391217
CAH61550

NP_269428

CAL25733

YP_242734

--------------------- MFQMAKRAFLSTTLTLGLLAGSALPFLPA
--------------------- MFQMAKRVLLSTTLTFSLLAGSALPFLPA
--------------------- MFKWTKRIILSTTLSFSLLAGSALPLFPA
-------------------------- MKRFMKLTAVWTLWLSLTLGLL—-
-------------------------- MKRFMKLTANMWLWLSLTLGLL--
-------------------------- MKKFLKSTAALALGLSLTFGLF--
-------------------------- MKSRYKRLTSLALSLSMALGIS--
-------------------------- MRRWLSLVLSMSFVFSAIFIVSDT
------------------------- VFRKLLCTLVTIITLSAWIVSHGGE
------------------- MINKKNSIGKAICICLSILLLFGVLSIFQPV
------------------------- -MRELHIKTYKLLTKSAVLLGLISF
------------------------ MTMNRFMKKLFSMFLALALIVGYTAA
MAGRATDLRAGDRRLEPDRGRCVRGAGPKRPGRAMMRSVLMAAMLLYSGA

SAVYADP ------ DTAVTNKQSFSTDVIYQVFTDRFLDGNPSNNPTGA--
SAIYADA ------ DTAVTNKQNFSTDVIYQVFTDRFLDGNPSNNPTGA--
ASVFADA ------ DTAVSNKQNFSTDVIYQVFTDRFLDGNPSNNPTGG--
SPVHAAP ------ DTSVSNKQNFSTDVIYQIFTDRFSDGNPANNPTGA--
SPVHAAP ------ DTSVSNKQNFSTDVIYQIFTDRFSDGNPANNPTGA—-
SPAQAAP ------ DTSVSNKQNFSTDVIYQIFTDRFSDGNPANNPTGA--
LPAWASP ------ DTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGD--
QKVTVEA ------ AGNLN-KVNFTSDVVWQIVVDRFVDGNTSNNPSGA--
VHASN -------- ATNDLSNVNYAEEVIYHIVTDRFKDGDPDNNPQGQ--
TNATQNSLEHIKEHTSVNNQVNYATDVIYQIVTDRFLDGDKYNNPTCEN-
PLTVSAAD ----- NASVTNKADFSTDTIYQIVTDRFNDGNTSNNGKTD~-
YPLPAVAA ----- ASGQSLGPVTSKDVIYQILTDRFYDGDHANNIPPGTP
ACAAPAP ------ GDYYGTLEPFAADAVYFVVTDRFVNGDTGNDHRDQGG
. a a“ o Vii! cit- it.

48

CAA48401
CAA33763
PI—C t

AAR3 682
ABGOZZBl
CAA55023
AAA22298
E05456

6A391217

CAEZ 5733
vp_242734

CAA48401
CAA33763
PI—Cgt
AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456
BAB91217
CAH61550
NP_269428
CAL25733
YP_242734

CAA48401
CAA33763
PI—Cgt
AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456
5A591217
CAH61550
NP_269428
CAL25733
YP_242734

CAA48401
CAA33763
PI—cgt

AAR32682
ABGOZZBl
CAA55023

8A891217
CAH61550
NP_269428
CAL25733
YP_242734

————————— AYDAILSNLKLYCGGDWQGLINKINDNYFSDLGVTALWISQ
--------- AFDGTCSNLKLYCGGDwQGLVNKINDNYFSDLGVTALWISQ
————————— AYDASCSNLKLYCGGDNQGLINKINDNYFSDLGITALWISQ
————————— AFDGSCTNLRLYCGGDWQGIINKINDGYLTGMGITAIWISQ
--------- AFDGSCTNLRLYCGGDHQGIINKINDGYLTGMGITAIWISQ
————————— AFDGTCTNLRLYCGGDWQGIINKINDGYLTGMGVTAIWISQ
————————— AFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQ
————————— LFSSGCTNLRKYCGGDWQGIINKINDGYLTDMGVTAIwISQ
————————— LFSNGCSDLTKYCGGDNQGIIDEIESGYLPDMGITALWISP
--------- LYSEDGADLRKYLGGDWRGIIQKIEDGYLPDMGISAIWISS
————————— VFDKN——DLKKYHGGDWQGIIAKIKDGYLTDMGISAIWISS
PELFNDDNGDGRGDGTDLNKYQGGDWKGIQEKIP——YLKNMGITAVWISA
AHRSFDVPTPCDGGVGDNIGYLGGDFKGIVDHAD--YIRGLGFGAVWITP

. "::*: . *: .:*. . .
PVEN ---------- IFATINYSGVTNTAYHGYWARDFKKTNPYFG-TMAD
PVEN —————————— IFATINYSGVTNTAYHGYWARDFKKTNPYFG—TMTD
PVEN —————————— IYSLINYSGVNNTAYHGYWARDFKKTNPAFG—TMTD
PVEN —————————— IYSVINYSGVHNTAYHGYWARDFKKTNPAYG—TMQD
PVEN ---------- IYSVINYSGVHNTAYHGYWARDFKKTNPAYG-TMQD
PVEN —————————— IYSIINYSGVNNTAYHGYwARDFKKTNPAYG—TIAD
PVEN ---------- ITSVIKYSGVNNTSYHGYwARDFKQTNDAFG—DFAD
PVEN —————————— VFSVMN—DASGSASYHGYWARDFKKPNPFFG—TLSD
PVEN —————————— VFDLHP—-—EGFSSYHGYWARDFKKTNPFFG—DFDD
PVEN —————————— IYAVHP-—-QFGTSYHGYWARDFKRNNPFFG—DLND
PVEN —————————— IDSIDP——SNGSAAYHGYWAKDFFKTNQHFG—TEAD

PYEN ---------- RENLIAG---MYASYHGYHARNYFATNPHFG-KMQD
IVDNPDEAFTGGKPITCESTLSDHGKTGYHGYWGVNFYRLDEHLPSPGLD
-w . a

- 011414" - -
- u . .

NLITTAHAKGIKIVIDFAPNHTSP ------------- AMETDTSFAEN
NLVTTAHAKGIKIIIDFAPNHTSP ————————————— AMETDTSFAEN
NLINTAHAKGIKVIIDFAPNHTSP ————————————— AMETDTSFAEN
KNLIDTAHAHNIKVIIDFAPNHTSP ------------- ASSDDPSFAEN
KNLIDTAHAHNIKVIIDFAPNH Juv.JrAEN
NLIAAAHAKNIKVIIDFAPNHTSP ————————————— ASSDQPSFAEN
NLIDTLTLITSRSDRLRPQPHVSG ————————————— RAGTNPGFAEN
RLVDAAHAKGIKVIIDFAPNHTSP ------------- ASETNPSYMEN
SRLIETAHAHDIKVVIDFVPNHTSP VDI ED
RELIAVANEHDIKVIIDFAPNHTSP ------------- AEVNNPNYAED
QLVKVAHQHHIKVVIDFAPNHTST ------------- AEKEGTTFKED
ALVDALHDNGIKVVIDFVTNHSGPRPDGDGVRXXPDRDSSGQSVFDPD

 
    
   
 
   
 

 

 

FTRSMHANDLKVVLDIVGNHGSP ------------- AY SMPVAQPGF
GRLYDNG ------ TLVGGYTNDTNGYFHHNGGSDFSSLENG-—IYKN D
GKLYDNG ------ NLVGGYTNDTNGYFHHNGGSDFSTLENG——IYKN g0
GKLYNNG —————— TLLGGYTNDTNKLFHHNGGSDFSTLENG——IYKN YD
GRLYDNG —————— NLLGGYTNDTQNLFHHYGGTDFSTIENG--IYKN YD
GRLYDNG —————— NLLGGYTNDTQNLFHHYGGTDFSTIENG--IYKN YD
GRLYDNG —————— TLLGGYTNDTQNLFHHNGGTDFSTTENG——IYKN YD
GALYDNG —————— SLLGAYSNDTAGLFHHNGGTDFSTIEDG——IYKN YD
GRLYDNG —————— TLLGGYTNDANMYFHHNGGTTFSSLEDG--IYRN FD
GALYDNG ------ TLLGHYSTDANNYFYNYGGSDFSDYENS--IYRN YD
GNLYNNG ------ EFVASYSNDLNEIFYHFGGTDFSTYEDS--IYRN FD
GALYKNG ------ KLVGKFSDDKDKIFNHESWTDFSTYENS--IYHS YG
GNPIDYNGDGKAENRIADILNDTNGFFHHEGNRPDSDTSQFGYRHKE KS
GKLYDAQG ————— RLVADHQNLAPAQLDPAHNPLHAFYNTS———-GG AE
I! u n .

 

49

CAA48401
CAA33763
PI-cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

BAB91217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI—cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

8A891217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI-C t

AAR3 682
ABGOZZBI
CAA55023
AAA22298
E05456

8A891217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI-cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

BABQlZl?
CAH61550

NP_269428

CAL25733

YP_242734

LADFNHNNATIDKYFKDAIKLWLDMGVDGIRVDAVKHMPLGWQKSWMSSI
LADLNHNNSTIDTYFKDAIKLWLDMGVDGIRVDAVKHMPQGWQKNWMSSI
LADLNHNNSTIDTYFKDAIKLWLDMGIDGIRVDAVKHMPMGWQKNWMSSI
LADLNHNNSSVDVYLKDAIKMWLDLGVDGIRVDAVKHMPFGWQKSFMSTI
LADLNHNNSSVDVYLKDAIKMWLDLGVDGIRVDAVKHMPFGWQKSFMSTI
LADLNHNNSTVDVYLKDAIKMWLDLGIDGIRMDAVKHMPFGWQKSFMAAV
LADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKQYPFGWQKSFVSSI
LADLNHQNPVIDRYLKDAVKMWIDMGIDGIRMDAVKHMPFGWQKSLMDEI
LASLNQQHSFIDKYLKESIQLWLDTGIDGIRVDAVAHMPLGWQKAFISSV
LAGLNLNNNFVDQYLRDSIKFWLDLGVDGIRVDAVKHMPLGWQKSFVDTI
LADLNNINPKVDQYMKEAIDKWLDLGVDGIRVDAVKHMSQGWQKNWLSHI
LADYSQENGVVIEHLEKAGKFWKAKGIDGFRHDATLHMNPAFVKGFKDAI
LSDLNEDNPAVLDYLAGAYLQWMEQGADAFRIDTIGWMPDRFWHAFVARI

Oil“:

YA--HKPVFTFGE LGS-AAPDADNTDFANESGMSLLDFRFNSAVRNVF
YG--YKPVFTFGEW§LGS-SASDADNTNFANQSGMSLLDJRFNNEVRNVF
NN--YKPVFTFGE LGV-NEISPEYHQFANESGMSLLDFRFAQKARQVF
NN--YKPVFTFGE LGV-NEISPEYHQFANESGMSLLDFRFAQKARQVF
NN--YKPVFTFGE LGV—NEVSPENHKFANESGMSLLDFRFAQKVRQVF
YGG-DHPVFTFGE LGA—DQTDGDNIKFANESGMNLLDFEYAQEVREVF
DN--YRPVFTFGE LSE-NEVDANNHYFANESGMSLLDFRFGQKLRQVL
YD--YNPVFTFGE GA-QGSN-HYHHFVNNSGMSALDFRYAQVAQDVL
YN--HKPVFVFGE LGK-DEYDPNYYHFANNSGMSLLDFEFAQTTRSVF
YE--KHNVFVFGE SGH-TDDDYDMTTFANNSGMGLLDFRFANAIRQLY
DSAPGGPVTHFGEF IGRPDPKYDEYRTFPDRTGVNNLDFEYYNANRQAF
REK- RPGVFMFGE DYD-—PAKIAGHTWARNAGVSVLDFPLKQQLSAVF

YA--HKPVFTFGEgELGS-AASDADNTDFANKSGMSLLDiRFNSAVRNVF

"if" 0*.

R-DNTSNMYALOSMINSTATDYNQVNoquFIDNHDMDRFKTSAVNNR-R
R—DNTSNMYALDSMLTATAADYNQVNDQVTFIDNHDMDRFKTSAVNNR-R
R-DNTSTMVALDSMITSTAADYAQVNDQVTFIDNHDMDRFKTSAVNNR-R
R-DNTDNMYGLKAMLEGSEVDYAQVNDQVTFIDNHDMERFHTSNGDRR-K
R-DNTDNMYGLKAMLEGSEVDYAQVNDQVTFIDNHOMERFHTSNGDRR-K
R-DNTDNMYGLKAMLEGSAADYAQVDDQVTFIDNHOMERFHASNANRR-K
R—DKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTR-A
R-NNSDNWNGFNQMIQDTASAYDEVLDQVTFIDNHDMDRFMIDGGDPR—K
R-NQKGTMHDIYDMLASTQLDYERPQDQVTFIDNHDIDRFTVEGRDTR-T
R-NHEKNMFDLYDMLKNTENNYERVVDQVTFIDNHDMDRFHYDGATKR-N
TGFSTFTMRDFYKVLENRDQVTNEVTDQVTFIDNHDMERFATKVANNQTA
G- -EFSRSMSDFGQMLVQTSADYMVENQAVTFIDNHDVSRFRYIQPNDK- -P

G-HKQAGFEQLATPLYLRKGPYGNPYELMSFYDNHOMARLDAS--DTG--

: ::i( “with: it.
LEQALAFTLTSRGVPAIYYGTEQYLT-GNGDPDN ----- RAKMPSFSKST
LEQALAFTLTSRGVPAIYYGTEQYLT-GNGOPDN ----- RGKMPSFSKST
LEQALAFTLTSRGVPAIYYGTEQYMT—GNGDPDN ----- RAKMPSFSKTT
LEQALAFTLTSRGVPAIYYGSEQYMS-GGNDPDN ----- RARIPSFSTTT
LEQALAFTLTSRGVPAIYYGSEQYMS-GGNDPDN ----- RARIPSFSTTT
LEQALAFTLTSRGVPAIYYGTEQYMS-GGTDPDN ----- RARIPSFSTST
TEQALALTLTSRGVPAIYYGTEQYMT-GDGDPNN ----- RAMMTSFNTGT
VDMALAVLLTSRGVPNIYYGTEQYMT-GNGDPNN ----- RKMMSSFNKNT
TDIGLAFLLTSRGVPAIYYGTENYMT-GKGDPGN ----- RKMMESFDQTT
VEIGLAFLLTSRGVPTIYYGTEQYLT-GNGDPYN ----- RKPMSSFDQNT
VNQAYALLLTSRGVPNIYYGTEQYAT-GDKDPNN ----- RGDMPSFNKES

YHASLAVLLTSRGIPNLYYGTEQYLNPGHGGSDAGRLFLQAAAPAFSEQT
FIDAHNWLFTARGIPVIYYGSETGFMRGRAEHAGNRNYFGEERVSNAPQS

:ﬂ:ﬂﬂ:ﬂ :***:*

50

CAA48401
CAA33763
PI—cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

BABQlZl?
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI-Cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

5A891217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
PI-Cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

8A891217
CAH61550

NP_269428

CAL25733

YP_242734

CAA48401
CAA33763
,PI-cgt

AAR32682
ABGOZZBl
CAA55023
AAA22298
E05456

BABQlZl?
CAH61550

NP_269428

CAL25733

YP_242734

TAFNVISKLAPLRKSNPAIAYGSTQQRWINNDVYVYERKFGKS--VAVVA
TAFNVISKLAPLRKSNPAIAYGSTQQRWINNDVYIYERKFGKS--VAVVA
TAFNVISKLAPLRKTNPAIAYGTTQQRWINNDVYVYERKFGNN——VAVVA
TAYQVIQKLAPLRKSNPAIAYGSTQERWINNDVIIYERKFGNN--VAVVA
TAYQVIQKLAPLRKSNPAIAYGSTQERWINNDVIIYERKFGNN--VAVVA
TAYQVIQKLAPLRKCNPAIAYGSTQERWINNDVLIYERKFGSN--VAVVA
TAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSS——AALVA
RAYQVIQKLSSLRRNNPALAYGDTEQRWINGDVYVYERQFGKD--VVLVA
TAYQVIQKLAPLRQENKAVVYGSTKERWINDDVLIYERSFNGD--YLLVA
KAYKIIQKLAPLRKSNPALAYGTTQERWLNNDVIIYERKFGNN--IVLVA
QAYKVISKLAPLRKQNQALAYGTTEQRWISDHVLVFERKFGNH--VALVA
VAYRLIGKLSALRQSNDALAYGTTDILFSNDDALVYKRQFFDK-—QVIVA
PIFGPLQRIATLRRNTPALQRGVQVDLQLRGDQAAFLRVYQHAGMTQTAL
. . .. «u. u. w u -
VNRNLSTSASITGLSTSLPTGSYTDVLGGVLNGNNITS----TNGSINNF
VNRNLTTPTSITNLNTSLPSGTYTDVLGGVLNGNNITS----SGGNISSF
VNRNLSTPTSISGLTTSLPSGTYNDVLAGALSGNNITS---—TGGNVANF
INRNMNTPASITGLVTSLPQGSYNDVLGGILNGNTLTVG-—-AGGAASNF
INRNMNTPASITGLVTSLPQGSYNDVLGGILNGNTLTVG—--AGGAASNF
VNRNLNAPASISGLVTSLPQGSYNDVLGGLLNGNTLSVG---SGGAASNF
INRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVG——-SGGAVTNF
VNRSSSSNYSITGLFTALPAGTYTDQLGGLLDGNTIQVG--—SNGSVNAF
INKNVNQAYTISGLLTEMPAQVYHDVLDSLLDGQSLAVK---ENGTVDSF
INRNLSQSYSITGLNTKLPEGYYYDELDGLLSGKSITVN--—PDGSVNQF
INRDQTNGYTITNAKTALPQNSYKDKLEGLLGGQELIVG---ADGTISSF
VNRQPDRTVSIPALTTTLPVGTYPDALDGLLYGRTMTVVNQNGALQIPAF
VLLNKGDAAADIAVSRLLQPGSWRDAFS ----------------------
o o a it o

TLAAGATAVWQYTTAE--TTPTIGHVGPVMGKPGNVVTIDGRGFGSTKGT
TLAAGATAVWQYTASE--TTPTIGHVGPVMGKPGNVVTIDGRGFGSAKGT
TLAAGATAVWQYTANT--TTPTIGHVGPVMGKAGNTVTIDGRGFGTTKGT
TLAPGGTAVWQYTTDA--TAPIIGNVGPMMAKPGVTITIDGR-ASARQGT
TLAPGGTAVWQYTTDA--TAPIIGNVGPMMAKPGVTITIDGRGFGSGKGT
TLAAGGTAVWQYTAAT-—ATPTIGHVGPMMAKPGVTITIDGRGFGSSKGT
TLAAGGTAVWQYTAPE--TSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGT
DLGPGEVGVWAYSATE--STPIIGHVGPMMGQVGHQVTIDGEGFGTNTGT
LLGPGEVSVWQHISESG-SAPVIGQVGPPMGKPGDAVKISGSGFGSEPGT
IINPGEVSIWQFAGET--ITPLIGQVGPIMGQVGNKVTISGVGFGDKKGT
ELGAGQVAVWTYEGED--KTPQLGDVDASVGIAGNKITISGQGFGNSKGQ
TLAGGEVSVWSHNPPADPAEPHIGEVISTMGRPRNTVYIYGTGLGDA-AA
--------------------------------- GEQVQVQGR--------
o a if

VYFGTTANTGAAITSWEDTQIKVTIPSVAAGNYAVKVA-ASGVNSNAYNN
VYFGTTAVTGSAITSWEDTQIKVTIPPVAGGDYAVKVA-ANGVNSNAYND
VYFGTTAVTGSAITSWEDTQIKVTIPAVAAGNYAVKVA-ASGVNSNTYNN
VYFGTTAVTGADIVAWEDTQIQVKILRVPGGIYDIRVANAAGAASNIYDN
VYFGTTAVTGADIVAWEDTQIQVKIPAVPGGIYDIRVANAAGAASNIYDN
VYFGTTAVSGADITSWEDTQIKVKIPAVAGGNYNIKVANAAGTASNVYDN
VYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKS
VKFGTTAAN---VVSWSNNQIVVAVPNVSPGKYNITVQSSSGQTSAAYDN
VYFRDTKID---VLTWDDETIVITLPETLGGKAQISVTNSDGVTSNGYD-
VNFGEIDAT---IISWTNSVIQIEIPSVPAGNYEITVSSEGGEKSNSYN-
VTFGEISAE---ILSWSDTLITLKVPTVPANYYNISVTTADKQTSNSYQA
VAFGSQQAA—-—VVSAQDNRIAAVVPNVQAGEYAITVT-KGGKTSNPFR-
-------------------- VTLQVPAHG-----VRVLLSDAPVT-----
o u o it o

51

CAA48401

PT I LTG DQVTVR FWN NASTTLGQNLY LTGNVAE LG WSTG STAI G — — — P

CAA33763 FTILSGDQVSVRFVINNATTALGENIYLTGNVSELGNWTTGAASIG---P
er-c t FTILSGNQVSVRFVINNASTTLGQNLYLTGNVAELGNWSTGPLAIG--—P
AARB 682 FEVLTGDQVTVRFVINNATTALGQNVFLTGNVSELGNWDP-NNAIG---P
ABGO2281 FEVLTGOQVTVREVINNATTALGQNVFLTGNVSELGNwoP—NNArG-——p
CAA55023 FEVLSGDQVSVRFVVNNATTALGQNVYLTGSVSELGNWDP-AKAIG---P
AAA22298 FNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGTWDP-NKAIG-—-P
E05456 FEVLTNDQVSVRFVVNNATTNLGQNIYIVGNVYELGNWDT-SKAIG---P
8A891217 FQLLTGKQESVRFVVDNAHTNYGENVYLVGNVPELGNWNP-ADAIG-—-P
CAH61550 FEVLTNKQIPVRFVVNNAYTSWGQNVYLVGNVHELGNWDP—NRAIG—-—P
NP_269428 FEVLTDKQIPVRLLINDFKTVPGEQLYLMGDVFEMGANDA—KNAVG-—-P
CAL25733 YQVLGGDQVQVIFHVNKXRSRDX -------------------- CLCRGxx
YP_242734 -DVALRKQLDAQMADQAARDARNK --------------------------
a it u o
CAA48401 AFN--QVIHQYPTWYYDVSVPAGKQLEFKFFKKNG—STITWESGSNHTFT
CAA33763 AFN--Qv1HAYPTwYYovsvaGKQLEFKFFKKNG-ArrerGGSNHTFT
PI—cgt AFN--QVIYSYPTWYYDVSVPAGTSLEFKFFKKNG-STITWENGNNHTFT
AAR32682 MYN-—QVVYQYPTWNYDVSVPAGQTIEFKFLKKQG—STVTWEGGANRTFT
ABGO2281 MYN—-QVVYQYPTWYYDVSVPAGQTIEFKFLKKQG-STVTWEGGANRTFT
CAA55023 MYN-—QVVYQYPNMNYDVSVPAGKTIEFKFLKKQG-STVTWEGGSNHTFT
AAA22298 MYN--QVIAKYPSWYYDVSVPAGTKLDFKFIKKGG-GTVTWEGGGNHTYT
E05456 MFN--QVVYSYPT“NIDVSVPEGKTIEFKFIKKDSQGNVTWESGSNHVYT
8A891217 MFN--QVVYSYPTWYYDVSVPADTALEFKFIIVDGNGNVTWESGGNHNYR
CAH61550 FFN--QVVYQYPTWNLDISVPADTTLEFKFIKIDESGNVIWQSGLNRVYT
NP_269428 LFNNTQTIAKYPNWFFDTHLPINKEIAVKLVKKDSIGNVLWTSPETYSIK
CAL25733 ----------- PNWGXGIRTX ------- asras -----------------
YP_242734 --------------------------------------------------

Figure 1.2. Alignment of CGTases. Sources of individual CGTases listed in Table 1 by

accession number. PI-cgt is designated by gray box. Stars, * denote identical residues, :

denotes conserved strong group residues, . denotes weak group conserved residues. PI-cgt

is outlined in gray, the four critical aromatic residues for CGTases are outlined in black.
The host strains for each of the included CGTases are shown (Table 1.1).

Table 1.1. Source organisms and references for CGTases included in Figure 1.1, and 1.2.

 

 

 

 

 

 

 

 

 

 

Accession . Major CD
Number Host Organism produced Reference
CAL25733 Bacillus halodurans NI Unpublished
. (Takano et
AAA22298 Baczllus macerans Ot-CD al., 1986)
CAA48401 Paenibacillus illinoinensis strain 8 B-CD (1:11tsilglggft
CAA33763 Bacillus lichenformis a-CD & B-CD (Hilégegfk
. . . This
Pl-cgtl Paembaczllus sp. strain C36 NI Dissertation
. .. (Takada et
BAB91217 Baczllus clarkn y-CD al., 2003)

 

 

 

52

 

 

 

 

 

 

 

 

 

 

 

 

 

Accession . Major CD
Number Host Organism produced Reference
Patent: JP
E0545.6 Geobacillus stearothermophilus cL-CD 1993244945-
(Translatron) A 1
CAA5 5023 Paenibacillus illinoinensis strain 251 B-CD (Lawson et
aL,1994)
ABG02281 Bacillus sp. N-227 B-CD Unpublished
.. (Thiemann
CAH61550 Anaerobranca gottschalkn NI et al., 2004)
AAR32682 Bacillus sp. I-5 Nl Unpublished
Xanthomonas campestris pv. (da Silva et
YP-242734 Campestris NI al., 2002)
NP_269428 Streptococcus pyogenes M1 GAS NI (Ferretti et
aL,2001)

 

PI-cgt clusters with most other Bacillus CGTases, and more speciﬁcally clusters
with the most similar group of CGTases from Bacillus lichenformis and P. illinoisensis
strain 8, although Pl-cgt is more different from both of these strains than they are from

each other.

Signal Peptide Analysis

CGTase is secreted extracellularly in bacterial strains harboring cgt. Since PI-cgt
is a novel CGTase and signal peptides are highly variable, SignalP software was used to
predict the cleavage location in Gram positive bacteria (the original host strain), Gram
negative bacteria and eukaryotic systems. The Gram positive Hidden Markov models
(HMM) predicted a cleavage site between position between 34 and 35 with a probability
of 0.96.

The Gram positive Neural Networks (NN) gave a slightly different position,

between residues 36 and 37 with all scores above the cutoff values, indicating high

53

probability of an accurate cleavage site prediction. With Gram negative HMM, a position
between 34 and 35 was predicted with a probability of 0.928. The Gram negative NN
gave low scores on the predicted cleavage site (C-score less than cutoff), but high scores
on the presence of a signal peptide (S-scores) with the cleavage site predicted to be
between positions 34 and 35. For eukaryotic HMM, a cleavage site between positions 34
and 35 was again predicted but with a probability of only 0.463. Eukaryotic NN predicted
a cleavage site between positions 23 and 24 with all scores above the cutoff values.

The different positions of the predicted eukaryotic versus bacterial cleavage sites are

shown in Figure 1.3.
MFKWTKRIILSTTLSFSLLAGSA"LPLFPAASVFADA*D
Figure 1.3. The predicted PI-cgt signal sequence. Arrow shows predicted eukaryotic

cleavage site of the PI-cgt signal peptide. The asterisk shows the predicted bacterial

cleavage site.

Clear Zone Formation
P. sp. C36, E. coli — PI-cgt and E. coli DHSa were all grown on the same plate

containing 1% starch basic medium (Figure 1.4).

54

  
 
 

P. sp. C36
‘—

E- 60” DHS; . . ‘ E. coli PI-cgt

Figure 1.4. Bacterial clear-zone formation. Shown clockwise from top: E. coli expressing
PI-cgt under control of the lac promoter, Paenibacillus sp. C36 parental strain and DH50L
negative control.

The plate was incubated ﬁrst at 30°C for 16hrs then at 37°C for an additional 16
hrs. After iodine staining, C36 showed large clear zones surrounding the colonies, E. coli
PI-cgt Showed smaller clear zones, and the untransformed E. coli strain, DHSct, did not
show any clear zones.

Enzymatic optimum temperature determination
CGTases are generally considered thermostable enzymes with typical temperature

optima in the range of 50-60°C. The temperature Optimum for PI-cgt was experimentally

55

determined for enzyme derived from the original host strain, P. sp. C36 via enzymatic
reaction with starch and analysis by phenolphthalein colorimetric determination. A set of
three replications of each temperature, 25, 30, 40, 50, and 60°C were included. The 0CD
production was measured at 6 hours but BCD levels in several treatments were still
relatively low, so reaction time was extended to 24 hours.

Complexation of BCD with the phenolphthalein dye results in an exponential
curve when absorbance at 550nm is related to [3CD concentration. To obtain a linear
relationship, both absorbance and concentration were log transformed (Figure 1.5). From
the graph, the lower detection limit for 0CD is approximately lSug/ml. The BCD
production over the tested temperatures was highest at 40 and 50°C yielding an average
of 667 and 594ug/ml BCD, respectively (Figure 1.6). BCD production dropped off
signiﬁcantly (P<0.1) at lower temperatures, 30°C and 25°C as well as the high
temperature, 60°C. There was no signiﬁcant difference between 40°C and 50°C

treatments .

Kinetic Studies of 0CD Production

The [3CD production increased over time in both P. sp. C36 and E. coli PI-cgt enzymatic
reactions with C36 increasing more rapidly than the E. coli strain (Figure 1.7). A spike in
[5CD concentration was noted at one hour of incubation in C36 but not in PI-cgt E. coli.

This same spike was observed in repetitions of the kinetic study.

56

 

  
 

 

 

 

D ? r5?)
0 %-—~-——o ——-—-- —-- 4 r*-——- -———9—— —-——--—-- --~—-- - -- -*r I
E- ’ _ _y=-4.2753X-0.3296 2* -9:
E RT=—OB785 e
U) m _E __ ......
1 ‘L
’2‘ E E L i - , _ E _,-_
0 I m i E L “w ._,_______ _ i EcL- -m .561-
9, z \
ge-e— —-++é+A-—---_ --———-—r
—' 9L o?”

l I o

-08 -06 -0.4 -02
Log(Abs) @ 550nm

Figure 1.5. Typical standard curve for colorimetric analysis of [3CD using
phenolphthalein. Log values were used to generate a linear relationship. The 0CD

concentrations used were: 15.6, 31, 62, 125, 250, 500, and 1000 ug/ml.

 

 

 

 

 

 

 

 

 

 

 

O

 

800—~ b
b
g 600:
é 400 -«
5:. l
E zoo-l a
s I a a
25 so 40 so so

Temperature in °C

Figure 1.6. Temperature optimum determination for P. sp. C36. Samples of 50111
supernatant were incubated at various temperatures with 200ml starch for 3hours.
Analysed for BCD content via the phenolphthalein method (n=3) Statistically similar

treatments (or < 0.05) are denoted with the same letter.

57

Quantitative Production of BCD by P. sp. C36 and E. coli PI-cgt

The BCD producing capabilities of both C36 and PI-cgt were measured over a set
period of six hours. Reaction conditions were the same as the enzymatic Optimum
determination, with the temperature being 50°C. P. sp C36 and E. coli PI-cgt strains
produced 7173 ng and 7231 ng [3CD per microliter of enzymatic solution respectively.
The average [3CD production per hour per mg cell dry mass was 897 ng and 1808 ng for

C36 and PI-cgt respectively.

TLC of bacterially produced CDS

Thin Layer Chromatography showed that the three cyclodextrins each had slightly
different RF values with or, B and y having 0.46, 0.42 and 0.38 respectively. However,
when run together the individual CDS merged into a single spot. Fused spots were found
in both bacterial enzymatic reactions at similar locations to the standards, implying that
all of the three major CDS were produced, or, B and y. No spots were produced by the E.
coli DHSa lacking the PI-cgt plasmid, correlating spot production with the presence of

PI-cgt in the bacterial host. Figure 1.8 Shows a representative TLC plate.

58

1200 , +picso‘ 636 I 2 g 2
I L _____ -g

  

 

 

Minutes of Incubation

Figure 1.7. CGTase Kinetic reaction Showing [3CD production over time. ug/ml [3CD is

given per mg of total solution protein, x axis Shows minutes of incubation at 55°C. (n =

3)

’ C36 PI- t
01 I3 Y coli? ‘ ‘ [cg 1);?“ 0.13.7

Figure 1.8. Thin layer chromatography analysis of CDS by CGTase producing bacteria.

0., l3, and yCD are shown as standards. C36, PI-cgt and DHSOI were incubated for 5 hours.

59

DISCUSSION

A novel CGTase has been cloned from P. sp C3 6. This gene clusters with the
similar CGTases from B. lichenformis and P. illinoisensis strain 8, although it is more
dissimilar from these two CGTases than they are from one another. The well
characterized CGTase from B. circulans strain 251 clusters more distantly but most of the
CGTases from Bacillus species form a single group. Bootstrap values for most branches
were high being 99 or 100. The lowest value near the base of the tree was 81. PI-cgt was
overall most Similar to or and BCGTases and least similar to yCGTases such as that from
Bacillus clarkia and more distant organisms such as Xanthomonas and Streptococcus.
The Bacillus halodurans CGTase also clustered very distantly from the other Bacillus
CGTases indicating alkaliphiles may impose very different evolutionary constraints on
CGTases in mesophilic species. The sequence similarities of PI-cgt to CGTases with
known CD products give a strong indication that PI-cgt probably produces primarily BCD
and aCD.

A signal peptide sequence was also detected in PI-Cgt that appears to be unique,
among CGTases, although signal sequences usually exhibit high variability (Bendtsen et
al., 2004). Cleavage site prediction showed the same location and a strong signal for both
Gram positive and Gram negative bacteria with much lower scores for eukaryotic
prediction. PI-Cgt Shares all of the strictly conserved domains found in other CGTases
including calcium binding residues present in A Domain, the circular B Domain and the
raw starch binding Domain E (Rahman et al., 2006). PI-cgt also contains the four
aromatic residues thought to be critical for CGTase function and speciﬁcity (N akamura et

aL,1994)

60

Starch plate clearing showed clear zones produced by both C36 and PI-cgt.
However clear zones produced by the original host strain, C36 were much larger than
those produced by the E. coli strain DHSct containing the cloned CGTase. This may be
partially due to the plate having been incubated at a sub-optimal temperature, 30°C for E.
coli. However, when the experiment was repeated with the bacteria growing separately at
their respective optima and preferred medium, the same difference in clear zone size was
noted. The smaller clear zone may be indicative of inefﬁcient secretion of Pl-cgt from E.
coli cells. Since PI-cgt originated from Paenibacillus, a Gram positive bacterium,
secretion through the more complex Gram negative membrane system may be difﬁcult,
and smaller clear zones might reﬂect a difference in secretion efﬁciency. Effective signal
peptide function in a heterologous host is somewhat unusual in CGTases though not
unheard of, which commonly Show little to no secretion in heterologous expression
systems (Lee et al., 2002). The signal sequence from Brevibacillus brevis CD162
CGTase could be secreted from E. coli cells (Kim et al., 1998). Signal peptide prediction
software, using HMM indicated a high probability for the Gram negative cleavage
location of PI-cgt to be identical to that of the Gram positive location, a strong indication
that the PI-cgt signal peptide would be functional in a Gram negative bacterium such as
E. coli.

PI-cgt showed a marked preference for higher temperatures in temperature
optimum studies, this is typical for CGTases being thermostable enzymes with PI-cgt
showing a similar temperature optimum to other CGTases derived from Bacillus species
(Rahman et al., 2006). However, it is important to note that under ﬁeld conditions

CGTase operates at substantially lower temperatures Since many CGTase producing

61

bacteria are mesophilic soil-dwelling organisms, and strain P. sp C36 was isolated from
temperate ﬁeld soil (Qi and Zimmerrnann, 2005). High temperature stability in CGTases
may reﬂect long-term stability in extracellular environment or perhaps a simple
coincidence in enzyme functionality and temperature stability.

The kinetic starch degradation studies showed increased production of [3CD over
time, with C36 Showing the highest rate followed by the recombinant, E. coli. It is
possible that the lower 0CD production by E. coli PI-cgt is simply due to lowered
secretion of CGTase rather than lower activity of the enzyme itself. The solution protein
levels from supematants both bacterial species used in enzymatic reactions were very
similar. Similar total protein concentration may not reﬂect the relative abundance of PI-
Cgt in that solution. Also of note was the very high levels of [3CD found in the medium at
1 hour which was immediately followed by lower concentrations measured at 2 hours.
This Spike is primarily due to a single high concentration sample of the three replications,
indicating the degree of increase in concentration is strongly exaggerated by this sample.
If this sample were removed, the remaining samples still Show an initial spike in
concentration somewhat higher than the second time point but not higher than the ﬁnal
time point. Previous experiments also showed a spike in 0CD concentration by C36 at
approximately the same reaction time. This observation may be because CGTases can
degrade CDS as well as synthesize them. CGTases often degrade larger CDS to smaller
CDS when incubated over longer periods (Schmid, 1989). The faster reaction seen in the
C36 strain could be due to the presence of an additional enzyme such as cr-amylase since
the enzyme extract was not speciﬁcally puriﬁed for CGTase. In steady state analysis of

the two enzymes over 6 hours, relatively similar BCD production was observed indicating

62

that while the initial reaction rate of C36 CGTase may be faster, E. coli PI-cgt can
eventually approach the BCD production of C36.

TLC of bacterial starch reactions showed that both the original host strain and
cloned E. coli CGTase were capable of producing all of the standard CDS. Lighter Spots
produced by PI-cgt versus P. sp. C36 probably reﬂect the lower CD production by the E.
coli expressed CGTase. This again may also simply reﬂect lower CGTase secretion by E.
coli cells. Careful examination of the intensity of Spots on the plate seem to indicate a in
intensity bias towards more quickly migrating CDS, possibly indicating that PI-cgt may
produce more (1 and B CD than yCD.

For improved production of PI-cgt in E. coli, modiﬁcation of the signal sequence
may be required. Additionally, the codons of PI-cgt may be sufﬁciently different from E.
coli to reduce expression somewhat, since the codon usage of Bacillus species and E. coli

are not identical.

CONCLUSION

In this study, a novel cgt gene was cloned from a Bacillus relative. Its sequence
was examined via several different comparison metrics and unique sequence features
were found within PI-cgt. Bacterially produced enzyme from both Paenibacillus sp. C36
and E. coli transformed with PI-cgt was capable of degrading starch and producing BCD
and other CDS. The clear zones produced by E. coli PI-cgt give strong evidence that the
signal sequence from P. sp. C36, a Gram positive bacterium can function in a Gram
negative species, conﬁrming signal peptide prediction. However, CD production by E.

coli was somewhat lower than that produced by strain C3 6, indicating that either the

63

Signal peptide may not efﬁciently translocate PI-Cgt through the E. coli membrane or the
E. coli produced enzyme may simply be less efﬁcient than the C36.

These bacterial studies are a proof of concept, showing that the cloned P. sp. C36
CGTase is ﬁrnctional in a heterologous host. Further characterization and manipulation of
PI-cgt would give a fuller picture of the nature of the enzyme and the precise ratio of or, B
and yCDs. Examination of the CD ratio produced by the P. sp. C36 CGTase could give
additional information for understanding how CGTase sequences relate to the production
of speciﬁc CDS. Future comparisons of other CGTases may allow for the isolation of

superior enzymes for in-situ CD production.

64

LITERATURE CITED

Apostolo, N. M., Brutti, C., Ferrarotti, S. A., Llorente, B. E., and Krymkiewicz, N.
(2001). Stimulation of root development with cyclodextrins on jojoba shoots in
vitro. In Vitro Cellular & Developmental Biology-Plant 3 7, 414-418.

Badr, T., Hanna, K., and de Brauer, C. (2004). Enhanced solubilization and removal of
naphthalene and phenanthrene by cyclodextrins from two contaminated soils.
Journal of Hazardous Materials 112, 215-223.

Bardi, L., Mattei, A., Steffan, S., and Marzona, M. (2000). Hydrocarbon degradation by a
soil microbial population with beta-cyclodextrin as surfactant to enhance
bioavailability. Enzyme and Microbial Technology 27, 709-713.

Bendtsen, J. D., Nielsen, H., von Heijne, G., and Brunak, S. (2004). Improved prediction
of signal peptides: SignalP 3.0. Journal Molecular Biology 340, 783-795.

Binder, F., Huber, 0., and Bock, A. (1986). Cyclodextrin glycosyltransferase from
Klebsiella pneumoniae M5A1 - cloning, nucleotide sequence and expression.
Gene 47, 269-277.

da Silva, A. C. R., Ferro, J. A., Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B.,
Monteiro-Vitorello, C. B., Van Sluys, M. A., Almeida, N. F ., Alves, L. M. 0, do
Amara], A. M., Bertolini, M. C., Carnargo, L. E. A., Carnarotte, G., Cannavan, F.,
Cardozo, J ., Chambergo, F ., Clapina, L. P., Cicarelli, R. M. B., Coutinho, L. L.,
Cursino-Santos, J. R., El-Dorry, H., Faria, J. B., Ferreira, A. J. S., Ferreira, R. C.
C., Ferro, M. I. T., Forrnighieri, E. F., Franco, M. C., Greggio, C. C., Gruber, A.,
Katsuyama, A. M., Kishi, L. T., Leite, R. P., Lemos, E. G. M., Lemos, M. V. F.,
Locali, E. C., Machado, M. A., Madeira, A., Martinez-Rossi, N. M., Martins, E.
C., Meidanis, J., Menck, C. F. M., Miyaki, C. Y., Moon, D. H., Moreira, L. M.,
Novo, M. T. M., 0kura, V. K., 01iveira, M. C., 01iveira, V. R., Pereira, H. A.,
Rossi, A., Sena, J. A. D., Silva, C., de Souza, R. F ., Spinola, L. A. F., Takita, M.
A., Tamura, R. E., Teixeira, E. C., Tezza, R. I. D., dos Santos, M. T., Trufﬁ, D.,
Tsai, S. M., White, F. F ., Setubal, J. C., and Kitajima, J. P. (2002). Comparison of
the genomes of two Xanthomonas pathogens with differing host speciﬁcities.
Nature 41 7, 459-463.

delRio, G., Morett, E., and Soberon, X. (1997). Did cyclodextrin glycosyltransferases
evolve from alpha- amylases? Febs Letters 416, 221-224.

65

Ferretti, J. J., McShan, W. M., Ajdic, D., Savic, D. J., Savic, G., Lyon, K., Primeaux, C.,
Sezate, S., Suvorov, A. N., Kenton, 8., Lai, H. 8., Lin, S. P., Qian, Y. D.,'Jia, H.
G., Najar, F. 2., Ren, Q., Zhu, H., Song, L., White, J., Yuan, X. L., Clifton, S. W.,
Roe, B. A., and McLaughlin, R. (2001). Complete genome sequence of an M1
strain of Streptococcus pyogenes. Proceedings of the National Academy of
Sciences USA 98, 4658-4663.

Gines, J. M., PerezMartinez, J. I., Arias, M. J ., Moyano, J. R., Morillo, E., RuizConde,
A., and SanchezSoto, P. J. (1996). Inclusion of the herbicide 2,4-
dichlorophenoxyacetic acid (2,4-D) with beta-cyclodextrin by different processing
methods. Chemosphere 33, 321-334.

Greenberg-Ofrath, N., Terespolosky, Y., Kahane, I., and Bar, R. (1993). Cyclodextrins as
carriers of cholesterol and fatty acids in cultivation of mycoplasma. Applied
Environmental Microbiology 59, 547-551.

Janecek, S., Svensson, B., and MacGregor, E. A. (2003). Relation between domain
evolution, speciﬁcity, and taxonomy of the alpha-amylase family members
containing a C-terrninal starch-binding domain. European Journal of

Biochemistry 270, 63 5-645.

Kamiya, M., and Nakamura, K. (1995). Cyclodextrin inclusion effects on
photodegradation rates of organophosphorus pesticides. Environment
International 21, 299-304.

Kaneko, T., Kato, T., Nakamura, N., and Horikoshi, K. (1987). Spectrophotometric
determination of cyclization activity of beta-cyclodextrin-forming
cyclomaltodextrin glucanotransferase. Journal of the Japanese Society of Starch

Science 34, 45-48.

Kilsdonk, E. P. C., Yancey, P. G., Stoudt, G. W., Bangerter, F. W., Johnson, W. J .,
Phillips, M. C., and Rothblat, G. H. (1995). Cellular cholesterol efﬂux mediated
by cyclodextrins. Journal of Biological Chemistry 270, 17250-17256.

Kim, M. Y., Sohn, C. B., and 0h, T. K. (1998). Cloning and sequencing of a cyclodextrin
glycosyltransferase gene from Brevibacillus brevis CD162 and its expression in
Escherichia coli. FEMS Microbiology Letters 164, 411-418.

66

Lee, K. W., Shin, H. D., and Lee, Y. H. (2002). Extracellular overproduction of beta-
cyclodextrin glucanotransferase in a recombinant E. coli using secretive
expression system. Journal of Microbiology and Biotechnology 12, 753-759.

Maniatis, T., F ritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor: Cold Spring Harbor Laboratory).

Molnar, M., Leitgib, L., Gruiz, K., Fenyvesi, E., Szaniszlo, N., Szejtli, J ., and Fava, F.
(2005). Enhanced biodegradation of transformer oil in soils with cyclodextrin:
From the laboratory to the ﬁeld. Biodegradation16, 159-168.

Nakamura, A., Haga, K., and Yamane, K. (1994). Four aromatic residues in the active
center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011:
Effects of replacements on substrate binding and cyclization characteristics.
Biochemistry 33, 9929-9936.

Nunez-Delicado, E., SanchezFerrer, A., and GarciaCarmona, F. (1997). Cyclodextrins as
secondary antioxidants: Synergism with ascorbic acid. Journal of Agricultural
and Food Chemistry 45, 2830-2835.

Qi, Q. S., and Zimmerrnann, W. (2005). Cyclodextrin glucanotransferase: From gene to
applications. Applied Microbiology and Biotechnology 66, 475-485.

Rahman, K., Illias, R. M., Hassan, 0., Mahmood, N. A. N., and Rashid, N. A. A. (2006).
Molecular cloning of a cyclodextrin glucanotransferase gene from alkalophilic
Bacillus Sp TS 1-1 and characterization of the recombinant enzyme. Enzyme and
Microbial Technology 39, 74-84.

Schmid, G. (1989). Cyclodextrin glycosyltransferase production - yield enhancement by
overexpression of cloned genes. Trends in Biotechnology 7, 244-248.

Settavongsin, R. (2005). Application and Transgenic Production of the Biosurfactant
Cyclodextrin for Enhanced Polyaromatic Hydrocarbon Phytoremediation. In Crop
and Soil Sciences (East Lansing: Michigan State University), pp. 175.

67

Shen, X. H., Belletete, M., and Durocher, G. (1998). Spectral and photophysical studies
of the 1 : 3 (guest/host) rotaxane-like inclusion complex formed by a 3H-indole
and beta-cyclodextrin. Journal of Physical Chemistry B 102, 1877-1883.

Sheremata, T. W., and Hawari, J. (2000). Cyclodextrins for desorption and solubilization
of 2,4,6- trinitrotoluene and its metabolites from soil. Enviromnental Science &
Technology 34, 3462-3468.

Starnes, R. L. (1990). Industrial potential of cyclodextrin glycosyl transferases.
CerealFoods World 35, 1094-1099.

Szejtli, J. (1988). Cyclodextrin Technology (Dordrecht ; Boston: Kluwar Academic
Publishers).

Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position speciﬁc gap penalties and weight matrix choice. Nucleic

Acids Research 22, 4673-4680.

Uekama, K., Hirayama, F ., and Irie, T. (1998). Cyclodextrin drug carrier systems.
Chemical Reviews 98, 2045-2076.

Uitdehaag, J. C. M., Kalk, K. H., van der Veen, B. A., Dijkhuizen, L., and Dijkstra, B. W.
(1999). The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase)

as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-angstrom
resolution. Journal of Biological Chemistry 274, 34868—34876.

Wang, J. M., Maier, R. M., and Brusseau, M. L. (2005). Inﬂuence of hydroxypropyl-
beta-cyclodextrin (HPCD) on the bioavailability and biodegradation of pyrene.
Chemosphere 60, 725-728.

Wind, R. D., Liebl, W., Buitelaar, R. M., Penninga, D., Spreinat, A., Dijkhuizen, L., and
Bahl, H. (1995). Cyclodextrin formation by the thermostable alpha-amylase of
T hermoanaerobacterium thermosulfurigenes Em] and reclassiﬁcation of the
enzyme as a cyclodextrin glycosyltransferase. Applied and Environmental

Microbiology 61, 1257-1265.

68

CHAPTER II

Plant expression of bacterial Cyclodextrin Glycosyltransferase
for cyclodextrin production

Sarah Kinder
Department of Crop and Soil Sciences

Michigan State University
INTRODUCTION

Biological production of industrial and pharmacological compounds in plants has
become an important emerging technology. While plants have been used primarily for the
sources of food and ﬁber, harnessing them as biological factories is a more recent
development. Plants have been used for the production of heterologous proteins with
pharmaceutical applications, such as antibodies and enzymes (Berberich et al., 2005;
Girard et al., 2006), industrial plastic precursors and other industrial compounds (Conrad,
2005). Production of bioactive proteins, bioplastics and other biologically active
materials in plants may be cleaner and more economical than animal cell cultures or
synthetic production.

One biological compound with a wide variety of industrial, food and cosmetic
uses is cyclodextrin (CD). Cyclodextrins are cyclic sugars synthesized by bacteria from
starch and capable of enhancing the solubility of various hydrophobic compounds
(Szejtli, 1988). CD molecules orient the primary and secondary sugar hydroxyls along the
outer edges of the torus-shaped molecule with the ether linkages positioned in the
interior, hydrophobic region. CDS are commonly composed of 6, 7, and 8 glucose units
(termed or, B, and 7CD, respectively) each capable of associating with different

compounds due to the distinct cavity sizes. Host molecules become complexed within the

69

cyclodextrin, which provides enhanced stability, protection from oxygenating factors and
light-induced decomposition as well as solubility and biological absorption (Kamiya and
Nakamura, 1995; Loukas et al., 1994; Uekama et al., 1998). CDS can also block bitter
tastes in medicines and foul odors in the environment by entrapment of the source
compounds and prevention of binding to taste and scent receptors (Szejtli and Szente,
2005). CD5 have been used as soil or solution amendments to accelerate environmental
remediation treatments, including soil washing and biodegradation applications (Bardi et
al., 2000; Molnar et al., 2005; Viglianti et al., 2006; Wang et al., 2005; Wang and
Brusseau, 1995).

Cyclodextrin is typically produced on industrial scales using the preparations of
the bacterial enzyme cyclodextrin glycosyltransferase (CGTase) in large vat reactors with
starch as a feedstock (Starnes, 1990). Starch is ﬁrst liqueﬁed using either heat or amylase
treatment followed by addition of CGTase. CGTases always produce a mixture of (1, [3,
and y cyclodextrin in ratios dependent on the CGTase source and reaction conditions, so
the production of a pure CD form can be difﬁcult. [3CD is the easiest to purify since it has
low water solubility compared to the other CDS and precipitates at high concentrations.
The more water soluble ctCD and yCD compounds must be separated by
chromatographic techniques to achieve pure reagent forms, though organic solvents may
be added as complexing agents to help separate the individual cyclodextrins by enhanced
precipitation (Lee and Kim, 1991). Commercial and industrial applications for CDS are
increasing with global CD production in excess of 10,000 tons per year and concurrent

economy of scale reducing [3CD costs to only a few dollars per kg (Szejtli, 2004).

70

An alternative to bacterial CGTase enzyme production is development and
utilization of transgenic plants or plant cell cultures. Plants may be capable of producing
higher quantities of CGTase and perhaps more efﬁciently than bacteria due to the ability
of plants to grow in less stringently controlled conditions than bacterial cultures. CD
produced by plants would be free of pathogenic contaminants present in animal based
culture systems and could require less post-synthesis processing.

For environmental remediation applications, in situ CD production would be an
effective alternative to labor intensive addition of CD amendments to the soil or
maintenance of CD-producing organisms. Since plant starch is typically found only
within the sub-cellular plastid compartment, direct plant secretion of CDS from roots
would likely involve considerable metabolic engineering. It may be advantageous to
engineer plants to secrete CGTase into the rhizosphere essentially in the same manner as
CD-producing bacteria. This strategy is in contrast to previous efforts to express CGTase
in potato tubers to produce CD within plant amyloplasts as a substitute for conventional
industrial CD sources, which resulted in ineffective CD accumulation (Oakes et al.,
1991). Rhizosecreted CGTase may offer a more manageable solution to CD production
using plants, rather than labor intensive addition to soil or addition of potentially
problematic bacterial strains. The similarity of prokaryotic signal peptides to those in
eukaryotes should allow plants engineered with the bacterial cgt gene to secrete CGTase
into the surrounding matrix without further modiﬁcation (Hall et al., 1990). Using a
bioreactor approach, extracellular CGTase would be able to degrade starch in hydroponic

system to provide an easily extractable CD.

71

AS a direct application to in situ contaminant remediation, CGTase secreting
plants could be planted directly in contaminant soils. Plant produced CGTase could
convert starch present in soil due to exogenous addition or released during root turnover
to cyclodextrin, potentially enhancing the degradation rate of persistent organic
pollutants. The objective of this study is to transfer a bacterial cgt gene into laboratory
plants and evaluate cgt transgenic expression and product function. If effective, this
approach could provide an efﬁcient alternative source of reagent grade cyclodextrin and

possibly a new tool for environmental bioremediation.

MATERIALS AND METHODS

Cgt Gene Vector Construction

The PI-cgt gene was cloned from Paenibacillus sp. strain C36 as previously
described (Settavongsin, 2005). The gene was subcloned from the bacterial expression
vector, pBS SK' using XhoI and Sac], into plant expression vectors, pAPC-9K and
pE1778. The expression cassettes of these vectors each contain different promoters such
as Arabidopsis Actin2 and the “super promoter” construct to drive expression of the cgt
gene in plants (An et al., 1986; An et al., 1996). The “super promoter” was derived from the
mannopine synthase promoter and multiple octopine enhancer elements originally found
in Agrobacterium spp. (Ni et al., 1995). Both promoters are constitutively expressed at
high levels in all tissues, though the “super promoter” is more active in root tissue. The
transcription terminator used in the Actin2 gene expression cassettes is PE21 from Citrus
sinensis cv. Valencia (sweet orange) pectinesterase gene (N aim et al., 1998). The

expression cassette was excised from pAPC-9K using SpeI, Xbal, and Xmal restriction

72

enzymes cloned into the binary vector pCAMBIA (Gene bank accession #AF234296)

(CAMBIA Institute, Canberra, Australia).

Spcl Notl Xhol Sacl Xmal

 

Actin-2 PE21

pAPC-9K

 

 

 

 

 

Xba I Xmal
3SS-term
+ {CaMV3531 hpt | r—*
RB "" LB

Figure 2.1. Diagram of the pAPC9K and pCAMBIA 1300 cloning vectors.

To create plant expression constructs using the Arabidopsis Actin2 promoter, PI-
cgt was excised from pBS-PI-cgt using NotI and SacI and inserted into pAPK-9K using
the same restriction enzymes, resulting in pAPC-9K-PI-cgt. Expression cassettes of
pAPC-9K containing PI-cgt were excised using SpeI and XmaI restriction enzymes and
inserted into pCAMBIA 1300 cut with XbaI and Xmal. The resulting construct was
named pCAMBIA-Act-PI-cgt. PE1778 constructs were created by excising PI-cgt from
pBS-PI-cgt using XhoI and Sac] restriction enzymes, pE1778 was cut with these same
two enzymes to allow the insertion of PI-cgt. Completed pCAMBIA and pE1778
constructs were transformed into electrocompetent Agrobacterium LBA4404 (Invitrogen,
Carlsbad, CA) via electroporation using a Bio-Rad Micropulser according to the

manufacturer’s recommendations (Bio-Rad, Hercules, CA). A grobacterium

73

transforrnants were screened using semi-solid YM plates containing, 0.4g/L yeast extract,
1% mannitol, 1.7mM NaCl, 0.5mM MgSO4'7H20, 2.2mM K2HP04, and 15g/L Bacto
agar (Sigma, St. Loius, M0) supplemented with 100mg/L kanamycin for pCAMBIA or
pE1778 selection and 100mg/L streptomycin for Ti plasmid selection. KanR, StrR
colonies were grown in liquid LB medium (lOg/L NaCl, 5g/L yeast extract, lOg/L Bacto
tryptone (Beckton Dickinson, Sparks, MD) for plasmid DNA extraction and analysis.
Agrobacterium colonies displaying appropriate enzyme digested DNA bands fragments
via gel electrophoresis analysis were utilized in subsequent plant culture transformation

procedures.

Plant Transformation

Tobacco transformation

Tobacco (Nicotiana tabacum) cv. Little Havana transformation was carried out
using a standard method for co-cultivation of leaf sections with A grobacterium LBA4404
(Invitrogen, Carlsbad, CA) strains harboring the plant expression vector containing the
cgt gene construct and the selectable marker genes.

Agrobacterium strains were grown in liquid culture for tobacco leaf sections
inoculation. Transformation was performed by growing the strain of interest for two days
in YM medium. Agrobacterium cell preparations were spun down and resuspended in
500p] of fresh YM medium. Semi-solid Murashige and Skoog medium (MSO: 25g
sucrose/L, B5 vitamins lOOmg/L myoinositol, 10mg/L thiamine-HCI, 1mg/L nicotinic
acid, 1mg/L pyroxidine-HCI and 7g/L phytagar) (Invitrogen, Carlsbad, CA) was utilized

as a standard basic tissue culture medium (Murashige and Skoog, 1962). Tobacco leaf

74

sections approximately l-2cm square were pro-incubated without Agrobacterium on
M80 medium for 1-2 days then transferred to 1.5mL microtubes containing the
resuspended A grobacterium solution, then vortexed for 30 seconds and allowed to stand
for approximately 5 minutes. Afterwards the tissue sections were blotted dry using sterile
ﬁlter paper and placed on non-selective, M80 medium. After a period of two days, the
leaf sections were transferred to semi-solid M80104 which contained, in addition to
M80 ingredients, plant growth regulators 1mg/L benzylaminopurine (BA), 0.1mg/L 1-
napthaleneacetic acid (N AA) (Invitrogen, Carlsbad, CA), and either 25mg/L hygromycin
(for pCAMBIA—PIcgt) or 300mg/L kanamycin (for pEl778—PI-cgt) as selection agents
and 400mg/L timentin (Smithkline-Beecham, Philadelphia, PA) to control the growth of
Agrobacterium.

After about one month shoots began to form and once true leaves had formed,
shoots were excised and placed on rooting media which consisted of M80 medium at
6g/L phytagar, without plant growth regulators, containing antibiotic selection of 25mg/L
hygromycin or 100mg/L kanamycin and 400mg/L timentin. Rooted plantlets were placed
in 1 gallon round pots containing free draining potting soil (Baccto High Porosity
Professional Potting Mix, Michigan Peat, Houston, TX) and grown in a temperature
controlled greenhouse (25-3 0°C) for seed production. Each plant was reproductively
isolated from the others via placement of an isolation bag over the ﬂowers before
opening. Entire inﬂorescences were removed from the plant once capsules were ﬁlled
and beginning to dry. Bags containing inﬂorescenses were stored under greenhouse

conditions until fully dry. One to two dry capsules were harvested from each

75

inﬂorescence, seeds placed in 1.5ml microtubes and stored at 4°C until use. These seeds

were called the T1 generation.

Arabidopsis Transformation

Arabidopsis thaliana (ecotype RLD, Lehle Seeds, Round Rock, TX) was
transformed using the vacurun inﬁltration method. Vacuum inﬁltration draws
Agrobacterium cells into Arabidopsis ﬂoral tissues, where DNA transfer occurs in
developing ovules (Ye et al., 1999). Vacuum inﬁltration was performed as follows: 4 1/2
inch pots were ﬁlled with wet potting soil (Baccto High Porosity Professional Potting
Mix, Michigan Peat, Houston, TX). Standard size window screen was cut into squares
and placed over the top of mounded soil, held in place by rubber bands. Arabidopsis
seeds were mixed with sand in an approximate 1:10 ratio, and placed in a salt shaker. The
sand and seed mixture was shaken over the pots to evenly distribute seed, with soil being
kept moist until seed germination. After approximately 1-2 weeks any excess plants were
removed, resulting in approximately 7-10 mature Arabidopsis plants growing in each pot.
The plants were allowed to mature until the primary inﬂorescenses began to emerge and
elongated to approximately 3-4 inches. At this stage primary inﬂorescenses were cut and
the plants placed in the plant growth chamber for 1-2 days prior to Arobacterium
transformation.

Agrobacterium cultured overnight in 5m] YM medium containing 100mg/L
streptomycin and 100mg/L kanamycin after which one rrrl culture was used to inoculate
500ml of the same selective YM medium and grown on orbital shaker 1-2 days at 28°C.

After incubation the culture was Spun down and resuspended in IL of inﬁltration medium

76

consisting of 1/2X Murashige and Skoog salt mix (Murashige and Skoog, 1962), 1X BS
vitamins (listed in M80 medium above), SOg/L sucrose, 0.5g 2-[N-
morpholinojethanesulfonic acid (MES) and 0.044uM BA. 200p] Silwet L-77 (Lehle
Seeds, Round Rock, TX) was added to the Agrobacterium suspension and mixed well.
The bacterial solution was placed in a 600m] beaker and pots containing Arabidopsis to
be inﬁltrated were placed upside down into the ﬁlled beakers. The beakers were placed
inside of an 18.9L polycarbonate vacuum chamber (N algene, Rochester, NY). Vacuum
was pulled to approximately 15mm Hg on the system for 5 minutes, then the vacuum
source was removed and pressure was held for another 2 minutes. Afterwards pressure
was released slowly and pots were removed from the beakers, laid on their sides in a
large tub covered with plastic wrap. Pots were left covered for one full day after which
they were set upright and watered well. Plants were allowed to develop normally until
seed set. Pots containing mature plants were placed on their side and inﬂorescences were
placed in bags until the entire plant was dry. Seeds were collected from inﬁltrated and
non-inﬁltrated control plants once the Siliques appeared fully mature and near dryness.
Seeds were then harvested via gentle compression of the bag followed by screening to
remove chaff and other dry contaminants. Collected Arabidopsis T1 generation seeds

were placed in 1.5ml microtubes and stored at 4°C.

Transgenic Seed Screening and Selection
Seeds of tobacco and Arabidopsis were sterilized with 15% household bleach
(6.15% sodium hypochlorite, Clorox Company, Oakland, CA) for 15 minutes followed

by two rinses with sterile water.

77

For Arabidopsis, plating of seed represented the initial screen for transformed
lines. Seedlings showing resistance to hygromycin had incorporated the marker hpt gene
and likely the cgt gene as well. Seeds were germinated on selective medium - MS salts
only with 7g/L phytagar plus 25mg/L hygromycin. Resistant seedlings were scored as
putative transforrnants and removed from selective medium and planted in 2 1/2 inch pots
as soon as they were recognized. Resistant seedlings were allowed to grow and set seed,
which was narrred the Arabidopsis T2 generation.

Ratios of resistant to susceptible seedlings in T1 tobacco seed lots were used to
estimate the copy number of the PI-cgt gene. A three to one ratio of resistant to
susceptible seedlings is consistent with a single site of genome integration. Resistant
seedlings were removed from selection promptly and greenhouse grown as previously
described, with the resulting seeds being labeled the tobacco T2 generation. Seeds from
subsequent generations were also screened on antibiotic containing media. The results of

seedling screening on Arabidopsis and tobacco are shown in Tables la and 1b.

Transgenic Plant Genomic PCR Screening

Genomic DNA extracts from putative transgenic plantlets of both Species were
screened for integration of the gene of interest using PIcgt speciﬁc primers in PCR.
Genomic DNA extracts were generated using the DNeasy plant kit (Qiagen, La Jolla,
CA) according to manufacturer instructions. PCR reactions were carried out using 0.5 —

1.0ul genomic DNA template, 2ul 10X PCR Buffer (SOOmM KCl, 100mM Tris*Cl (pH

8.8) 1% TritonX), 0.6111 50mM magnesium chloride, 0.5111 of 10pm forward and reverse

78

primers 0.25 pl of 100mM dNTPS, and 1.0rrl of crude Taq polymerase extract with the

total reaction volume being 20p].

Crude Taq polymerase was generated by growing Esherichia coli expressing Taq
polymerase in Super Broth, which contains (16g Tryptone, 10g Yeast Extract, 2.5g NaCl
2.5m] of NaOH with the ﬁnal volume being 500ml. Overnight cell cultures were spun
down and resuspended in Buffer A consisting of, 50mM Tris (pH 8.0), 50mM dextrose,
lmM Ethylenediaminetetraacetic acid (EDTA) pH8.0. Buffer B consisting of, lOmM Tris
pH 8.0, 50mM KCl, lmM EDTA pH 8.0, 0.5% Tween 20, 0.5% NP-40, was added to
lyse the cells, which were then incubated at 75°C for one hour. Cells were again spun
down with the supernatant being mixed 1:1 with storage buffer (50mM Tris pH 8.0,
100mM NaCl, 0,1mM EDTA pH 8.0, 0.5mM DTT, and 50% glycerol). This solution was
diluted again 1:1 with 80% glycerol, resulting in the working solution of crude Taq

polymerase which was stored at —20°C until use.

RT-PCR-expression analysis

RNA was extracted from leaves using the RNeasy kit (Qiagen, La Jolla, CA)
along with on-column DNase digestion using the RNase-free DNAse set. The cDNA
synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad,
CA) according to the manufacturer’s instructions. PCR reactions for cloning were
performed in reactions containing, 0.5 -— 1.0ul template, 2p] PCR buffer (500mM KCl,
100mM Tris-HCI (pH 8.8) 1% TritonX), 0.6111 50mM MgCl2, 0.5ul of 10pm forward and
reverse primers 0.25 pl of 100mM dNTPs, and 1.0111 of crude Taq polymerase extract

with the total reaction volume being 20ul. The thermocycler conditions were as follows:

79

A primary denaturation step, 94°C for 3 minutes followed by 40 cycles of 94°C 30
seconds, 55°C annealing temperature for 30 seconds and a 68°C extension temperature
for 45 seconds. RT-PCR products were run on agarose gels of concentration from 0.8 —
1% in Tris-acetate EDTA at 100 V for 1 hour. Reactions using primers designed for the
selectable marker gene were run in parallel as an expression and cDNA preparation
control. Gels were stained with 0.8mM ethidium bromide in water for 15 minutes and
visualized under UV light using a Bio-Rad Quantity One Gel Documentation system

(Bio-Rad, Hercules, CA).

Starch Agar Clearing

The simplest CGTase expression assay is the clearing of starch. Putative CGTase
expressing plants were placed in 1X Murashige and Skoog media containing 1.0-0.1%
starch and 7g/L Phytagar (Murashige and Skoog, 1962). After growing on the medium
for 2-3 weeks, starch agar is stained with a 1:30 aqueous dilution of an iodine solution
consisting of 10% potassium iodide 1% iodine and 50% ethanol by mass with water.
Starch forms a deep blue colored complex with iodine. Iodine dye does not form a
colored complex with cyclodextrin, causing areas of starch degradation and CD

production appear as clear regions on a blue-black background of agar.

Arabidopsis and Tobacco Plant Tissue Preparation
Tobacco plants were grown individually in 300ml Erlenmeyer ﬂasks ﬁlled with
1/5X MS salt medium. Plants were watered once weekly and allowed to grow to near

maturity. Samples of approximately 15ml of hydroponic medium were removed and

80

concentrated using a ﬁltration/concentration apparatus (Millipore, Billerica, MA) using a
centrifuge at 2000Xg for 20 min, resulting in approximately 500ul crude preparation
which was used directly in enzymatic reactions with starch.

Sterilized tobacco and Arabidopsis seeds in aqueous solution were added to 30mls
of sterile MS salts medium in sterile 150ml Erlenmeyer ﬂasks capped with aluminum
foil. These ﬂasks were placed on a rotary shaker inside of a growth chamber under 16h
day and 86 p.mol/s"‘m2 and approximately 25rpm. After 15 - 30 days of growth 15ml of
hydroponics medium were concentrated using the same method as for adult plants to
between 250ul and 500u1. Crude preparations were removed from the apparatus and used
immediately or stored at 4°C for future use. Plant tissue was removed from the ﬂask and
weighed to determine the biomass present in the system, plant tissue was dried in an oven

at 60°C and weighed again after drying to determine dry biomass.

Colorimetric [3CD Assay

Enzymatic reactions were carried out by placing 50p] of the concentrated plant
enzymatic solution in 200ul of 1.25% starch in pH 6.0 lmM phosphate buffer. The
enzymatic reactions were placed in a 50°C incubator shaking at 200 rpm for a minimum
of 2 hours up to several days. After the incubation time had elapsed a 50ul aliquot of the
enzymatic reaction was analyzed for phenolphthalein color reduction by addition of 25p]
of 0.4mM phenolphthalein and 20p] of 1M NaC04 mixed in a 96 well microplate. After
light shaking and standing for a few moments, the plates were read at 550nm with a
Spectra Max 190 (Molecular Devices, Sunnyvale, CA) spectrophotometer equipped with

a microplate reader. The [3CD produced was quantiﬁed via comparison of absorbance to a

81

standard curve of known quantities of 0CD. The curve produced was an exponential
reduction in color in response to increasing [3CD concentration. Both concentration and

absorbance were log transformed to result in a linear relationship.

Thin Layer Chromatography

Thin layer chromatography (TLC) was performed to separate and identify the
different CDS. 2rrl of starch reaction was spotted 4 times to a 10cm X 20cm silica gel
60/Kieselguhr 1:254 aluminum TLC sheet (EM Science, Gibbstown, NJ). or, [3, and yCD
(Sigma, St. Lois, M0) were used as standards and spotted from 1% solutions to run
adjacent to starch reactions, these standards were spotted only once. The mobile phase
was acetonitrile-water-ammoniurn hydroxide (6:3:1) with plates being run in a sealed'
glass TLC tank. Completed TLC plates were sprayed with Vaugh’s solution (1 g
Ce(804)2, 24g (N H4)2M004, 50ml concentrated H2804 and 450ml H20) using a TLC

sprayer and developed by heating on a hot plate until blue spots appeared.

RESULTS
Selection of cgt Plant lines
Both the Super Promoter and Actin2 Promoter constructs were transformed into
tobacco (Nicotiana tabacum) cv. Little Havana. The transformation resulted in the
production of 12 independent transgenic lines with seven of these being selected for
ﬁrrther screening. These lines were allowed to grow and set seed. Using the appropriate

antibiotic marker, seed lots were screened by sterile plating. Most transgenic lines

82

displayed segregation ratios consistent with a Single-copy transgene integration (Table
2.1a).
Table 2.1a. Selective marker segregation analysis for T2 generation tobacco seeds. PC

lines use hygromycin selection, PE lines use kanamycin selection.

 

 

 

 

 

 

 

 

Line Hyg/KanR Hyg/KanS Segregation ratio
PC8A7 55 12 ~3 :1
PC 1B2 56 0 All positive
PE3A1 39 14 ~3 :1
PE] 1A1 49 17 ~3 :1
PE] 1A4 50 14 ~3 :l
PE13A2 53 15 ~3:1
PE2B4 54 15 ~3 :1

 

 

 

 

 

 

Kan“: Transformed lines resistant to kanamycin

KanS= Transformed lines susceptible to kanamycin

Only the Actin2 promoter construct was utilized in Arabidopsis transformation.
Six lines of cgt-Arabidopsis were generated. Of these lines, only one was found to be
homozygous after the T2 generation, cgt1-5 (Table 1b).

Table 2.1b. Selective marker segregation analysis for T2 generation Arabidopsis seeds

 

 

 

 

 

 

 

 

Line Hygft HygS Segr_egation ratio
PIcgt1-3 27 15 ~3:1
PIcgtl-S 3 1 0 All positive
PIcgt13 18 10 ~32]
PIcgtl4 19 10 ~3:1
PIcgtl-6 15 10 3:2
PIcgtl-l 15 26 1:1

 

 

 

 

 

 

Hng= Transformed lines resistant to hygromycin
Hygs= Transformed lines susceptible to hygromycin
This result may indicate multiple insertion events in line cgtl-5 but conﬁrms

single integration events for the cgtl-l, cgt14, and cgtl3 with ratios of resistant to

83

susceptible seedlings near 3: 1. Several lines exhibited low ratios, with some lines such as

cgtl-l Showing a 1:1 ratio.

Starch Clearing

Several transgenic lines of both tobacco and Arabidopsis have exhibited enhanced
ability to degrade starch by the formation of a zone of clearing in the blue iodine-starch
region of the medium. Wild type strains of either species either produced only faint clear
zones or none at all. Arabidopsis transforrnants appeared to be capable of forming larger
. clear zones than tobacco plants of Similar Size. Clear zone formation by Arabidopsis
seedlings also appeared to be both more rapid and extensive than with tobacco seedlings.
Interestingly, smaller Arabidopsis seedlings also seemed capable of forming larger clear

zones than their more developed counterparts (Figure 2.2).

PCR and RT-PCR

All seven lines chosen for further testing have been shown to be positive for
hygromycin or kanamycin resistance. Six of these tobacco lines were Show to be positive
for genomic PCR. The seven tobacco lines were tested in RT-PCR, with the 5 tested
Showing a positive reaction. Out of 3 Arabidopsis lines tested for RT-PCR, 2 showed a
positive result for PIcgt expression and hptII expression. RT-PCR results from
Arabidopsis correlated well with the observed quantity of cyclodextrin produced with
those exhibiting a positive result in RT-PCR also producing detectable quantities of BCD.
Example gels of tobacco and Arabidopsis RT-PCR reactions are shown in Figures 2.3a

and 2.3b.

84

      

wt cgtl-S

Figure 2.2. Clear zone formation by cgt-arabidopsis. Upper plate shows before iodine
staining. Lower plate shows clear zones in 0.1% starch agar formed by Cgt-expressing

Arabidopsis line, cgtl-S

85

Figure 2.3a. RT-PCR of Arabidopsis lines. Left gel is with cgt primers Right gel is with

hygromycin control primers. Arrows denote expected fragment size

    

RNA controls cgt cDNA RNA controls cgt cDNA
cgt primers hyg primers

Figure 2.3b. RT-PCR of tobacco lines. Leﬁ gel contains hygromycin primers, right gel is

cgt primers. Arrows denote expected fragment sizes.

 

RNA controls PC lines cDNA RNA controls PC lines cDNA

hyg Primers cgt primers

86

BCD production

Adult tobacco hydroponics failed to produce detectable quantities of 0CD.
Concentrated Arabidopsis seedling hydroponic solution did produce detectable quantities
of BCD after 48 hours of incubation. Quantities of BCD continued to increase over
extended incubation time in Arabidopsis seedling exudates. 0f 6 tested tobacco lines,
seedling hydroponics also failed to produce detectable quantities of 0CD even after 48
hours of incubation. Only one tested tobacco line did produce detectable quantities of
[3CD despite positive RT-PCR reactions from all lines. Lines cgt1-5 and cgt13 produced
the highest quantities of [1CD with cgt1-3 producing little to no BCD conﬁrming the
negative RT-PCR result in this line (Figure 2.4). The quantity of 0CD produced using the
enzymatic assay was 5300ng/mg dry tissue averaged across functional arabidopsis lines
at 120 hrs of incubation and 1120ng/mg tissue in the single functional tobacco line at 72
hrs. Both values are much higher than the reported in-tuber production of transgenic
potatoes, which was approximately 5 - 25ng/mg dry tissue (Oakes et al., 1991). However,
comparisons between the two types of transgenic plants may be unfair due to the facts of
in-tuber production versus an in-vitro incubation at the optimal temperature for the
enzyme.

The TLC assay for the various CDS showed that plant produced CGTase is
capable of synthesizing all of the usual CDS as evidenced by the multiple fused spots
found in plant enzymatic reactions. The Rf values were determined for each of the CDS
when rrm separately, 0.49, 0.42 and 0.40 for or, B and yCD. However, when run together
the CDS fused into a single long Spot. There did appear to be less quantity ofctCD

compared to the other CDS as Shown by the reduced spot intensity at the highest RF.

87

.__...

pg BCD l mg tissue

 

 

 

120

#315.

 

“9 [3CD I mg tissue

 

 

 

     

72 120
Hours of Incubation

Figure 2.4. Production of BCD by Arabidopsis seedlings as shown by colorimetric assay.
WT is the parental RLD strain. Cgt lines are independently transformed lines all under
control of the Actin promoter. Values are shown as micrograms of 0CD per mg of plant

tissue. Samples marked with a star are higher than wt control for that time point, 0. 5 0.1.

88

However the presence of spots in the transgenic Arabidopsis lines, cgtl-l and cgtl4 and
lack of spots in RLD provide additional evidence that cgt Arabidopsis are capable of
producing CDS. Tobacco line PEI lA-l also displayed a fused spot on TLC plates with
spots being absent in lines which did not produce detectable [5CD as well as the
hygromycin only transformed control line P1-1 (Figure 2.5).

Table 2.2a and 2.2b show the compiled results of antibiotic resistance testing,
genomic PCR, RT-PCR and phenolphthalein analysis methods for all Arabidopsis and
tobacco lines.

Table 2.2a. Gene integration, expression, function summary of Tobacco lines, y =

positive result, it = negative result, nt = not tested

 

 

 

 

 

 

 

 

Line Hyg/KarTR gPCR RT-PCR BCD positive
positive Positive (phenolphthalei

lI!)

PC8A7 y y y n
PC 1 B2 y y y n
PE3A1 y y y n
PEl 1A1 y y y y
PE] 1 A4 y y nt n
PEl 3A2 y nt y n
PE2B4 y y nt n

 

 

 

 

 

 

 

Table 2.2b. Gene integration, expression, function summary of Arabidopsis lines, y =

positive result, 11 = negative result, nt = not tested

 

 

 

 

 

 

 

 

 

 

 

 

Line Hyg/KanR gPCR RT-PCR BCD positive
positive Positive (phenolphthalei

11)
PIcgt1-3 y y n “
PIcgt 1 -5 y y y y
PIcgtl 3 y y y y
PIcgt14 y nt nt y
PIcgt 1 -6 y nt nt y
PIcgtl-l y nt nt y

 

 

89

.TE 75:8 388%wa 2: use mmﬂum
.32 Em 8:: 0838 2: Beam oTw 853 .312 352$» 2: use #4336;ng woe: GREEEEV .mQO m :m we 23me 3:3 as
maﬁa 25 av 05 59> mDU maﬁa $.28— v 8E 05 E 850% 8a 6.8935 .megxono—ozo 305054.33 mo maimed 01E. .md oSwE

1 a. X“ x. a. w. . a. .
YE «Eon. Sim; Sm 350 9:8 35 > a e

90

DISCUSSION

The production of a biologically based surfactant via extracellular secretion of a
bacterial enzyme has potential utility for a wide variety of applications. From a
contaminated soil remediation standpoint, in-situ production of a relatively non-toxic
compound with surfactant—like properties will, along with the expression of novel genes
for contaminant degradation, enhance the efﬁcacy of plant-based remediation.
Arabidopsis production of CGTase is a step towards ﬁeld implementation of plant-
produced solubilizing compounds.

Several challenges are still in place for cgt-expressing plants. In vitro production
of BCD from plant-generated enzyme was generally low, but appeared to increase slowly
over incubation time. Despite the presence of positive bands in RT-PCR reactions of
almost all tobacco lines, [3CD could be detected via the phenolphthalein method in only
one line, PE11A1. This line was able to produce detectable quantities of 0CD from
seedling exudates. The failure of so many tobacco lines to produce CGTase that was
active under standard enzymatic conditions, despite production of cgt-mRNA may
indicate a basic problem with CGTase expression in tobacco speciﬁcally. The fact that
two different promoters gave similar results in tobacco makes it unlikely that the
promoter itself is the major limitation. It could be that tobacco plants may produce
functional transcript in many cases but nonfunctional CGTase protein. Modiﬁcation of
foreign proteins in plant systems through the attachment of various sugars, glycosylation
has been documented and might account for the difference in the two species (Samyn-
Petit et al., 2003). It is also possible that in tobacco CGTase is poorly expressed in or

transported through root epidermal tissues. Arabidopsis has much ﬁner roots than tobacco

91

and the increased surface to volume ratio may allow for enhanced protein diffusion to
culture media. Different temporal or spatial expression between Arabidopsis and tobacco
may cause differences in detectable CGTase activity. 0r tobacco may contain a
nonspeciﬁc protease, released in hydroponic solution that degrades CGTase, which is
lacking in Arabidopsis. Degradation of foreign proteins expressed in plant systems,
especially when secreted into growth media, has also been a frequent occurrence (Doran,
2006)

The CGTase signal peptide sequence also seems to indicate the potential for low
efﬁciency secretion of CGTase due to a sub-optimal signal peptide for eukaryotic
systems, due lack of clarity in splice location. Replacement or modiﬁcation of the signal
peptide to a more plant-like version might assist in further increasing the quantity of
secreted CGTase. The cgt gene itself, being originally from a bacterial host, may need to
be optimized for better expression. While genes from Bacillus sp. are generally of
relatively high AT content, and usually less of a problem than those from high GC
bacteria, they can contain regions that are detrimental to expression such as sequences
that appear to be intron Splice sites, repeated ATTTA sequences, or a very extreme AT
skew (Perlak et al., 1991). PI-cgt is 51% AT and may be relatively suitable as is. But
optimization of the main cgt sequence may still enhance cgt expression to levels capable
of providing more CD production.

The low ratios of viable seeds in seedling antibiotic resistance screening, observed
in some Arabidopsis lines may be due to an overall weakness in the seed lots tested, since
hygromycin is a very strong selection agent and genetically resistant but physiologically

weak seedlings still might be overcome by the added stress of the selective agent.

92

Once optimized for efﬁcient production, hydroponically produced CGTase could
also be a cheaper, alternative source of industrial enzyme for the commercial production
of cyclodextrins. The current bacterial enzymatic systems may continue to out-compete
plant produced enzyme until higher expression and/or activity levels can be achieved.

Modiﬁcation of PIcgt or use of other CGTases with altered CD production
proﬁles, biased towards yCD or chD may be useful for increasing the range of
contaminants that may solubilized by cgt-expressing plants. With greater manipulation of
the plant biochemical machinery and utilization of more bacterial genes, direct secretion
of cyclodextrin into soil may be possible — thus eliminating bacterial competition with
freely available exogenous starch substrate. CD uptake mechanisms could also be cc-
Opted from bacteria and inserted into plants to allow for plant uptake of the intact CD-
contaminant complex. Used in concert with intracellular CD and contaminant
degradation genes, plants could become an improved system for organic contaminant
degradation or metal sequestration. Uptake of intact CDS also might allow for very
speciﬁc nutrient, pesticide or growth regulator delivery to plants via plant absorption of
complexed agents added to the soil. Cyclodextrin technologies will continue to grow and
evolve, and plants capable of producing cyclodextrins may have applications to very

diverse ﬁelds.

CONCLUSION

C gt-Arabidopsis is capable of expression and secretion of bacterial CGTase.
Some tobacco transforrnants are capable of secreting CGTase, but the BCD production is

lacking or low in most lines. Plant produced CGTase is capable of producing [3CD from

93

starch. Plants expressing CGTase may be useful in environmental restoration and
production of industrial or food grade CGTase enzyme. Future work can enhance the
production of CDS from plant sources and potentially combine the beneﬁts of CGTase
production with the expression of genes for contaminant degradation, creating a self
contained system for biologically based remediation. Further genetic manipulation should

yield improved expression and function of the PI-cgt gene in plants.

94

LITERATURE CITED

An, G., Ebert, P. R., Yi, B. Y., and Choi, C. H. (1986). Both TATA box and upstream
regions are required for the nopaline synthase promoter activity in transformed
tobacco cells. Molecular & General Genetics 203, 245-250.

An, Y. Q., McDowell, J. M., Huang, 8. R., McKinney, E. C., Chambliss, 8., and
Meagher, R. B. (1996). Strong, constitutive expression of the Arabidopsis
ACT2/ACT8 actin subclass in vegetative tissues. Plant Journal 10, 107-121.

Bardi, L., Mattei, A., Steffan, S., and Marzona, M. (2000). Hydrocarbon degradation by a
soil microbial population with beta-cyclodextrin as surfactant to enhance
bioavailability. Enzyme and Microbial Technology 27, 709-713.

Berberich, T., Takagi, T., Miyazaki, A., Otani, M., Shimada, T., and Kusano, T. (2005).
Production of mouse adiponectin, an anti-diabetic protein, in transgenic sweet
potato plants. Journal of Plant Physiology 162, 1169-1176.

Conrad, U. (2005). Polymers from plants to develop biodegradable plastics. Trends in
Plant Science 10, 511-512.

Doran, P. M. (2006). Foreign protein degradation and instability in plants and plant tissue
cultures. TRENDS in Biotechnology In press.

Girard, L. 8., Fabis, M. J ., Bastin, M., Courtois, D., Petiard, V., and Koprowski, H.
(2006). Expression of a human anti-rabies virus monoclonal antibody in tobacco
cell culture. Biochemical and Biophysical Research Communications 345, 602-

607.

Hall, J., Hazlewood, G. P., Surani, M. A., Hirst, B. H., and Gilbert, H. J. (1990).
Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial
endoglucanase by mammalian cells. Journal of Biological Chemistry 265, 19996-
19999.

Kamiya, M., and Nakamura, K. (1995). Cyclodextrin inclusion effects on
photodegradation rates of organophosphorus pesticides. Environment
International 21, 299-304.

95

Lee, Y. D., and Kim, H. 8. (1991). Enhancement of enzymatic production of
cyclodextrins by organic solvents. Enzyme and Microbial Technology 13, 499-

503.

Loukas, Y. L., Antoniadouvyza, E., Papadakivaliraki, A., and Machera, K. G. (1994).
Gamma-cyclodextrin inclusion complex of a new organophosphorus insecticide -
Determination of stability constant with HPLC. Journal of Agricultural and Food
Chemistry 42, 944-948.

Molnar, M., Leitgib, L., Gruiz, K., F enyvesi, E., Szaniszlo, N., Szejtli, J ., and F ava, F.
(2005). Enhanced biodegradation of transformer oil in soils with cyclodextrin -
from the laboratory to the ﬁeld. Biodegradation 16, 159-168.

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays
with tobacco tissue cultures. Physiologia plantarum 15, 473-497.

Naim, C. J ., Lewandowski, D. J ., and Burns, J. K. (1998). Genetics and expression of two
pectinesterase genes in Valencia orange. Physiologia Plantarum 102, 226-235.

Ni, M., Cui, D., Einstein, J ., Narasimhulu, 8., Vergara, C. E., and Gelvin, S. B. (1995).
Strength and tissue Speciﬁcity of chimeric promoters derived from the octopine
and mannopine synthase genes. Plant Journal 7, 661-676.

Oakes, J. V., Shewmaker, C. K., and Stalker, D. M. (1991). Production of cyclodextrins,
a novel carbohydrate, in the tubers of transgenic potato plants. Bio-Technology 9,
982-986.

Perlak, F. J., Fuchs, R. L., Dean, D. A., McPherson, S. L., and F ischhoff, D. A. (1991).
Modiﬁcation of the coding sequence enhances plant expression of insect control
protein genes. Proceedings of the National Academy of Sciences of the United
States of America 88, 3324-3328.

8amyn-Petit, B., Dubos, J. P. W., Chirat, F., Coddeville, B., Demaizieres, G., Farrer, S.,
Slomianny, M. C., Theisen, M., and Delannoy, P. (2003). Comparative analysis of
the site-speciﬁc N-glycosylation of human lactoferrin produced in maize and
tobacco plants. European Journal of Biochemistry 270, 3235-3242.

96

Settavongsin, R. (2005). Application and Transgenic Production of the Biosurfactant
Cyclodextrin for Enhanced Polyaromatic Hydrocarbon Phytoremediation. In Crop
and Soil Sciences (East Lansing: Michigan State University), pp. 175.

Starnes, R. L. (1990). Industrial potential of cyclodextrin glycosyl transferases.
CerealFoods World 35, 1094-1099.

Szejtli, J. (1988). Cyclodextrin Technology (Dordrecht ; Boston: Kluwar Academic
Publishers).

Szejtli, J. (2004). Past present and future of cyclodextrin research. IUPAC Pure and
Applied Chemistry 76, 1825-1845.

Szejtli, J ., and Szente, L. (2005). Elimination of bitter, disgusting tastes of drugs and
foods by cyclodextrins. European Journal of Pharmaceutics and Biopharrnaceutics
61, 115-125.

Uekama, K., Hirayama, F., and Irie, T. (1998). Cyclodextrin drug carrier systems.
Chemical Reviews 98, 2045-2076.

Viglianti, 0, Hanna, K., de Brauer, C., and Germain, P. (2006). Removal of polycyclic
aromatic hydrocarbons from aged-contaminated soil using cyclodextrins:
Experimental study. Environmental Pollution 140, 427-435.

Wang, J. M., Maier, R. M., and Brusseau, M. L. (2005). Inﬂuence of hydroxypropyl-
beta-cyclodextrin (HPCD) on the bioavailability and biodegradation of pyrene.
Chemosphere 60, 725-728.

Wang, X. J ., and Brusseau, M. L. (1995). Simultaneous complexation of organic
compounds and heavy metals by a modiﬁed cyclodextrin. Environmental Science
& Technology 29, 2632-2635.

Ye, G. N., Stone, D., Pang, 8. Z., Creely, W., Gonzalez, K., and Hinchee, M. (1999).
Arabidopsis ovule is the target for Agrobacterium in planta vacuum inﬁltration
transformation. Plant Journal 19, 249-257.

97

CHAPTER III

Effect of Cyclodextrin Glycosyl Transferase expressing plants on the remediation of
PAHS and PCBS

Sarah Kinder

Department of Crop and Soil Sciences
Michigan State University

INTRODUCTION

Persistent organic pollutants (POPS) are widespread environmental toxicants and
important threats to the global ecosystem. POPS are capable of global atmospheric
transport as well as long-terrrr persistence in the environment (Rodan et al., 1999). POPS
are strongly hydrophobic and resistant to most forms of degradation, remaining in the
environment for decades after deposition partly due to a strong tendency to sorb or
dissolve into the organic fraction of the soil matrix. This Similarity of chemical properties
to large portions of the soil itself contributes to the pollutants’ resistance to solar radiation
and chemical reactions.

Among the most more notoriously toxic and damaging POPS are the polyaromatic
hydrocarbons (PAHS) and polychlorinated biphenyls (PCBS). PAHS occur if organic
material iS burned with insufﬁcient oxygen and PCBS are synthetically created via
chlorination of biphenyl molecules. High molecular weight PAHS are considered both
carcinogenic and mutagenic, particularly after some photoconversion processes
(Alexander et al., 2002; Brown et al., 1999; Yan et al., 2004). PCBS are thought to be
toxic to the endocrine systems of many creatures due to similarity to hormones, causing
abnormal developmental effects (Colbom et al., 1993). Both PCBS and high molecular

weight PAHS are capable of accumulating in food chains through the process of

98

biomagniﬁcation in which each trophic level concentrates a contaminant at a higher level
than the one before (Gobas et al., 1999).

Persistent organic contaminants are most commonly remediated by standard
engineering-based techniques such as excavation and off-site disposal, which is labor
intensive, expensive, and ecologically disruptive to the treated site (Sellers, 1999). One
alternative to standard remediation practices is biologically-based remediation. Unlike
engineering-based techniques, biological degradation can transform organic contaminants
to less harmful compounds or even mineralize organic pollutants to non-toxic
components such as CO2, chloride and water. A wide variety of microbes have been
isolated that are capable of degrading many POPS, including both PCBS and PAHS. Low
molecular weight PAHS, such as those with three benzene rings or less, may serve as a
carbon source for microbes (Ahn et al., 1999; Daane et al., 2001). PAH degradation
becomes more difﬁcult with increasing molecular weight and typically proceeds co-
metabolically, requiring the presence of additional substrates for microbial growth (HO et
al., 2000). Highly chlorinated PCBS cannot generally be used as a carbon substrate for
microbial metabolism (Boyle et al., 1992; Quensen and Tiedje, 1997).

Plants have been proposed as a potential enhancer of biologically based
environmental restoration. Plants have been used to treat a wide variety of pollutants
including metals and organic compounds of various types, including PCBS and PAHS
(Arthur et al., 2005; Cunningham and 0w, 1996). Phytoextraction is a process in which
plants remove and concentrate soluble metals within their tissues. Alternatively, plants

may be used to stabilize inorganic contaminants in the soil matrix through the process of

99

phytostabilization (Berti and Cunningham, 2000). In phytodegradation applications,
organic pollutants are transformed to less toxic products after uptake into plant tissues.

Low contaminant bioavailability can severely reduce contaminant biodegradation
by plants and microbes (F eng et al., 2000). Bioavailability is a measure of the
accessibility of a contaminant to biological systems and is typically related to its water
solubility. Microbes, with their high surface area to volume ratios, can more easily utilize
compounds with low bioavailability by adsorption to solid phase pollutants and rapid
uptake of solubilized molecules (Johnsen and Karlson, 2004).

Many strategies have been proposed to help overcome bioavailability limitations
of organic contaminants including various soil amendments intended to liberate strongly
sorbed organic molecules. Surfactants, or surface-active agents, are compounds used for
the enhancement of hydrophobic compound bioavailability. Surfactants contain a charged
hydrophilic portion, which is soluble in water, and a hydrophobic “tail”, which is
chemically Similar to nonpolar organic contaminants. The head to tail organization allows
the organic contaminant to associate with the hydrophobic portion of the surfactant, while
the hydrophilic portion of the molecule pulls the complex into the aqueous phase, making
the compound soluble and therefore bioavailable. Most surfactant molecules must be
present in a solution at or above a certain concentration called the critical micelle
concentration (CMC) for maximum effectiveness in solubilization (Kile and Chiou,
1989). Micelles are Spherical arrangements of surfactant molecules with the charged
“head” groups facing outwards towards the aqueous solution and the non-polar “tail”
groups facing inwards, creating a hydrophobic cavity to contain hydrophobic pollutant

compounds.

100

Several studies have Shown that surfactants can enhance biological degradation of
organic pollutants (Bury and Miller, 1993; F ava and Di Gioia, 1998). However, the
effects of surfactants on biological degradation can be variable. While micelles can raise
the apparent concentration of organic contaminants in the aqueous phase, surfactant-
solubilized compounds may still be inaccessible to bacteria and bacterial enzymes
(Makkar and Rockne, 2003). Surfactants themselves may cause bacterial toxicity and
enhanced movement of toxic compounds through soil, potentially limiting the
effectiveness of surfactants (Berselli et al., 2004).

Biologically synthesized surfactants, called biosurfactants, may be a more
environmentally friendly alternative to synthetic surfactants (Cameotra and Makkar,
2004). Biosurfactants are generally classed into groups such as glycolipids,
phospholipids, fatty acids, surface active antibiotics and polymeric microbial surfactants
(Maier, 2003). Biosurfactants, such as rhamnolipids, have been Shown to enhance the
biological degradation of organic contaminants (Herman et al., 1997; Zhang et al., 1997).

Cyclodextrins (CDS) are another group of biologically synthesized compounds
that have been examined for solubilizing effects on contaminants in soil. Cyclodextrins
are not true surfactants but are unique cyclic oligosaccharide compounds synthesized by
bacteria from starch, capable of enhancing the solubility of hydrophobic compounds in a
similar fashion to surfactant micelles (Szejtli, 1988). Unlike surfactants, CDS do not exist
as monomers and have no critical micelle concentration, making them largely non-toxic
to both plants and bacteria (Apostolo et al., 2001; Bar and Ulitzur, 1994). CDS are
enzymatically formed from starch in a mixture of products primarily made up of three

CD forms composed of 6, 7, and 8 glucose units, classiﬁed as a—, B—, and yCD,

101

respectively. CDS are structured in such a way that the hydroxyl groups of the glucose
subunits face outward leaving the interior of the molecule relatively hydrophobic. While
surfactant micelles can grow to almost any Size, cyclodextrins are limited by the numbers
of glucose units forming the hydrophobic cavity consequently limiting the size of
inclusion molecule. Cyclodextrins have been used to enhance the dissolution and
biological degradation of various contaminant compounds, including PCBS and PAHS
(Fava et al., 1998; McCray and Brusseau, 1998; Wang et al., 1998). Previous work has
shown BCD can reduce soil sorption of the PAH phenanthrene (Settavongsin, 2005). CDS
have been tested for their ability to enhance desorption and biological degradation of
various organic contaminants. Chemically modiﬁed CDS are commonly used for applied
soil treatments due to the relatively low water solubility of naturally occurring CDS.
However, at biologically relevant concentrations, natural CDS have sufﬁcient water
solubility to perform as complexing agents (Gao et al., 1998).

Many bacteria are capable of producing cyclodextrins via extracellular secretion
of the CD biosynthetic enzyme cyclodextrin glycosyltransferase (CGTase) (Binder et al.,
1986; Larsen et al., 1998; Takano et al., 1986). Bacterially produced CGTase creates
cyclodextrins by degradation and circularization of starch molecules. For cyclodextrins to
be a useful in-situ treatment for contaminated soil, starch and CD-producing organisms
need to be present in sufﬁcient quantities or CD must be added exogenously. Direct
addition of CD can be expensive and labor intensive. [3CD has become relatively
inexpensive due to easier chemical production, though the price of or and yCD is still
relatively high. [3CD may have a more limited range of compound solubilization than a

mixture of all three CDS. In-situ production of cyclodextrins at a contaminated Site could

102

be an effective method of enhancing biologically-based Site cleanup if the process of
[3CD production could be controlled and enhanced. CD producing bacteria may be
present, but may be insufﬁcient to produce the needed quantities of cyclodextrin. Starch
addition may not promote bacterial CGTase production Since cgt, like the expression of
many secondary bacterial metabolic genes, is tightly regulated (N ishida et al., 1997). The
presence of alternative substrates in soil, possibly due to starch degradation by other
microbes, may inhibit cgt expression, CGTase production, and subsequent CD
production. Addition of CGTase producing microbes to soil is subject to the same
difﬁculties of other types of bioaugmentation, including strain persistence and preferred
metabolic activity (vanVeen et al., 1997).

We propose that plant secretion of CGTase into the rhizosphere for in-situ
production of CD would be a cost effective alternative to microbial bioaugmentation or
direct addition of CD. Bacterial CGTase is secreted into the soil environment and plants
engineered to express this gene may also secrete CGTase. Plant roots are a source of
starch in the rhizosphere and could be easily available on a contaminated site, remaining
in place for the duration of treatment. If root-produced starch were found to be
insufﬁcient, exogenous starch could be added to the soil at considerably lower cost
relative to addition of CD. In an effort to generate an alternative in-situ source of
CGTase, transgenic tobacco and Arabidopsis plants capable of secreting CGTase from
their roots were generated through biotechnological procedures. Genetically engineered
cgt-plants were tested for their effect on PCB and PAH biodegradation rates in

contaminated soil.

103

MATERIALS AND METHODS

PAH Soil Phytoremediation
PAH soil X Tobam

PAH-contaminated soil was obtained from the Rouge Manufacturing Complex
(Dearbom, MI) coke oven facility. The “PAH soil” contained, 13% organic matter, 81%
sand, 11% Silt and 8% clay as analyzed by A&L Great Lakes Analytical Labs (Fort
Wayne, IN). PAH soil was sieved through a stainless steel mesh (2.36 mm) to remove
large rocks and debris and thoroughly homogenized. The resulting PAH Soil contained
approximately 2000ppm total PAHS (tPAH) — the sum of concentrations of 15 of the EPA
priority PAHS found in detectable concentrations. The PAH 8011 was placed in 25 X 150
mm glass test tubes, bottom wrapped with aluminum foil to exclude light from the roots.
A 10” Pasteur pipet was placed in the test tube before soil addition to facilitate bottom
watering. Tubes were well watered and planted with a single 2-week-old tobacco
seedling of either wild type or one of two cgt-tobacco lines PE13A1 (Line A) or PC1B2
(Line B). Unplanted controls were also included. Plastic wrap was loosely wrapped
around loaded test tube racks placed in a plant growth chamber maintained at 25° +/-3°C
with 16-hour day length (150—230uE"‘S'l *m'z). Treatments were Split in half with one
half being watered weekly with 2ml of autoclaved 1% starch and the other half being
watered with distilled, deionized water. Test tube treatments were harvested
approximately 50 days after planting with total plant shoot tissue and approximately 40cc

of soil from each tube collected.

104

Soil PAH analysis

For soil PAH determination, 6g of soil were measured into a 40ml vial to which
6ml of aqueous saturated KCl were added, followed by 20ml dichloromethane. Vials
were then capped, using Teﬂon liners, vortexed for 20 seconds, sonicated in an ultrasonic
water bath for 10 minutes and then shaken on a rotary Shaker at 150 RPM for 16 hours.
After overnight shaking the vials were removed and set upright for 10- 20 minutes. A
portion of the lower layer of solvent but above the settled soil was removed with a
Pasteur pipet and ﬁltered through a glass wool stuffed Pasteur pipet. Filtrate was placed
in GC vials and analyzed via an Agilent GC 6890 equipped with an Agilent 3396 B/C
integrator and Agilent 7683 8L8 auto sampler and injector, ICB PAH column and ﬂame
ionization detector. The GC conditions were: ICB-PAH capillary column 15m in length
250nm id 0.15 pm ﬁlm thickness (J&K scientiﬁc, Milton, Ontario), with helium carrier
gas at 41Kpa constant pressure, 270°C inlet temperature, ﬂame ionization detector
maintained at 330°C. Column initial temperature was 80°C, followed by elevation to
220°C at 40°C per minute, and a second ramp including elevation to 285°C at 8°C per
minute. The injected volume was 4rrl with a split ratio of 7:1. The PAHS measured and
summed to determine the total soil PAH concentration (tPAH), listed in order of
increasing molecular weight and retention time were, napthalene, acenapthylene,
ﬂuoranthene, phenanthrene, anthracene, ﬂuorene, pyrene, benzo[a]anthracene, chrysene,
benzo[b]ﬂuoranthene, benzo[k]ﬂuoranthene, benzo[a]pyrene, indeno [1,2,3 -CD]pyrene,
and benzo[ghi]perylene. tPAH is the sum of the 14 compounds typically found in the

Rouge soil in highly unequal proportions (Table 3.1).

105

Table 3.1. Percentage of relative concentration of each of the individual PAH compounds

in reference to the total PAH content of the soil.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Compound Percentage of tPAH
napthalene 1 .8
acenapthylene 3.6
ﬂuoranthene 0.9
phenanthrene 5.7
anthracene 2.8
ﬂuorene 15.3
pyrene 1 5. l
benzo[a]anthracene 9.7
chrysene 6.6
benzo[b]ﬂuoranthene 1 1.5
benzo[k]ﬂuoranthene 4.8
benzo[a]pyrene 1 1.9
indeno[1 ,2,3-CD]pyrene 2.5
benzo[ghi]perylene 7.7

 

 

 

 

106

Plant Biomass and Soil Moisture determination

Fresh weight of plant aboveground tissues was determined immediately after
sample harvest and dry biomass was determined after drying to static weight in a 60° C
oven. Soil moisture determination was accomplished by weighing 5 g treatment soil
subsamples into aluminum weigh pans, oven drying at 105°C for 24 hrs, and reweighed

for dry weight.

PCB Soil Phytoremediation
PCB soil X Arabidopsis

Industrially contaminated PCB soil was obtained from the Kalamazoo River
Basin Superfund site (EPA ID# MID006007306). The collected soil was stored in steel
cans at room temperature for approximately 6 months prior to use. In preparation for the
experiment, soil was sieved ﬁrst through 5mm steel mesh and then through 2.36 mm
stainless steel mesh. Soil was thoroughly homogenized and poured into aluminum foil
wrapped 25 X 150 mm glass test tubes. Soil was watered and tubes planted with wildtype
or cgt-Arabidopsis seedlings or left as unplanted controls. Plastic wrap was loosely
wrapped around loaded test tube racks placed in a plant growth chamber maintained at
25° +/-3°C with 16-hour day length (150-230I1E1‘s'l *m'z). Test tubes were watered with
2ml autoclaved 1% starch in water 10 days after planting (DAP), 1/4 X MS salts at 19
DAP, 3m] 1% starch at 33 DAP. Two treatment times were used prior to sampling; 72

DAP (N = 4 tubes each treatment) and 92 DAP (N = 7 tubes).

107

Spiked PCB Soil X Tobacco

Soil was obtained from Wayne County, Michigan and classiﬁed as a Hoytville
series, a silty clay loam. This soil was spiked with PCB by addition of Aroclor 1248
diluted in acetone resulting in a calculated ﬁnal concentration of 100ppm. The Spiked
PCB soil was stored dry at room temperature for a period of 2 months and then added to
25 X 150 mm glass test tubes. Transgenic cgt- or wild type tobacco seedlings were
planted in each tube with left Implanted. Plastic wrap was loosely wrapped around loaded
test tube racks placed in a plant growth chamber maintained at 25° +/-3°C with 16-hour
day length (150-23OIJ.E*S'l *m'z). At 4 days after planting (DAP) all tubes were watered
with 2ml 1% starch. All treatments were destructively sampled after 74 DAP.

In order to examine the effect of bioaugmentation with PCB soil extract inoculum
on the spiked PCB treatments, a soil extract was made by washing an aliquot of
Kalamazoo River Superfund Site soil in 1% tetrasodium phosphate buffer (1:1000 ::
soilzbuffer). To determine total colony forming units (CF Us), soil extract samples were
plated on YEPG agar (Per liter: 1 g glucose, 2g polypeptone, 0.2g yeast extract, 0.2g
NH4N03, and 15g Bactoagar) and visible colonies enumerated after 12d incubation at

25°C.

PCB an_alysis

PCB extraction was conducted using an accelerated solvent extractor, ASE 200
(Dionex, Sunnyvale, CA). Ten grams of dry soil were mixed with an equal amount of
sodium sulfate (Sigma, St. Louis, MO) and placed into a 33ml extraction cell with a glass

ﬁber ﬁlter (P/N 047017, Dionex, Sunnyvale, CA) on the bottom Side of the cell. Void

108

space in the cell was ﬁlled with Ottawa sand (823-3, Fisher Scientiﬁc, Pittsburg, PA).
Cells were capped and placed on the machine with pre-weighed 60ml glass collection
vials for retention of solvent extract. The ASE program was: 1 minute preheat, 5 min
heat, 5 min static time, 60% ﬂush and a 60 second purge. The extraction solvent was 50%
acetone and 50% hexane. After extraction, the collection vials were weighed to determine
extract mass and recapped with undamaged septa. ASE samples were transferred to GC
vials and analyzed on an Agilent GC 6890 (Agilent, Santa Clara, CA) equipped with a
Supelco Equity 5 column (Supelco, Bellefonte, PA), 30m in length 320nm id. and

0.25 pm ﬁhn thickness. The GC program consisted of a 4rd splitless injection with the
inlet temperature at 250°C, helium carrier gas with pressure set at 59KPa. The initial run
temperature was 120°C increased by 9°C per minute to 300°C. The detector was an
Agilent Micro ECD set at 310°C with 30ml/min nitrogen makeup. Ten representative
peaks were choSen based on fraction of total area and uniqueness in the Aroclor 1248
standard as compared to the Aroclor 1254 standard (Supelco, Bellefonte, PA). The sum

of these peaks was taken to represent the total PCB content of the soil.

RESULTS and DISCUSSION

PAH Soil Phytoremediation

The effects of cgt-tobacco plants with and without supplemental starch addition on soil
PAH degradation were examined. Starch appeared to enhance tPAH reduction in two of
the nine treatments: unplanted (26.3% soil [tPAH] reduction) and cgt-tobacco line B

(24.9% soil [tPAH] reduction) compared with the untreated control soil (Figure 3.1).

109

 

 

 

 

 

 

AtPAH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

‘1 .o.‘ - This?! lit. .
. II .. llll4r| Ill

<<Z CZ wz mm cm

43935:»

Ema—8 w. L. my»: 8:82 om mo: H.408 For: we: Omﬂmrﬁoaaaﬁmaos @8353? 44835:» ooaom” > n OmAIHocmooomE PPM. w H
Omdéocmooomnﬂww. << n 13:360. C H Calming. Z u 20 maﬁa: 2552.. m H manor magma. HNHCERSSQ 855:. Day; £62m
vanomsgma 253% 5 8: Eva: 8595 8 sandman 838—. $3385 madame. £85838 3 A ome m8 ganged SE. 90 8.80
533.

 

 

 

 

 

 

 

110

However, these two treatments were also statistically similar to the unarnended and
Implanted treatments. Unarnended cgt-tobacco line B also showed a signiﬁcant reduction
in tPAH content (28.1%). Other than these three treatments, no signiﬁcant reduction in
tPAH content was observed in any other plant genotype-arnendment treatment
combinations compared to untreated control.

While most individual compounds exhibited similar patterns of reduction to that
shown by tPAH, a few compounds displayed interesting differences. Benzo[ghi]perylene
(BGHP), the highest molecular weight PAH measured by our methods, also showed a
similar pattern with all treatments being signiﬁcantly lower than the untreated (Figure

3.2).

 

 

Abghp

     

AN UN
Treatment

Figure 3.2. Benzo[ghi]perylene (BGHP) content of soil PAH Soil Cgt-Phytoremediation
treatments. Treatment codes: A = Cgt-Tobacco-PE13A2 B = Cgt—Tobacco-PC1B2 W =

Wildtype U = Unplanted N = No starch addition 8 = Starch added. Abghp shows

111

percentage change in soil [BGHP] relative to untreated control. Statistically similar
treatments (or 5 0.05) are denoted with the same letter.

Starch-watered cgt-tobacco line B showed a Signiﬁcantly lower content of BGHP than
Implanted and wildtype treatments both starch treated and water only, with a 55, 53, 43
and 51 percent reduction, respectively. Napthalene, the lowest molecular weight PAH,

had signiﬁcantly reduced levels in all treatments when compared to the untreated control

(Figure 3.3).

 

Treatment

Figure 3.3. Napthalene content of soil PAH Soil Cgt-Phytoremediation treatments.
Treatment codes: A = Cgt—Tobacco-PE13A2 B = Cgt-Tobacco-PC1B2 W = Wildtype U
= Unplanted N = No starch addition 8 = Starch added. ANapth shows percentage change
in soil [naphthalene] relative to untreated control. Statistically similar treatments (or _<_

0.05) are denoted with the same letter.

112

Napthelene was most likely lost by a combination of volatilization and biodegradation,
since its low molecular weight makes it more susceptible to biological degradation and
loss by volatilization.

The high levels of PAHS and poor agronomic qualities of the Rouge soil resulted
in highly stressed plants in all treatments, with yellow leaves and accelerated leaf
senescence, although this appeared to be less of a problem in treatments with starch
addition, potentially indicating some protective effects of starch on plant health.
Interestingly the dry biomass of line B cgt and wildtype treatments seemed to positively
correlate, although not signiﬁcantly, with the addition of starch. However, dry biomass
between treatments showed no Signiﬁcant differences between treatments of the same

genotype, or the same treatment between genotypes (Figure 3.4).

 

0.35

 

0.30 , , .
s 0.25 -
2
B020
'3
g 0.15 .
2
0.
5,010

E
0.05

 

 

 

0.00 .

AN AS BS BN WN WS

Figure 3.4. Plant dry biomass from soil PAH Soil Phytoremediation treatments.
Treatment codes: A = Cgt-Tobacco-PE13A2, B = Cgt-Tobacco-PC1B2, W = Wildtype,
U = Unplanted, N = No starch addition, 8 = Starch added. No signiﬁcant differences

were observed (or 5 0.05).

113

Plant survival rate appeared to be enhanced in the starch amended wild type
treatment. Starch amended wild type had 17 surviving plants versus only 9 in the
unamended treatment. Both transgenic tobacco lines showed similar survival rates in
starch treated and unamended treatments.

One problem associated with this study was the choice of tobacco lines. Neither
PC1B2 (Line B) nor PE13A1 (Line A) have been shown to produce detectable quantities
of CD under enzymatic digest conditions. It is possible that both lines were producing
small amounts of CGTase at levels, which are undetectable by direct enzymatic analysis.
Or in-vitro enzymatic analysis may also not reﬂect the ability of plant produced CGTase
to ﬁmction in soil, or the potential for plant production of CGTase when grown in soil.
cgt line B was shown to produce light clear zones on starch containing media. However,

. it is likely that the quantity of CDs produced by these tobacco lines in soil is quite low. A
signiﬁcant decrease in contaminant levels by a starch watered cgt line B was observed for
at least one high molecular weight PAH (BGHP), compared to both unplanted and wild
type treatments. It is possible that low levels of plant-produced CD promoted the
degradation of BGHP, perhaps by partly solubilizing the high molecular weight
compound. The extremely low water solubility of BGHP might be such that even a slight
increase in bioavailability would result in a large increase in biodegradation and a
subsequently noticeable reduction in concentration. The reductions seen in BGHP
concentration by starch treated cgt-tobacco line B may hint that positive effects on other
PAHS may be possible with increased CD concentration or incubation time. The presence
of cyclodextrin alongside the effects of starch may still provide beneﬁts for degradation

even if not quantitatively observable for most of the PAH compounds.

114

Starch addition seemed to be a contributing factor in promoting tPAH degradation
in Rouge soil, especially notable in the unplanted treatment. The highest reduction in
tPAH and BGHP speciﬁcally were seen in starch-amended treatments. Enhancement of
biodegradation promoted by the addition of a substrate such as starch has been seen in
many other treatment studies when carbon sources are added to impoverished soils (Haby
and Crowley, 1996; Leigh et al., 2002; Rao et al., 1995).

The effect from starch alone on tPAH reduction was similar to any effects of
cyclodextrin produced from starch, at least by cgt-tobacco line B, in total PAH
degradation. Even so, the presence of cyclodextrin alongside the effects of starch may
still provide beneﬁts even if not quantitatively observable for the majority of PAH
compounds. Starch combined with plants was not always effective as both cgt-tobacco
line A and wild type plants treated with starch showed no signiﬁcant reduction of tPAH
when compared with untreated soils. It is possible that these plant lines were in some way
presenting a hindrance to PAH degrading microbes, perhaps by maintaining unfavorable
soil conditions, when compared to cgt-tobacco line B. These lines may have been
producing exudates that were unfavorable to microbes while cgt-tobacco line B was not,
and the CD production of this line was able to mitigate the apparent negative effects of
tobacco on PAH degradation.

Future analysis of the effects of starch and CD producing plants on the microbial
community, speciﬁcally starch and PAH degrading microbes might provide additional
information on the effects of CGTase expressing plants on PAH impacted soil and

clariﬁcation of the results of the experiments. The use of single spiked compounds might

115

be another way of teasing apart the interactions of the plants, cyclodextrin, and PAH

molecules.

PCB Soil Phﬂoremediation

Cgt-Arabidopsis was tested with supplemental starch addition for enhancement of
PCB biodegradation. The cgt-Arabidopsis line chosen for the PCB phytoremediation
experiment was demonstrated to produce CGTase with subsequent conversion of starch
to CD in bioindicator assays (as described in Chapter 2). These observations make it
likely cgt-Arabidopsis line 14 was producing [3CD in-situ for some if not all of the
treatment period.

Aﬁer 72 days of growth, all of the treatments had signiﬁcantly lower PCB content
than the untreated control. However, no individual treatment was signiﬁcantly different
from any other treatment, with soil [PCB] reductions of 34%, 32%, and 32% for cgt-

Arabidopsis, wild type Arabidopsis, and unplanted treatments, respectively (Figure 3.5).

mg plant dry matter

 

untrt
Figure 3.5. PCB content of soil from PCB Soil Cgt—Phytoremediation treatments.

Arabidopsis cgt14, wildtype, unplanted and untreated are listed from left to right. Values
are after 72 days of plant growth. Statistically similar treatments (0. < 0.05) are denoted

with the same letter.

116

Plants would have to make a very large difference over unplanted treatments in
what is already a miniscule amount of contaminant with many individual PCB peaks
being less than one ppm in soil concentration. Additionally, the chronic contamination of
the soil may be very resistant to large changes in contaminant content, due to long-term
sorption of some proportion of the PCBS. Low levels of CDs produced by cgt-
Arabidopsis may have partly counteracted the plant induced effect resulting in the
slightly lower PCB content in cgt-Arabidopsis treatments. The effects of cgt-plants on
PCB degradation appears to be minimal in this experiment, in either time point as
unplanted samples were lower and statistically indistinguishable from planted treatments.
The simple mixing of soil and the addition of water may explain the overall loss of PCBs.
These actions may have stimulated preexisting microbes, by increased exposure to
oxygen due to breakup of large aggregates through sieving. The low levels of PCBS in the
Kalamazoo soil may have contributed to the inability to detect signiﬁcant differences
between the treatments.

Overall, resulting plant biomass of the cgt-Arabidopsis line was signiﬁcantly
lower (25% less dry mass) than that of the wild type control plants (Figure 3.6). This may
be explained by the presence of the transgene causing pleiotropic effects or possibly
lower seed vigor from the seed stocks used due to previous antibiotic selection of the

parental plants.

Spiked PCB Soil Phytoremediation

The spiked PCB experiment was distinct from the other trials in that it used

freshly spiked soil rather than aged, industrially contaminated soil. The relatively low

117

levels of PCBs observed in the untreated sample (25 ppm) compared to the calculated
concentration of PCBs (100 ppm) was likely due to volatilization during the aging period.

The spiked PCB soil treatments were highly variable in contaminant removal,
though bioaugmentation with microbial extract from industrially contaminated soil
appeared to improve effectiveness (Figure 3.7).

Extract-inoculated soils of the unplanted and cgt-tobacco line B planted
treatments displayed greater [PCB] reduction that uninoculated soils. These results
suggest that the soil extract supplied PCB biodegrading microbes to the inoculated soils,
which may have been lacking in the “clean” ﬁeld soil initially spiked in this study. Soil
PCB reduction was signiﬁcant in one treatment with a 45% reduction by inoculated cgt-
tobacco. However, the inoculated unplanted treatment was statistically similar to
inoculated cgt treatments suggesting that soil inoculation may have a larger impact on
PCB reduction than the presence of plants or the production of CGTase. Given the
probability of low levels of CGTase present in the soil and the potential for a nearly total
lack of PCB degrading organisms in spiked soil, this result may be somewhat expected.
Soil spiking very often results in a signiﬁcant mortality in soil microbes such that few
native microbes may have remained to carry out degradation (Brinch et al., 2002). The
total number of culturable microbes added from the soil inoculum was 7746 per tube, 193
per gram based on an average of 40 grams (DW) soil per tube. Since the spiked soil was
maintained in a dry state for several years and was never contaminated, it is also likely
the overall microbial population as well as potential PCB degraders was very low. A
sudden appearance of PCB degrading microbes in contact with relatively labile

contaminants, due to “fresh” spiking, might have resulted in relatively rapid degradation.

118

 

 

Figure 3.6. Biomass of Arabidopsis shoot tissues in PCB Soil Cgt-Phytoremediation
treatments, 92 days after planting. Dry (Dwt) and fresh (Fwt) biomass are shown. Stars

denote treatments signiﬁcantly lower biomass (0t < 0.05).

 

 

 

15 W, 7 —— —
l b

10 an

51 -
m

a

a 0. ~
<1 Bl

 

 

 

 

Treatment
Figure 3.7. Percentage change in soil PCB content from spiked PCB Soil Cgt-

Phytoremediation treatments as compared to untreated control. U = unplanted, W =
wildtype, B = PC1B2 cgt-tobacco, I = inoculated, O = not inoculated. Statistically similar

treatments (on < 0.05) are denoted with the same letter.

119

Unequal dispersal of these microbes in soil inoculum might have resulted in the observed
scattered, but high degradation rates in some samples.

In terms of plant dry biomass, none of the treatments of any time point showed
any signiﬁcant difference between the others, however the plant biomass did increase
signiﬁcantly between the two time points, indicating that the presence of PCBS in the soil
did not excessively hinder plant growth (Figure 3.8).

The lack of difference between the treatments shows that neither added starch nor

the presence of the transgene in tobacco affected plant growth.

CONCLUSION

Several studies on two different contaminant classes, PCBS and PAHS, were done
to examine the effects of cgt-expressing plants, starch addition, and bioaugmentation for
soil remediation. All but one of the tested soils was chronically contaminated and such
soils are less likely to see large reductions in contaminant levels due to strong, long term
sorption of contaminants to soil components and high variability of contaminant
concentration within those soils (Burgos etal., 1996; Carmichael et al., 1997).
Additionally, the cgt-expressing plants used in this study are ﬁrst generation transgenics
and may not produce sufﬁcient CGTase to make substantial quantities of CD in-situ.
Despite these limitations, several of the studies yielded some interesting results in soil
contaminant loss and plant biomass production.

In this study cgt-Arabidopsis and cgt-tobacco were tested for their effects on PAH
and PCB remediation. The results of the biodegradation experiments showed positive

effects by one tobacco line on at least one PAH compound and under speciﬁc treatment

120

 

 

 

 

 

 

 

Figure 3.8. Tobacco fresh and dry biomass in spiked PCB Soil Cgt-Phytoremediation

ulated. Stars denote

treatments. W = Wildtype, B = PC1B2, I = Inoculated, O = Not incc

< 0.05

treatments signiﬁcantly lower at or

121

conditions for PCBs. Starch addition was beneﬁcial for soil PAH reduction and
contaminated soil extract inoculation appeared to enhance PCB biodegradation. Cgt-
tobacco enhanced dissipation of the highest molecular weight PAH compound,
benzo[g,h,i]perylene, in the phytoremediation study, though most of the other PAH
compounds did not show clear reduction. The lack of strength in the results may be due
to a large number of factors including low expression/production of CD in soil. The high
variability of contaminants in many of the soils also may have hampered efforts to show
signiﬁcant reductions on contaminant concentration that might be a direct result of
transgenic plants. Future experiments may be able to show the positive effects of cgt-
plants on a wider range of compounds and conﬁrm the effects of cgt-plants on BGHP.
Given the difﬁculty in showing quantitative loss of organic pollutants in soil, a direct
assessment of mutagenicity or toxicity reduction in treated contaminated soil may be a
better measure of phytoremediation success. In addition to being less constrained by
contaminant variability, direct toxicity/mutagenicity experiments would more completely
show whether remediation, in terms of true reduction of the toxic qualities of the soil, is
occurring. Information about the microbial community might also be useful in ﬁnding out
what sort of effect CDs and plant produced CGTase might have on numbers of microbial
degraders. Hopefully, with better lines and expression, coupled with additional
experiments the effects of cgt-plants on the degradation of contaminants in soil will

become clearer.

122

LITERATURE CITED

Ahn, Y., Sanseverino, J ., and Sayler, G. (1999). Analyses of polycyclic aromatic
hydrocarbon-degrading bacteria isolated from contaminated soils. Biodegradation
10, 149-157.

Alexander, R. R., Tang, J. X., and Alexander, M. (2002). Genotoxicity is unrelated to
total concentration of priority carcinogenic polycyclic aromatic hydrocarbons in
soils undergoing biological treatment. Journal of Environmental Quality 31, 150-
154.

Apostolo, N. M., Brutti, C., Ferrarotti, S. A., Llorente, B. E., and Krymkiewicz, N.
(2001). Stimulation of root development with cyclodextrins on jojoba shoots in
vitro. In Vitro Cellular & Developmental Biology-Plant 3 7, 414-418.

Arthur, E. L., Rice, P. J ., Anderson, T. A., Baladi, S. M., Henderson, K. L. D., and Coats,
J. R. (2005). Phytoremediation - An overview. Critical Reviews in Plant Sciences
24, 109-122.

Bar, R., and Ulitzur, S. (1994). Bacterial toxicity of cyclodextrins - Luminous
Escherichia coli as a model. Applied Microbiology and Biotechnology 41, 574-
577.

Berselli, S., Milone, G., Canepa, P., Di Gioia, D., and Fava, F. (2004). Effects of
cyclodextrins, humic substances, and rhamnolipids on the washing of a
historically contaminated soil and on the aerobic bioremediation of the resulting
efﬂuents. Biotechnology and Bioengineering 88, 111-120.

Berti, W. R., and Cunningham, S. D. (2000). Phytostabilization of metals. In
Phytoremediation of Toxic Metals: Using Plants to Clean Up the Environment, 1.
Raskin and B. D. Ensley, eds.: John Wiley & Sons, Inc.), pp. 71-88.

Binder, F., Huber, O., and Bock, A. (1986). Cyclodextrin glycosyltransferase from
Klebsiella pneumoniae M5A1 - cloning, nucleotide sequence and expression.
Gene 47, 269-277.

Boyle, A. W., Silvin, C. J., Hassett, J. P., Nakas, J. P., and Tanenbaum, S. W. (1992).
Bacterial PCB biodegradation. Biodegradation 3, 285-298.

123

Brinch, U. C., Ekelund, F., and Jacobsen, C. S. (2002). Method for spiking soil samples
with organic compounds. Applied and Environmental Microbiology 68, 1808-
1816.

Brown, D. G., Knightes, C. D., and Peters, C. A. (1999). Risk assessment for polycyclic
aromatic hydrocarbon NAPLS using component fractions. Environmental Science
& Technology 33, 4357-4363.

Burgos, W. D., Novak, J. T., and Berry, D. F. (1996). Reversible sorption and irreversible
binding of naphthalene and alpha-naphthol to soil: Elucidation of processes.
Environmental Science & Technology 30, 1205-1211.

Bury, S. J ., and Miller, C. A. (1993). Effect of micellar solubilization on biodegradation
rates of hydrocarbons. Environmental Science & Technology 27, 104-110.

Cameotra, S. S., and Makkar, R. S. (2004). Recent applications of biosurfactants as
biological and immunological molecules. Current Opinion in Microbiology 7,
262-266.

Carmichael, L. M., Christman, R. F ., and Pfaender, F. K. (1997). Desorption and
mineralization kinetics of phenanthrene and chrysene in contaminated soils.
Environmental Science & Technology 31 , 126-132.

Colbom, T., Saal, F. S. V., and Soto, A. M. (1993). Developmental effects of endocrine-
disrupting chemicals in wildlife and humans. Environmental Health Perspectives
[01, 378-384.

Cunningham, S. D., and Ow, D. W. (1996). Promises and prospects of phytoremediation.
Plant Physiology 110, 715-719.

Daane, L. L., Harjono, I., Zylstra, G. J ., and Haggblom, M. M. (2001). Isolation and
characterization of polycyclic aromatic hydrocarbon-degrading bacteria
associated with the rhizosphere of salt marsh plants. Applied and Environmental
Microbiology 67, 2683-2691.

124

Fava, F., and Di Gioia, D. (1998). Effects of Triton X-100 and Quillaya Saponin on the
ex situ bioremediation of a chronically polychlorobiphenyl-contaminated soil.

Applied Microbiology and Biotechnology 50, 623-630.

F ava, F., Di Gioia, D., and Marchetti, L. (1998). Cyclodextrin effects on the ex-situ
bioremediation of a chronically polychlorobiphenyl-contaminated soil.

Biotechnology and Bioengineering 58, 345-355.

Feng, Y. C., Park, J. H., Voice, T. C., and Boyd, S. A. (2000). Bioavailability of soil-
sorbed biphenyl to bacteria. Environmental Science & Technology 34, 1977-

1984.

Gao, S. X., Wang, L. S., Huang, Q. G., and Han, S. K. (1998). Solubilization of
polycyclic aromatic hydrocarbons by beta-cyclodextrin and carboxymethyl—beta—

cyclodextrin. Chemosphere 3 7, 1299-1305.

Gobas, F. A. P. C., Wilcockson, J. B., Russell, R. W., and Haffner, G. D. (1999).
Mechanism of biomagniﬁcation in ﬁsh under laboratory and ﬁeld conditions.

Environmental Science & Technology 33, 133-141.

Haby, P. A., and Crowley, D. E. (1996). Biodegradation of 3-chlorobenzoate as affected
by rhizodeposition and selected carbon substrates. Journal of Environmental

Quality 25, 304-310.
Herman, D. C., Zhang, Y. M., and Miller, R. M. (1997). Rhamnolipid (biosurfactant)

effects on cell aggregation and biodegradation of residual hexadecane under
saturated ﬂow conditions. Applied and Environmental Microbiology 63, 3622-

3627.

Ho, Y., Jackson, M., Yang, Y., Mueller, J. G., and Pritchard, P. H. (2000).
Characterization of fluoranthene- and pyrene-degrading bacteria isolated from
PAH-contaminated soils and sediments. Journal of Industrial Microbiology &

Biotechnology 24, lOO-112.

Johnsen, A. R., and Karlson, U. (2004). Evaluation of bacterial strategies to promote the
bioavailability of polycyclic aromatic hydrocarbons. Applied Microbiology and

Biotechnology 63, 452-459.

125

Kile, D. E., and Chiou, C. T. (1989). Water solubility enhancements of DDT and
Trichlorobenzene by some surfactants below and above the critical micelle
concentration. Environmental Science & Technology 23, 832-838.

Larsen, K. L., Christensen, H. J. S., Mathiesen, L. H., Pedersen, L. H., and Zimmerrnann,
W. (1998). Production of cyclomaltononaose (ES-cyclodextrin) by cyclodextrin
glycosyltransferases from Bacillus spp. and bacterial isolates. Applied
Microbiology and Biotechnology 50, 314-317.

Leigh, M. B., Fletcher, J. S., Fu, X. 0., and Schmitz, F. J. (2002). Root turnover: An
important source of microbial substrates in rhizosphere remediation of recalcitrant
contaminants. Environmental Science & Technology 3 6, 1579-1583.

Maier, R. M. (2003). Biosurfactants: Evolution and diversity in bacteria. Advances in
Applied Microbiology 52, 101-121.

Makkar, R. S., and Rockne, K. J. (2003). Comparison of synthetic surfactants and
biosurfactants in enhancing biodegradation of polycyclic aromatic hydrocarbons.
Environmental Toxicology and Chemistry 22, 2280-2292.

McCray, J. E., and Brusseau, M. L. (1998). Cyclodextrin-enhanced in situ ﬂushing of
multiple-component immiscible organic liquid contamination at the ﬁeld scale:
Mass removal effectiveness. Environmental Science & Technology 32, 1285-

1293.

Nishida, T., Nakamura, A., Masaki, H., and Uozumi, T. (1997). Regulation of
cyclodextrin glucanotransferase synthesis in Bacillus ohbensis. Ferns
Microbiology Letters 149, 221-226.

Quensen, J ., and Tiedje, J. (1997). Methods for evaluation of PCB dechlorination in
sediments. In Bioremediation Protocols, D. Sheehan, ed. (Totowa, NJ: Humana
Press Inc.), pp. 241-253.

Rao, N., Grethlein, H. E., and Reddy, C. A. (1995). Mineralization of atrazine during
composting with untreated and pretreated lignocellulosic substrates. Compost
Science & Utilization 3, 38-46.

126

Rodan, B. D., Pennington, D. W., Eckley, N., and Boethling, R. S. (1999). Screening for
persistent organic pollutants: Techniques to provide a scientiﬁc basis for POPs
criteria in international negotiations. Environmental Science & Technology 33,
3482-3488.

Sellers, K. (1999). Fundamentals of Hazardous Waste Site Remediation (Boca Raton, FL:
CRC Press).

Settavongsin, R. (2005). Application and Transgenic Production of the Biosurfactant
Cyclodextrin for Enhanced Polyaromatic Hydrocarbon Phytoremediation. In Crop
and Soil Sciences (East Lansing: Michigan State University), pp. 175.

Szejtli, J. (1988). Cyclodextrin Technology (Dordrecht ; Boston: Kluwar Academic
Publishers).

Takano, T., Fukuda, M., Monma, M., Kobayashi, S., Kainuma, K., and Yamane, K.
(1986). Molecular cloning, DNA nucleotide sequencing, and expression in
Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase
gene. Journal of Bacteriology [66, 1118-1122.

vanVeen, J. A., vanOverbeek, L. S., and vanElsas, J. D. (1997). Fate and activity of
microorganisms introduced into soil. Microbiology and Molecular Biology
Reviews 61, 121-&.

Wang, J. M., Marlowe, E. M., Miller-Maier, R. M., and Brusseau, M. L. (1998).
Cyclodextrin-enhanced biodegradation of phenanthrene. Environmental Science
& Technology 32, 1907-1912.

Yan, J., Wang, L., Fu, P. P., and Yu, H. T. (2004). Photomutagenicity of 16 polycyclic
aromatic hydrocarbons from the US EPA priority pollutant list Mutation
Research-Genetic Toxicology and Environmental Mutagenesis 55 7, 99-108.

Zhang, Y. M., Maier, W. J ., and Miller, R. M. (1997). Effect of rhamnolipids on the
dissolution, bioavailability and biodegradation of phenanthrene. Environmental
Science & Technology 31 , 2211-2217.

127

CHAPTER IV
Is Phytoremediation Safe? A Comparison of Risks and Management Strategies of Plant-

Based Environmental Remediation Technologies and Their Engineering-Based
Counterparts

Sarah Kinder
Department of Crop and Soil Sciences
Michigan State University

INTRODUCTION

Phytoremediation is the use of plants to remove, stabilize and/or detoxify soil or
aqueous contaminants. Phytoremediation is a relatively young technology initially
focused on the removal of toxic metals from soil but broadened to include organic
contaminants (Baker et al., 1994; Bell, 1992). Public perception of phytoremediation as
an ecologically compatible, cost effective and aesthetically pleasing alternative to more
disruptive standard remediation approaches has helped fuel and even driven the growth of
the technology.

Engineering-based remediation which, are technologies that use purely physical
and abiotic chemical means to stabilize, destroy, remove or contain pollutants, is the most
commonly implemented treatment for contaminated sites. While the potential risks of
most of these traditional treatments have been thoroughly evaluated (Wickramanayake et
al., 2000), risks associated with biologically based technologies, especially
phytoremediation, have received relatively limited consideration (Angle and Linacre,
2005; Linacre et al., 2003). Because public perception of phytoremediation is almost
invariably positive, this paper will focus on actual risks posed by the technology rather
than perceived risks. This paper will address the risks and beneﬁts associated with the

various phytoremediation technologies by elaborating on an approach to risk-beneﬁt

128

considerations that should be taken into account when deciding on a technology.
Exploration of potential hazards inherent in applied phytoremediation will allow
preemptive management and containment of those risks, while maximizing its
effectiveness as an environmental rehabilitation tool. In thus study we will deﬁne
phytoremediation as “safe” if it can be determined to be at least as safe as widely

accepted and utilized engineering based approaches.

RISK ASSESSMENT AND RISK MANAGEMENT

Risk has been deﬁned in various ways, and has generally been thought of as both
a chance for a bad outcome and the bad outcome itself. Multiplying a numerical
probability of a particular risk by the potential severity of the risk mathematically derives
a standard measure of risk. Risk analysis is a tool that looks for the approach that presents
the lowest overall risk. However, a more obviously logical approach can be the risk
beneﬁt analysis, which combines the assessment of risks alongside of the beneﬁts and the
probabilities of those beneﬁts. A risk beneﬁt approach will be used to compare
phytoremediation and engineering based technologies. Exposure is the key component of
risk on a contaminated site. If there is no route of exposure, risk from contaminants is
minimized. A technology’s effects on routes, frequency, duration and degree of exposure
of the pollutants to receptors are all important factors in determining the nature of a risk
from a particular contaminated site. Considerable research has been done on methods of
environmental risk assessment for quantitative risk analysis, which utilize numerical
measurements of probability usually in very speciﬁc situations (Alexander, 2000; Al-

Yousﬁ et al., 2000; Oberg and Bergback, 2005).

129

Despite the obvious ease in decision making when utilizing numeric comparisons
of risk, there are instances where quantiﬁable probabilities of bad outcomes are difﬁcult
to obtain. Locations of high levels of pollutants are frequently completely unpredictable,
especially when they are the result of individual spillage events. Even after thorough
mixing individual soil particles may hold chunks of highly concentrated contaminant.
Complicating remediation, the histories of contaminated sites may be unknown and new
impacted areas, higher levels of contamination or new pollutant types may be uncovered
during remediation. These factors make it difﬁcult to obtain a precise value for risk

probability from phytoremediation installations.

RISKS AND BENEFITS FROM STANDARD REMEDIATION TECHNOLOGIES
Contaminated sites are currently most frequently remediated via standard
engineering based techniques. Engineering based remediation techniques can be broken '

into two groups, treatments that remove or destroy pollutants in soil, sediment or water
and those that stabilize contaminants within the matrix. Technologies that stabilize
contaminants in soil are, excavation and off site disposal, stabilization and solidiﬁcation.
Excavation and off site disposal is the most commonly used treatment for contaminated
soils, which involves removal of the soil and disposal at an approved landﬁll. But this
type of treatment generates some new risks; disturbance of contaminated soil and
sediment during excavation operations may, at least in the short term, increase wind and
rain erosion of hazardous particulates or bulk soil material. Excavation activities can also
pose a signiﬁcant risk to workers dealing with the contaminated soil (Cohen et al., 1997;

Proctor et al., 1997). Landﬁlling of excavated wastes is not a permanent solution for

130

treatment of soil contamination. At some point hazardous waste landﬁlls will leak and
pose recurring and complex hazards to adjacent communities and natural resources.
Many sites awaiting remediation are former landﬁlls, with over 200 of the approximately
2000 sites listed on the EPA national priorities list, being former landﬁlls (EPA, 2006).
Methods for removing or destroying contaminants in soil sediment and water
include, chemical extraction, soil washing, soil incineration, thermal desorption, soil
vapor extraction, air sparging, pump and treat, reactive barriers and bioremediation.
Table 4.1 contains a list of accepted technologies, along with potential risks, costs and

target contaminants.

Beneﬁts of Standard Remediation Technologie_s

Engineering technologies have their own set of beneﬁts and advantages, these
tend to vary within individual technologies. The main beneﬁt of most engineering based
operations and especially that of excavation based techniques is the nearly complete and
immediate removal of risk from a site, this particular quality is the primary reason for the
popularity of excavation as a treatment solution. Some engineering based technologies
are capable of completely removing a contaminant from the soil, usually resulting in

destruction of or damage to the soil as a result of treatment.

RISKS AND BENEFITS FROM PHYTOREMEDIATION
Phytoremediation technologies are divided into two basic approaches based on
target pollutant chemistry. An organic contaminant can be broken down into its

constituent elements. PCBs, for example, can be degraded to carbon dioxide and chloride

131

Haw—o AL. min—:85. on >888.— 5838338: Hen—uue—ommam 28 8803.8 $3.33»— Earn

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8:

 

 

 

 

883338 8 w:

 

Han—.889. Conn—8:8: Hwnmon » mas—5:8 088 am a 538::— Emru ”one—.288
0.333333 :2. 8.: 2. m2. :5: 5
. . . 8:38:38: 888 .38
12.98 8. 38 :8 888 88 8 a: c 8 A838 8 on. mm 39.88—
3.982.88 888 83538 8:833" 22:1. HE: . m 8 . . . 93:2. 303
. @883 <83. 3.88: 858
8:08:38: . .
$33. o_omm_:m
mo: <82 988a: ”0305: o». 83:0 8:83:38 <3 . .
65w 833:. cm 8388 a: 220:. 1.: 28 883mg .03 cm 8:88:38 8 w: Amw? moo:
. . . Dom—.880: ow 9333338 3. wow. «5:. :3? 8.0818 0880
$8838.80: 3081: m<OO. :0: 3.8 388mg 13:3" ow c.8303 Amy? Noe:
. ”033.38 083:8. 87.8».
938 £533 832.». 3 8855.5: 3 8:2: 8:. SE. 2 5.33 888.5822: 2%? 8:
0x888 m<OO .
9.: 03:0: 8 80m :08
. . H8338" o». @8338 8:. 8838:. wow. 12:. 43:. 28:05 2:38:88 ow
303238: 88 o a E s 8:: 2:33 83:: m<OO. :01 83 988%; 8:83:88 8 p: Amw? moo—v
>= 8: :8 «<38. 83. 2. 38:88».
rm8m:3m mxow<8o.:. :8 8:80: 8 8n8<8 88303.. a: ooémoo 28.8 8.58 98338:. cum? 30.:
m8: m2 355 2. 95m: . 8838 2.8% m83 58::
8:83:38 38
08:82an 83:3m 8:928 3. awn 32mm.“ 8::— 83 84 7288 o». 938
3.3: m... :8" . . 22:. 58 mp. 38:83:32 . . Amw? woo:
35.38— 2 803.8. 8338" #888 0833303 3:8."
C80~®8=8 53.84 £33m" F838
W888 :83 8:38:88 I 80988 0.. ES. 23:. 3.8 :2 58 mm: Qommmg 2. mum—Ed S. :89. 3882.. N88
. . . . 98828:. 838
3309—38
. 383208
. . . . >= 8: m8 . . . .
8:383: 5895.8 8835.5... J. 2538 seas: 85-8.8 5838828.. $258.. 88
8:9:88: . 98893 o». 8858
8.83388 .
332::
. W288. 3. 8:83:38 3‘ 81.88:: ES. wow. :2... F838 om 8:38:36
83.833 $838 8332: w<OO 28-38 ”033:3 843033 3889.. woo»:
. . 20x85 338:03 om
525838 :8. 88....an 328 4082: E 8:85.52... 28-88 Sausage a a. 58528 a; .8:
883m 88588: 2. 8: $3038 3 805.328 8: . . . .
3383820:
.2538 8338: 8 8:8 Zoxmocm 33088.4. 2.
:53:— Uomoazo: 853830: c». 8:3338G :83 wow. 32:. :0: 2098: $838: . Amw? moo:

 

 

.. 023.2. M 888 8:78:85 0:80 :88. I: n 88% 38:. Z>0 u 88:83:58 838889 733. n 838805 :38 :88. Z: n 8503 8:58" A9. :Eomo: E8
888885v. 1.»: H 8:383:53 388888. wow u “co—8:81:38 E881. .20: u 88 8:283 388888. m<OO 0 83:28.0 2830 83838

132

ions, but inorganic contaminants such as lead and mercury cannot be changed and can

only be converted to alternate valence states or integrated into different compounds.

Inorganic Phytoremediation

Phytoextraction is one of the favored approaches in phytoremediation of metals,
due to the unique ability of some plants, which are capable of extracting and
concentrating metals within their tissues. Plants that naturally accumulate up to 2% of a
metal in their leaf tissues are called hyperaccumulators (Baker et al., 1994). In
phytoextraction, plants capable of concentrating metals to some degree, preferably
hyperaccumulators, are planted on a contaminated site. After a period of growth and
biomass accumulation, the plant material can be subsequently harvested and either
landfilled or the metals reclaimed in cases where it is economically viable (Li et al.,
2003). Despite the very high tissue concentrations some hyperaccumulators can achieve,
the rate of remediation can still be quite slow. An early study using Thlaspi caerulescens
showed it would take a minimum of 13 years to remediate the soil under treatment (Baker
etaL,l994)

Consumption of contaminated plant material by wildlife is another potential risk
of phytoextraction using hyperaccumulators. Herbivory from large animals can be
controlled by means of a fence, however more stringent means might be necessary to
control insect feeding and prevent dissemination of toxins through the food chain.

In most studies, given a choice, herbivores tend to reject contaminated plant material if
uncontaminated plants are available. This pattern has been observed plant material

containing Zinc and Selenium (Hanson et al., 2004; Pollard and Baker, 1997). But the

133

degree of feeding choice within an industrial setting, where many contaminated sites are
found, might be very low, with very little to no other surrounding vegetation. One
potential mitigating factor in the potential for hyperaccumulator escape is that many
hyperaccumulating species are speciﬁcally adapted to grow on metal enriched soils and
do not thrive elsewhere (Baker and Brooks, 1989). This makes the likelihood of a plant
species being capable of both high metal content and vigorous growth on uncontaminated
soils relatively low.

Increased bioavailability is another potential risk from phytoremediation, but
exposure via direct contact might actually be reduced, since metals in soil may be present
in the aqueous soil solution whereas plant tissues usually sequester metals within the
vacuole of the cell using various transport proteins (Elbaz et al., 2006; Kupper et al.,
1999). Although the likelihood of exposure from direct contact and inhalation may be
reduced due to plant coverage of the soil and moisture retention, remaining leaf litter
would provide a more concentrated source of the contaminant and a new risk when
compared to the unaltered soil.

After hyperaccumulation has been utilized in the ﬁeld and the plants harvested,
there still exists the problem of dealing with the plant tissue. In situations where the
removed metals are economically valuable, incineration or pyrolysis may be used to
release the stored metals from dry plant tissue so that they may be recycled and utilized
for manufacture (Li et al., 2003). In other situations where metals are not intended for
reclamation, the dry tissue may be stored or landﬁlled.

Some metals are difﬁcult for plants to take up naturally into the above ground

tissues. Chelators such as ethylenediaminetetraactetic acid or EDTA can enhance plant

134

accumulation of insoluble metals. In lead phytoremediation, chelators are typically
utilized to help solubilize the lead and allow plants to take up the metal (Cooper et al.,
1999). When chelators enhance the water solubility of metals they also increase mobility
in soil and bioavailability. The enhanced bioavailability of metals will also cause any
fauna that ingests the metal contaminated soil to be at higher risk due to the EDTA
facilitating absorption. Chelators may also cause the movement of metals lower into the
soil proﬁle and even into groundwater. Movement of lead into groundwater has been a
documented occurrence on at least one chelator assisted phytoremediation installation,
however the problem of leaching on this particular site was probably exacerbated by the
short growing season and poor plant growth (ESTCP, 2001). This study shows chelator
enhanced phytoremediation should be carefully managed and planned, taking into
account rainfall events, sorption of contaminants to soil particles, preferential ﬂow of
water through the soil and distance to groundwater. The application of a chelator may
not only enhance the mobility of the target metal it may also enhance the movement of
other metals in the soil. This necessitates that analyses be performed on other metals so
that their movement into groundwater would not go unobserved. Technological
combinations such as phytoextraction with permeable reactive barriers could reduce the
risk of chelate-induced leaching considerably, by over 60 times in one lead
phytoremediation study (K03 and Lestan, 2003).

An alternative treatment strategy for inorganic contaminants is phytostabilization,
in which plants stabilize the elements within the soil and prevent their dissemination by
wind and rain. Plants can also alter the pH of soil by secreting organic acids, potentially

affecting the sorption of contaminants (Neumann and Romheld, 1999; White et al., 2003;

135

Zhou et al., 2003). These alterations may in turn provide metal resistance or aid in metal
uptake by plants (Ma et al., 2000; Tolra et al., 1996). Phytostabilization may be at highest
risk from attractive nuisance since phytostabilization is typically permanent and
potentially subject to less management and monitoring.

Phytovolatilization, or the delivery of soil-based pollutants to the atmosphere via
plant transpiration, is a component of some phytoremediation installations by design,
however it may potentially occur in any situation where volatile contaminants are
present. Phytovolatilization can pose risks similar to those found in hyperaccumulation
and phytoextraction since the contaminant is moved from the soil to another medium.
Rather than being contained within the plant tissue, phytovolatilization moves the
contaminants into the air. For phytovolatilization to considered an appropriate solution, a
signiﬁcant reduction in toxicity must be expected or a very high dilution effect.
Conversion of methyl mercury in the soil to elemental mercury in transgenic plant tissue
and subsequent release the atmosphere may be an acceptable reduction in toxicity, due to
the extreme toxicity of methylmercury and the much lower toxicity of elemental mercury
(Bizily et al., 2000; Rugh et al., 1996). Volatilization and atmospheric transport of
selenium will simply cause dispersal of an overly concentrated nutrient to areas where
selenium may be deﬁcient (De Souza et al., 2000). In cases of uncertainty actual
contaminant levels in the atmosphere, leaves and stems could be analyzed to determine if
phytovolatilization has the potential to produce a high concentration in the atmosphere
surrounding the site such that it would be overly risky to be within or near the site.
However atmospheric dilution would probably be able to compensate for relatively low

levels of contaminants released from plant leaves.

136

Q_rganic Phytoremediation

Phytoremediation of organic contaminants can be accomplished by the plant alone
(phytodegradation) or in concert with soil bacteria (phytostimulation) and in some cases
phytovolatilization. Water-soluble compounds may be moved from the soil into plant
roots or even aboveground tissues and degraded by plant-produced enzymes or
volatilized. Alternatively, extracellularly secreted enzymes may degrade less water-
soluble compounds. This has been especially well documented in TCE phytoremediation
in which TCE can be converted to the less toxic dichloroethylene or trichloroethanol
within plant cells (Shang et al., 2001). Quantitative studies of the amount of TCE
transpired by poplar trees growing on TCE contaminated aquifers have been conducted
which showed that the release of TCE from leaf tissue was less than 9% of the total
quantity of uptaken TCE (Newman et al., 1999).

Phytodegradation is the transformation or degradation of organic compounds by
plants. Hydroxylation of xenobiotic compounds or conjugation with varying sugars is a
frequent result of exposure studies using plant cell cultures (Huckelhoven et al., 1997;
Wilken et al., 1995). However, uptake through the transpiration stream might result in a
somewhat different set of metabolites than direct cell culture exposure. Some studies
have already begun to characterize these metabolites and future studies should help
clarify xenobiotic metabolic pathways for a wider variety of compounds (Subramanian et
al., 2006). Chemical modiﬁcations can transform organic compounds to be more water-
soluble and therefore more mobile or possibly more toxic. Transformation to more toxic

compounds is known to occur in microbes that degrade trichloroethylene (TCE). When

137

degraded anaerobically, TCE can be converted to the more toxic and carcinogenic vinyl
chloride (McCarty, 1997). It is not impossible that similar increases in toxicity could
occur in plants. The optimal outcome for the degradation of organics is mineralization or
conversion back to basic elements such as C02, chlorine and water. Even if contaminants
are uptaken and modiﬁed, their loss from intact plant tissue via volatilization, or
mineralization may be variable and likely to not be 100%, with the maximum observed
for RDX being 25% in one study (Yoon et al., 2006).

Ideally the major metabolites produced by phytoremediating plants be identiﬁed
and assessed for their individual impact. Barring actual data, potential metabolites could
be imagined based on typical plant metabolic patterns and estimated for toxicity. While
the potential for plant-induced changes to organic chemicals to be harmful, the variety of
plant produced metabolites is likely to be high meaning that the concentration of any one
metabolite is likely to be below thresholds for toxicity. Although the combination of
many metabolites could be more toxic than any individual compound or an individual
compound could be at extremely increased toxicity, this seems to be relatively low
likelihood. Direct toxicity tests of exposed plant tissue and soils post-treatment on
indicator species would likely be considerably easier and more appropriate than chemical
analysis of metabolites, and should provide sufﬁcient proof of lowered toxicity. If it is
found that plants are increasing toxicity through their metabolic activities they may need
to be removed and alternative remediation methods found, or methods of reducing
bioavailability through chemical addition could be implemented (Chen et al., 2000).
Comparisons to contact toxicity from the soil or medium itself may be useful in

determining the potential costs and beneﬁts of the phytoremediation technology.

138

Metric Phytoremediation

Transgenic plants are those that are transformed with bacterial genes for enhanced
contaminant degradation, solubilization or stabilization. All of the risks from non-
transgenic phytoremediation are also a factor in remediation installations using transgenic
plants as the processes are typically the same as with non-transgenic plants, including the
potential for genetic escape. Any introduction of a non-native organism poses a potential
risk to the local region and the environment as a whole. Instances of damaging invasive
species resulting from well-intentioned introductions are not unusual and invasive species
are now considered a major threat to the global ecosystem (Vitousek et al., 1997).
However, the simple introduction of a non-native species or transgenic plants does not
necessarily spell disaster, since many thousands of plant species have been imported with
little effect, but those that do cause problems may do so on a massive scale. The severity
of this risk is largely dependent on the gene and plant species involved. The degree of
risk posed by any particular transgene is highly variable and depends on the nature of the
transgene. The escape of a gene for PCB degradation might have a very different
ecological impact than a gene to enable arsenic hyperaccumulation.

The presence of compatible relatives for the introduced plants to potentially cross
with or permissible conditions for pollen or seed dissemination can enhance the risk.
Lack of compatible relatives nearby to a site of implementation greatly reduces potential
risk of gene escape but does not eliminate it, if the transgenic plant is able to propagate
through viable seeds or vegetatively such as suckers, rhizomes or runners. Transgenic

plants may also have new properties not present in unmodiﬁed plant species, posing some

139

risk to wildlife simply by being placed on a site. Plants containing and secreting new
compounds for uptake of novel contaminants represent unique risks over non-transgenic
phytoremediation. Engineered mercury volatilization, overexpression of phytochelatins
has shown that the creation of novel hyperaccumulators is possible (Pilon-Smits and
Pilon, 2002). The chemistries in these plants could bypass normal avoidance mechanisms
by herbivores, leading to increased risk over natural hyperaccumulator species.

An already proposed method of assessing the risks presented by transgenic crops
could be applied to the assessment of plants genetically modiﬁed for enhanced
phytoremediation and even the introduction of new species (Hancock, 2003). Variables
such as dispersal distance, potential for crossing to native relatives and
weedy/invasiveness of the transgenic or novel species can be weighed as to importance in
a particular instance of implementation. The chance that a known plant species that is
non-invasive will be made invasive via transgenic manipulation for phytoremediation is
probably quite low, since only 1-2 genes are typically added to the transgenic plant.

But the risk of escape cannot be discounted entirely and the consequences for a
single escape could be very damaging to public perception of phytoremediation even if a
documented escape does not actually pose a high risk to the community or environment.
There are quite a number of methods to reduce the risk of plant escape, primarily the use
of sterile cultivars so that dissemination via seed or pollen is prevented or cultural
practices such as harvesting plants before ﬂowering can occur (Singh et al., 2006). There
may still be situations in which the potential risk from the introduction of a new species
or a genetically modiﬁed plant would still be overly risky from the standpoint of escape.

In these cases phytoremediation, using species that need to be avoided, can be conducted

140

ex-situ in greenhouses, or avoided altogether. Any new introductions to an outdoor area,
transgenic or no, should be carefully examined for ecological impact prior to large scale
planting. Table 4.2 lists the costs and associated risks of most phytoremediation

approaches.

Beneﬁts of Phytoremediation

Phytoremediation has a number of advantages and beneﬁts over engineering
based remediation approaches. Phytoremediation is typically lower cost than engineering
operations as the planting of plants on soil is usually considerably cheaper than
excavation or intensive soil treatment. Phytoremediation is generally considered
environmentally friendly and aesthetically pleasing, potentially capable of restoring
habitat when implemented in degraded locations. Phytoremediation may also be the only
technology, which is suitable for certain types of contaminated sites, large regions such
mine tailings or locations downwind from smelter operations would beneﬁt from
phytostabilization installations, since excavation and other forms of treatment are
frequently economically unviable in very large areas of contamination. Phytoremediation
also has unique capabilities for the speciﬁc removal and containment of certain metals,
including the concentration of metals into a much smaller volume and the potential for
metal reclamation from plant tissue. The combination of lowered cost and greater public
acceptance of phytoremediation technologies can also lead to a more pro-active cleanup

effort by responsible parties.

141

H.020 Ab. 9.3303. 0». 05.810.509.33: H00ru0_0mm0m Ea >0000ma~0n 0000:»?— Eur:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. . 000m: mug—:30.— 038 am a . .
adage—05. 5000:0200 000933030. 00.. 00.: 0.. 00.. :5: 5 00.0.50. 326 ”002.0000:
. . 00033303 000035030? Awﬂmrﬁn 0. 0...
00:05.98 £020.30 0.50:0: 0m 3303. 0.03 :0? Z0. 85.. _0<0_m 0.. 090.. a. .MQ—nma 9.0.. _o 0... 00303303 0.80.00 38 03.0.. 300“ mum—.00.
00.3 005.000 00:833mb 0020:
$0839.88 _ 33
30:8 :00: mm 0 35.2 $030. 00:83.35. 000030.020:
20.035? 0033. 003035300 90.30%...0. Z>0F :Z 39.0 30 @3988 . 90>. ~88
00:83.33 80000
305030.:
0:5. 0330020: 0.. 05030 2: - an mun 00 0:9: 008:2”: 8.30:0: 0». 850 “Muﬁmwmrwwow.
03.80%..3020: 8:833:30 8 :0:-8x.0 20,0? m<00. Z>O . . . 0 m 3.0309300 0n 009.3538: 0.. . .
0: 0:0 2:0 0:0 930:5. . U050: 0. 0...
33m. Saga—000 308:0. Noomv
203 :038 0:: 3:0. 00:833...." 30.02530: 3. 300:0 :00
0340053010: 000030530: «0. 10200. 0:: :7... 20. 0mm. 000 «3-30 00a 000.0 «:3 0099.000 :32 0.. 9.1:m 0:105..- .003 0 .
a; 000— 0.. 80 0:: 000.080 302.0%. $010.5?
. . . 20:. 3002335: 0*. 8.; 3. 8.8 . 2 be 00.. 0:30 00:83:03 002.3533: 803.3%.»3 0.
03000395020: 0:03.00— m:0\0q : £00. 3003 ES 3080 35.090 3.5.500 m... .03?
0.0:. ~00" c0238 30 0010— 2.: - MN SN 00 3:: 00303.0: 0., £301mozco30 8:300. 0. 3..
03432033030: 3.025020: 0m 5530 Z>0F m0. :m . . . 00 m 8:833:30 8 05.030 .0qu 0050: 0.
0: 30 2:0 0:0 0.30:5. .
m0==§G 00:35:32.3 a; noowv
. 0:53 000030 003033300 .50
. . 0.0:. 3.9 :0Sme01020: 0m 3.: 00: 90:00:: 3:3 .00 0.00800 0.. 68:03.0... 0.
5:83.320: 003033.56 .403 «$50 $08: :3. Z>O . .
mE—oa 0m £30.. WE. 9; "00—30—09. .0 500:0. :0» a... .003
00.023 .
:05 30.030300
0:5. 808 53508 000810. . 30030—08 gang—20:. A0050: 0. 0:.
”0300235020: 0033.35. 8 gnaw...” Z>O. 02.00? 000. m3... - «NbNN 0000355 00:03:30.: on 00303303 noon. .9300. 0.
. 00:. w<OO 0: 0:0 030 30 05.850. . . .
8:533:30 38.30930 08:35:00: 0... Moo:
. . 00303303 000035030? .
E0010: 02.100 W322 8 3:03. 2.03 :0:-00=: 20. _0s.‘ _0<0_w 0% 02.0.. m3boo I 398° 00: 00:33:53 000000 3 8 050.. 3508-003...
83.08 8:83.338 :20 208305000 0. 0... ~83

 

 

142

.. UZ>0F n 0030 3.150005 0:30 :05? I: n 3005. 308—. Z>O n 32.02.0330 00300.30? Z>0r n :0:.B..00:m 0:80 :03... 20 u 3.30:. 00:50.: A2 :50m0: .30
0:80—8:95. 0>I n 020083020 3.080030? 00w n 00.06.100.380 90:03:. 000 M 00330:. 05:30 00:08:». 00: u 88. 00229.3 39.00030? w<OO u 00310—030
08030 0030033

IS PHYTOREMEDIATION SAFE?
Is Phytoremediation Suitable?

The ﬁrst step in an assessment of any technological application is to determine if
it is suitable for a particular impacted site. Suitability assessment includes an implicit risk
reduction strategy, since technological failure due to placement in an improper location,
would almost certainly cause increased risk, even over no action at all. A decision tree to
determine phytoremediation suitability has already been constructed by the Interstate
Technology and Regulatory Cooperation (ITRC) (ITRC, 2000). The general limitations
of phytoremediation are; long treatment times, shallow treatment depth (13 feet or less
usually) poor performance in short growing seasons or arid conditions, and high
contaminant levels (potential plant toxicity). Once it has been determined that
phytoremediation is an otherwise viable option, the safety implications of its

implementation on a particular site can be considered.

Is Phytoremediation Safe?

To deﬁne an approach or a technology as “safe”, probabilities of different
outcomes can be used as a basis, but they do not work in every circumstance nor can they
be applied to every situation, especially in cases where uncertainty is high as is often the
case in phytoremediation installations. While some determination of exposure can be
made, the nature of a phytoremediation installation makes these factors hard to quantify.
The degree of contaminant uptake by plants may be difﬁcult to predict in some instances
and the degree to which plants reduce or accelerate contaminant dispersal may also be

highly variable. Because of the difﬁculty in assigning numerical values to risks from

143

phytoremediation we will compare standard engineering practices to phytoremediation
practices. Since these technologies are currently in use, these technologies are generally
considered by regulatory bodies as well as the public at large as “safe”. If
phytoremediation is “safe” it should have similar or fewer risks and potentials for risk to
standard engineering technologies. A simpliﬁed tabular comparison of basic risks of
phytoremediation and engineering based approaches is shown in (Table 4.3).

One basic difference between the most common form of engineering based
remediation, excavation, and off site disposal is the nature of contaminant treatment.
While immediate risk to an area or group of people is removed via excavation, ultimately
the risk has been transferred spatially and temporally to a controlled location.
Phytoremediation, especially of organic contaminants and phytoextraction of metals

Table 4.3. Direct comparison of exposure based risks from remediation technologies.

 

 

 

 

 

 

 

 

 

Risk Phytoremediation Engineering based
technologies
Reduced Risk in most cases due Enhanced by excavation
. to plant root containment, except
Leaching . . based treatments, reduced by
in chelate or surfactant ass15ted . . .
. . stabilization
phytoremediation
Soil Particle Reduced risk, some contribution Risk increased by
Dissemination by planting operations excavation/soil disruption
. May increase risk due to
. . . May decrease except in . . . .
Volatilization h tovolatilization disturbance or volatilization
p y based technology
Potential risk of almost any
Altered . . . . . .
. phytoremediation installation, Generally unlikely except in
Contaminant . . .
. . lower for contaminants With low cases of chemical treatment
Tox1c1ty . .
water solubility.
. Phytoremediation often Technologies typically well
Failure of unpredictable. Failure or longer . .
. . . known, risk of failure
Technology than antic1pated treatment times
generally low
common

 

 

 

Engineering based

Risk Phytoremediation technologies

 

Only in technologies designed
for concentration such as soil
washing

Concentration of By design in phytoaccumulation,
Contaminants potential for increased risk

 

Dissemination of .
Only occurs in the case of

 

 

aSSimilated Risk speCIﬁc to phytoremediation pre-exis ting biota
compounds
Plant Escape Risk speciﬁc to phytoremediation Not Applicable

 

 

 

actually cleanses the soil by removing or destroying the contaminants in the soil while
not damaging the soil and even potentially improving the soil’s agronomic qualities.
Despite this seemingly overwhelming advantage in terms of risk removal rather than risk
transfer, phytoremediation cannot always perform complete remediation. Sometimes
plants may not be able to remove or detoxify contaminants up to a regulatory standpoint
that is sufﬁcient to reduce risk to an acceptable level.

The decision between phytoremediation and engineering technologies comes
down to a choice between removing all or most of the risk from a particular area
immediately with the understanding that the risk may reoccur at some other place and
time, or accepting some level of ongoing risk in the hopes that over a long period of time
the risk will be substantially reduced or removed completely. In most cases regulatory
agencies ﬁnd the idea of immediate risk removal to be superior, especially in cases where
contamination levels are high. However, if a site is not an immediate threat to a
surrounding area, phytoremediation as a potential permanent solution should gain favor.
In terms of overall risk reduction risk removal is preferable to temporal or spatial risk

transfer. Although the risk from gene and plant escape stands out as a novel and

145

 

somewhat unknown risk, it is potentially highly manageable with sterile clones and
cultural practices. The sum total of risks and beneﬁts from phytoremediation make it no
less safe than engineering practices, when implemented under appropriate circumstances.
Phytoremediation should by no means replace engineering approaches, as there are
situations where phytoremediation is of limited utility. However especially in situations

of low level contaminants, phytoremediation can be particularly safe and effective.

CONCLUSION

Phytoremediation is still an emerging treatment technology, slowly gaining
market share and becoming more broadly implemented. Understanding the risks posed by
a phytoremediation installation is an important step forward for responsible choices
concerning phytoremediation and increased regulatory acceptance of the technology.
Phytoremediation is not devoid of risk and bears some different risks than standard
engineering-based remediation technologies. Careful analysis and comparisons of risks
from both technological types shows that, when properly implemented, phytoremediation
does not appear to pose any more risk than currently accepted technologies. However,
like any remediation technology careful management and monitoring is necessary to
avoid unforeseen complications and unnecessaiy risks.

Several of the special risks posed by phytoremediation are worthy of
consideration for further study to clarify and obtain quantiﬁable probabilities where
possible. These areas include the risks posed by phytoremediative gene escape and the
potential for phytoremediation species to become invasive. More methods for preventing

gene escape and the production of sterile clones will allow for the implementation of

146

genes or plant species that might pose an overly high risk without stringent controls.
Information concerning uptake and transformation of various environmental toxicants
would be especially valuable in assisting in assessment of risks of contaminant
conversion for particular pollutant and plant species combinations.

Assuming careful management and appropriate implementation of
phytoremediation, even given some gaps in knowledge is no less safe than currently
accepted remediation technologies and may offer the public a superior level of protection
in some situations. Continued research and creation of improved plant varieties with
reduced potential for escape will ensure the future of phytoremediation as a safe and

effective technology.

147

LITERATURE CITED

Alexander, M. (2000). Aging, bioavailability, and overestimation of risk from
environmental pollutants. Environmental Science and Technology 34, 4259-4265.

Al-Yousﬁ, A. B., Hannak, P. G., Strunk, J. F., Davies, W. V., and Shah, S. I. (2000).
Risk—based zoning strategy for soil remediation at an industrial site. Journal of
Soil Contamination 9, 1-12.

Angle, J. S., and Linacre, N. A. (2005). Metal phytoextraction - A survey of potential
risks. International Journal of Phytoremediation 7, 241-254.

Baker, A. J. M., and Brooks, R. R. (1989). Terrestrial higher plants which hyper-
accumulate metallic elements: A review of their distribution, ecology and
phytochemistry. Biorecovery 1 , 81-126.

Baker, A. J. M., McGrath, S. P., Sidoli, C. M. D., and Reeves, R. D. (1994). The
possibility of in situ heavy metal decontamination of polluted soils using crops of
metal-accumulating plants. Resources Conservation and Recycling [1, 41-49.

Bell, R. M. (1992). Higher plant accumulation of organic pollutants from soils. USEPA,
Risk Reduction Engineering Lab EPA 600-SR-92-138.

Berghage, R. D., MacNeal, E. P., Wheeler, E. F., and Zachritz, W. H. (1999). "Green"
water treatment for the green industries: Opportunities for bioﬁltration of
greenhouse and nursery irrigation water and runoff with constructed wetlands.
Hortscience 34, 50-54.

Bizily, S. P., Rugh, C. L., and Meagher, R. B. (2000). Phytodetoxiﬁcation of hazardous
organomercurials by genetically engineered plants. Nature Biotechnology 18,
213-217.

Blanco-Canqui, H., Gantzer, C. J ., Anderson, S. H., Alberts, E. E., and Thompson, A. L.
(2004). Grass barrier and vegetative ﬁlter strip effectiveness in reducing runoff,
sediment, nitrogen, and phosphorus loss. Soil Science Society of America Journal
68, 1670-1678.

148

Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S., and Baker,
A. J. M. (1997). Phytoremediation of soil metals. Current Opinion in
Biotechnology 8, 279-284.

Chen, H. M., Zheng, C. R., Tu, C., and Shen, Z. G. (2000). Chemical methods and
phytoremediation of soil contaminated with heavy metals. Chemosphere 41, 229-
234.

Cohen, J. T., Beck, B. D., and Rudel, R. (1997). Life years lost at hazardous waste sites:
Remediation worker fatalities vs. cancer deaths to nearby residents. Risk Analysis
1 7, 419-425.

Cooper, E. M., Sims, J. T., Cunningham, S. D., Huang, J. W., and Berti, W. R. (1999).
Chelate-assisted phytoextraction of lead from contaminated soils. Journal of
Environmental Quality 28, 1709-1719.

Cunningham, S. D., and Berti, W. R. (1993). Remediation of contaminated soils with
green plants - An overview. In Vitro Cellular & Developmental Biology-Plant
29P, 207-212.

Cunningham, S. D., Berti, W. R., and Huang, J. W. W. (1995). Phytoremediation of
contaminated soils. Trends in Biotechnology 13, 393-397.

De Souza, M. P., Pilon-Smits, E. A. H., and Terry, N. (2000). The Physiology and
biochemistry of selenium volatilization by plants. In Phytoremediation of Toxic
Metals: Using Plants to Clean Up the Environment, 1. Raskin and B. D. Ensley,
eds. (New York: John Wiley & Sons, Inc.), pp. 171-190.

Deuren, J. V., Lloyd, T., Chhetry, S., Liou, R., and Peck, J. (2002). Remediation
Technologies Screening Matrix and Reference Guide, 4th Edition: Federal
Remediation Technologies Roundtable).

Dushenkov, S., Vasudev, D., Kapulnik, Y., Gleba, D., Fleisher, D., Ting, K. C., and
Ensley, B. (1997). Removal of Uranium from water using terrestrial plants.
Environmental Science & Technology 31, 3468-3474.

149

Edenspace. (1998). Edenspace Home: Products & Services: Statement of Qualiﬁcation:
Representative Projects. http://www.edenspace.com/qualiﬁcation.html

Elbaz, B., Shoshani-Knaani, N., David-Assael, O., Mizrachy-Dagri, T., Mizrahi, K., Saul,
H., Brook, E., Berezin, I., and Shaul, O. (2006). High expression in leaves of the
zinc hyperaccumulator Arabidopsis halleri of AhMHX, a homolog of an
Arabidopsis thaliana vacuolar metal/proton exchanger. Plant Cell and
Environment 29, 1179-1190.

EPA. (2006). National Priorities List: United States Environmental Protection Agency).

EPA. (2000). US Environmental Protection Agency - Introduction to Phytoremediation.

EPA. (1997). US Environmental Protection Agency - Management of Polychlorinated
Biphenyls in the United States: Ofﬁce of Pollution Prevention and Toxics).

EPA. (2001). US Environmental Protection Agency - Remediation Technology Cost
Compendium - Year 2000. In EPA 542-R-01-OO9, U. E. P. Agency, ed.

ESTCP. (2001). Phytoremediation of Lead-Contaminated Soil: Environmental Security
Technology Certiﬁcation Program), pp. 1.

ESTCP. (1999). The Use of Constructed Wetlands Phytoremediation Explosives-
Contarninated Groundwater at the Milan Army Ammunition Plant, Milan,
Tennesse: ESTCP Cost and Performance Report: Environmental Security
Technology Certiﬁcation Program), pp. 1.

Hancock, J. F. (2003). A framework for assessing the risk of transgenic crops. Bioscience
53, 512-519.

Hanson, B., Lindblom, S. D., Loefﬂer, M. L., and Pilon—Smits, E. A. H. (2004). Selenium
protects plants from phloem-feeding aphids due to both deterrence and toxicity.
New Phytologist 162, 655-662.

Huckelhoven, R., Schuphan, I., Thiede, B., and Schmidt, B. (1997). Biotransformation of
pyrene by cell cultures of soybean (Glycine max L), wheat (Triticum aestivum L),

150

jimsonweed (Datura stramonium L), and purple foxglove (Digitalis purpurea L).
Journal of Agricultural and Food Chemistry 45 , 263-269.

ITRC. (2000). Phytoremediation Online Decision Tree Document: Interstate Technology
and Regulatory Cooperation Work Group).

Kos, B., and Lestan, D. (2003). Induced phytoextraction/soil washing of lead using
biodegradable chelate and permeable barriers. Environmental Science &
Technology 3 7, 624-629.

Kuiper, I., Bloemberg, G. V., and Lugtenberg, B. J. J. (2001). Selection of a plant-
bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic
hydrocarbon-degrading bacteria. Molecular Plant—Microbe Interactions 14, 1197-

1205.

Kupper, H., Zhao, F. J., and McGrath, S. P. (1999). Cellular compartmentation of zinc in
leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiology 119, 305-
311.

Li, Y. M., Chaney, R., Brewer, E., Roseberg, R., Angle, J. S., Baker, A., Reeves, R., and
Nelkin, J. (2003). Development of a technology for commercial phytoextraction
of nickel: economic and technical considerations. Plant and Soil 249, 107-115.

Linacre, N. A., Whiting, S. N., Baker, A. J. M., Angle, J. S., and Ades, P. K. (2003).
Transgenics and phytoremediation: the need for an integrated risk assessment,
management, and communication strategy. International Journal of
Phytoremediation 5, 181-185.

Ma, J. F., Taketa, S., and Yang, Z. M. (2000). Aluminum tolerance genes on the short
arm of chromosome 3R are linked to organic acid release in triticale. Plant
Physiology 122, 687-694.

McCarty, P. L. (1997). Microbiology - Breathing with chlorinated solvents. Science 276,
1521-1522.

Miller, R. R. (1996). Air Sparging. In Technology Overview Report: Ground-Water
Remediation Technologies Analysis Center), pp. 1-18.

151

Neumann, G., and Romheld, V. (1999). Root excretion of carboxylic acids and protons in
phosphorus-deﬁcient plants. Plant and Soil 211, 121-130.

Newman, L. A., Wang, X. P., Muiznieks, I. A., Ekuan, G., Ruszaj, M., Cortellucci, R.,
Domroes, D., Karscig, G., Newman, T., Crampton, R. S., Hashmonay, R. A.,
Yost, M. G., Heilman, P. E., Duffy, J., Gordon, M. P., and Strand, S. E. (1999).
Remediation of trichloroethylene in an artiﬁcial aquifer with trees: A controlled
ﬁeld study. Environmental Science & Technology 33, 2257-2265.

Oberg, T., and Bergback, B. (2005). A review of probabilistic risk assessment of
contaminated land. Journal of Soils and Sediments 5, 213-224.

Pilon-Smits, E., and Pilon, M. (2002). Phytoremediation of metals using transgenic
plants. Critical Reviews in Plant Sciences 21, 439-456.

Pollard, A. J ., and Baker, A. J. M. (1997). Deterrence of herbivory by zinc
hyperaccumulation in Thlaspi caerulescens (Brassicaceae). New Phytologist 135,
655-658.

Proctor, D. H., Zak, M. A., and Finley, B. L. (1997). Resolving uncertainties associated
with the construction worker soil ingestion rate: A proposal for risk-based
remediation goals. Human and Ecological Risk Assessment 3, 299-303.

Rugh, C. L., Wilde, H. D., Stack, N. M., Thompson, D. M., Summers, A. 0., and
Meagher, R. B. (1996). Mercuric ion reduction and resistance in transgenic
Arabidopsis thaliana plants expressing a modiﬁed bacterial merA gene.
Proceedings of the National Academy of Sciences USA 93 , 3182-7.

Schnoor, J. L. (2002). Phytoremediation of soil and groundwater. In Technology
Evaluation report: Ground-Water Remediation Technologies Analysis Center),
pp. 1-52.

Shang, T. Q., Doty, S. L., Wilson, A. M., Howald, W. N., and Gordon, M. P. (2001).
Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway.
Phytochemistry 58, 1055-1065.

152

Singh, 0. V., Ghai, S., Paul, D., and Jain, R. K. (2006). Genetically modiﬁed crops:
success, safety assessment, and public concern. Applied Microbiology and
Biotechnology 71, 598-607.

Subramanian, M., Oliver, D. J ., and Shanks, J. V. (2006). TNT phytotransforrnation
pathway characteristics in Arabidopsis: Role of aromatic hydroxylamines.
Biotechnology Progress 22, 208-216.

Tolra, R. P., Poschenrieder, C., and Barcelo, J. (1996). Zinc hyperaccumulation in
Thlaspi caerulescens 11. Inﬂuence on organic acids. Journal of Plant Nutrition 19,
1541-1550.

Vitousek, P. M., Dantonio, C. M., Loope, L. L., Rejmanek, M., and Westbrooks, R.
(1997). Introduced species: A signiﬁcant component of human-caused global
change. New Zealand Journal of Ecology 21, 1-16.

White, J. C., Mattina, M. L, Lee, W. Y., Eitzer, B. D., and Iannucci-Berger, W. (2003).
Role of organic acids in enhancing the desorption and uptake of weathered p,p '-
DDE by Cucurbita pepo. Environmental Pollution 124, 71-80.

Wickramanayake, G. B., Gavaskar, A. R., Kelley, M. E., and Nehring, K. W. (2000).
Risk, Regulatory, and Monitoring Considerations. In Remediation of Chlorinated
and Recalcitrant Compounds (Columbus, OH: Battelle Press), pp. 438.

Wilken, A., Bock, C., Bokem, M., and Harms, H. (1995). Metabolism of different PCB
congeners in plant cell cultures. Environmental Toxicology and Chemistry 14,
2017-2022.

Yoon, J. M., Van Aken, B., and Schnoor, J. L. (2006). Leaching of contaminated leaves
following uptake and phytoremediation of RDX, HMX, and TNT by poplar.
International Journal of Phytoremediation 8, 81-94.

Zhou, D. M., Chen, H. M., Wang, S. Q., and Zheng, C. R. (2003). Effects of organic
acids, o-phenylenediamine and pyrocatechol on cadmium adsorption and
desorption in soil. Water Air and Soil Pollution 145, 109-121.

153

 

 

   

ulililljﬂijlillljljlli11111111