THE CHARACTERIZATION OF METAL BIOTRANSFORMATION

THE CHARACTERIZATION OF METAL BIOTRANSFORMATION

CAPABILITIES OF PHOTOSYNTHETIC MICROORGANISMS

By

Chad Donald Edwards

A thesis submitted to the Department of Biology

In conformity with the requirements for

the degree of Masters of Science

Queen’s University

Kingston, Ontario, Canada

(April, 2010)

Copyright ©Chad Donald Edwards, 2010

ii

Abstract

Industrial activity over the last two centuries have increased heavy metal contamination

worldwide and has led to greater human interaction with these contaminants. Cadmium, copper and zinc

are particularly common industrial effluents and are highly toxic in elevated concentrations. Due to the

hazards of heavy metal pollution, there is a growing demand for cost effective and efficient means to

remove heavy metals. Photoautotrophic microbes can biotransform heavy metals into the relatively

biologically unavailable and insoluble metal-sulfides. By characterizing the sulfur assimilation pathways’

role in heavy metal biotransformation, more effective heavy metal bioremediation processes may be

developed. Chlamydomonas reinhardtii, Synechoccocus leopoliensis and Cyanidioschyzon merolae were

exposed to cadmium, copper, and zinc and their abilities to synthesize metal-sulfides were assessed.

Sulfate, sulfite and cysteine were enriched in the respective media of each species to determine the role of

sulfur metabolites in enhancing metal tolerance and metal-sulfide formation. The activities of cysteine

desulfhydrase, serine acetyltransferase and O-acetylserine(thiol)lyase activity were analyzed under the

stress of Cd(II), Cu(II) and Zn(II). C. merolae supplemented with sulfate yielded a significant increase in

Cd(II), Cu(II), and Zn(II) tolerance, higher sulfide formation, and increased enzyme activity. C.

reinhardtii and S. leopoliensis supplemented with sulfate yielded significantly more biomass than the

sulfite and cysteine treatments when exposed to Cd(II), Cu(II) and Zn(II). In all species the addition of

sulfite and cysteine was found to be detrimental to cell growth. C. reinhardtii had an average increase of

5-8 times the initial cysteine desulfhydrase and serine acetyltransferase activity for each metal treatment.

Cd(II), Cu(II) and Zn(II) proved to have a limiting effect on S. leopoliensis’ enzyme activity. The

potential limitations in the sulfur assimilation pathway will be discussed. C. merolae may prove to be a

successful candidate for metal bioremediation. Utilizing supplemental sulfate nutrition may improve the

rate of biotransformation in aerobic microbes.

iii

Acknowledgements

I would first like to personally thank Dr. Daniel D. Lefebvre for his invaluable guidance, patience, good

will and friendship. Without his supervision and advice, this research would not have been possible.

Thank you, Dan, for this astounding opportunity.

A special thanks to Anthony Silva and Melanie Columbus for their spirited attitudes and passion towards

research; you both were strong inspirations.

Many thanks to my committee members, Dr. Kenton Ko and Dr. Gema Olivo, for their experience and

thoughtful insight.

Thank you to Jackoline Loiselle, Joseph Beatty, Caleb Van Ry, Katya Vlassov, Antoine Hnain, Stefans

Jackson, Sybil Machler, Winny Li, Iona Coza, and Mitangi Parekh for the pleasant attitude they provided

in the lab and the assistance that they offered to the project.

I would like to thank my parents, Donald and Tannis, for their direction, love and encouragement.

Lastly, I would like to extend my personal gratitude to Natalie Paquin for her everlasting support, and

irreplaceable company.

iv

Table of Contents

Abstract ......................................................................................................................................................... ii

Acknowledgements ...................................................................................................................................... iii

List of Figures………………………………………………………………………..……………….…...vii

List of Tables………………………………………………………………………………………..……viii

List of Abbreviations………………………………………………………………………………………ix

Chapter 1 General Introduction..................................................................................................................... 1

1.1 Introduction ......................................................................................................................................... 1

1.2 Membrane transport of heavy metals: ................................................................................................. 3

1.3 Toxicity of heavy metals ..................................................................................................................... 7

1.4 Uptake and assimilation of sulfate ...................................................................................................... 9

1.4.1 Sulfate transporters: ................................................................................................................... 12

1.4.2 Gene regulation during metal and sulfate starvation .................................................................. 14

1.5 Metallothioneins ............................................................................................................................... 16

1.5.1 Class II metallothioneins ............................................................................................................ 17

1.5.2 Class III metallothioneins .......................................................................................................... 17

1.5.3 Labile and non-labile phases of metals ...................................................................................... 18

1.5.4 Sequestration and compartmentalization of phytochelatins ....................................................... 19

1.5.5 Cellular exportation of phytochelatins ....................................................................................... 20

1.5.6 Anaerobic metal-sulfide production ........................................................................................... 20

1.5.7 Aerobic metal-sulfide biotransformation ................................................................................... 21

1.6 Objectives ......................................................................................................................................... 23

Chapter 2 Materials and Methods ............................................................................................................... 24

2.1 Culture sources and growth conditions ............................................................................................. 24

2.2 Sulfate, sulfite, and L-cysteine treatments ........................................................................................ 25

2.3 Supplemental treatment groups ......................................................................................................... 26

2.4 Heavy metal treatments:.................................................................................................................... 27

2.5 Metal toxicity .................................................................................................................................... 27

2.6 Bradford protein assays ..................................................................................................................... 28

2.7 Enzyme assay sample preparation .................................................................................................... 28

2.8 Acid labile sulfide analysis ............................................................................................................... 29

v

2.9 Cysteine desulfhydrase activity ........................................................................................................ 30

2.10 Serine acetyltransferase and O-acetylserine(thiol)lyase activity .................................................... 30

2.11 Statistics .......................................................................................................................................... 31

Chapter 3 Determining the detoxification capabilities of photosynthetic microorganisms to cadmium .... 32

3.1 Introduction: ...................................................................................................................................... 32

3.2 Results ............................................................................................................................................... 33

3.2.1 Cadmium tolerance .................................................................................................................... 33

3.2.2 Sulfide production ...................................................................................................................... 33

3.2.3 Cysteine desulfhydrase .............................................................................................................. 34

3.2.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity ............................................... 35

3.3 Discussion ......................................................................................................................................... 40

3.3.1 Cd(II) tolerance and supplemental sulfate, sulfite and cysteine. ................................................ 40


Chapter 4 The examination of zinc biotransformation by photosynthetic microbes .................................. 43

4.1 Introduction ....................................................................................................................................... 43

4.2 Results ............................................................................................................................................... 44

4.2.1 Zinc resistance ........................................................................................................................... 44

4.2.2 Acid labile sulfide analysis ........................................................................................................ 46


4.2.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity ............................................... 46

4.3 Discussion ......................................................................................................................................... 51

4.3.1 Zinc tolerance ............................................................................................................................. 51

4.3.2 C. reinhardtii and C. merolae sulfur assimilation ...................................................................... 52

Chapter 5 Copper biotransformation and the role of the sulfur assimilation pathway in photoautotrophic

microbes ...................................................................................................................................................... 54

5.1 Introduction ....................................................................................................................................... 54

5.2 Results: .............................................................................................................................................. 55

5.2.1 Copper tolerance and supplemental sulfur nutrition .................................................................. 55



5.2.3 Serine acetyltransferase activity and O-acetylserine(thiol)lyase activity................................... 58

5.3 Discussion ......................................................................................................................................... 62

vi

5.3.1 Cu(II) tolerance .......................................................................................................................... 62

5.3.2 Sulfide production under Cu(II) stress and the sulfur assimilation pathway ............................. 63

Chapter 6 General discussion ...................................................................................................................... 65

6.1 Growth and heavy metal tolerance .................................................................................................... 65

6.2 Heavy metal-sulfides ........................................................................................................................ 68

6.3 Cysteine desulfhydrase ..................................................................................................................... 71

6.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity ...................................................... 73

6.5 Future directions ............................................................................................................................... 74

Summary…………………………………………………………………………………………………..83

References…………………………………………………………………………………………………85

Appendix A: Microorganism quantification and unit conversions ........................................................... 107

Appendix B: Trace metal concentrations present in algal growth media. ................................................ 100

Appendix C: Metal concentrations in media employed for growth analysis ............................................ 101

Appendix D: Cellular counts vs optical density ........................................................................................ 102

Appendix E: Chlorophyll extractions ....................................................................................................... 103

Appendix F: Bradford BSA assay standard curves ................................................................................... 104

Appendix G: Acid labile sulfide analysis standard curve ......................................................................... 105

Appendix H: Cysteine desulfhydrase enzyme activity standard curve ..................................................... 106

Appendix I: Serine acetyltransferase and Serine acetyl-transferase and O-acetylserine(thiol)lyase activity

.................................................................................................................................................................. 107

vii

List of Figures

Figure 1 Flow diagram of sulfate reduction pathway in C. reinhardtii ...................................................... 11

Figure 2 Hypothetical structure of the sulfate permease holocomplex ....................................................... 14

Figure 3 Hypothetical sulfate starvation regulation model in C. reinhardtii. .............................................. 24

Figure 4 Model of bioreactor used for the culture of algae and cyanobacteria. .......................................... 25

Figure 5 Cadmium tolerance growth analyses ............................................................................................ 36

Figure 6 Cadmium induction of sulfide formation. .................................................................................... 37

Figure 7 Effects of cadmium on cysteine desulfhydrase activity ............................................................... 38

Figure 8 Effects of cadmium on serine acetyltransferase and O-acetylserine(thiol)lyase activity ............ 39

Figure 9 Zinc tolerance growth analyses .................................................................................................... 45

Figure 10 Zinc induction of sulfide formation ............................................................................................ 48

Figure 11 Effect of zinc on cysteine desulfhydrase activity ...................................................................... 49

Figure 12 Effect of zinc on serine acetyltransferase/ O-acetylserine(thiol)lyase activity ........................... 50

Figure 13 Copper tolerance growth analyses .............................................................................................. 57

Figure 14 Copper induction of sulfide formation. ...................................................................................... 59

Figure 15 Effect of copper on cysteine desulfhydrase activity ................................................................... 60

Figure 16 The effect of copper on serine acetyltransferase and O-acetylserine(thiol)lyase ...................... 61

viii

List of Tables

Table 1Heavy metal mimicry and membrane transport ................................................................................ 6

Table 2 The concentration of sulfur in the base algae growth media ......................................................... 26

Table 3 Heavy metal concentrations created for enzyme analysis.............................................................. 29

ix

List of Abbreviations

Ascorbate peroxidase…………………………………………………….………….………………(APX)

ATP binding cassette………………………………………………………………………………(ABC)

ATP sulfuralase………………………………………………………..……………………………(APS)

Adenylylsulfate reductase………………………………………………………………..…………..(APR)

Glutathione……………………………………………………………………………..…………....(GSH)

High salt medium…………………………………………………………………………..………..(HSM)

O-acetylserine(thiol)lyase……………………………………………………………....………...(OASTL)

Reactive oxygen species………………………………………………………….….……………....(ROS)

Serine acetyltransferase…………………………………………………………….………..………(SAT)

Sulfate ATP binding cassette………………………………………………………………….……..(sabc)

Sulfate binding protein…………………………………………………..…………………………….(sbc)

Sulfate permease……………………………………………………………………..………………(SulP)

Super oxidase dismutase……………………………………………………………..………………(SOD)

1

Chapter 1

General Introduction

1.1 Introduction

Despite the concern associated with their adverse health effects, heavy metal contamination

continues to increase worldwide to the present level of 4000 times that of 150 years ago (Jarup, 2003).

Living organisms have developed successful strategies to sustain appropriate intracellular metal

concentrations but may also have o woke with excess. Heavy metals are commonly defined as having a

specific density above 5g cm-3

. In this regard, cells are normally challenged with the task of absorbing

sufficient levels of essential metals while having to cope with harmful non-essential metals (Cobbett &

Goldsbrough, 2002). In the event the cell is not capable of performing these tasks, cellular processes

could be severely hindered with cell death a possible end result (Rijstenbil et al., 1994; Stauber &

Florence, 1987). Additionally, even the excessive absorption of essential heavy metals can have harmful

consequences (Stauber & Florence, 1987).

Elevated levels of heavy metals often occur naturally; and anthropogenic sources of heavy metals

have also accumulated because of industrial activities. Sources include industrial effluents, sewage

treatment, mining, refining of fossil fuels, and urban and agricultural runoff. As a consequence,

detrimental effects are now witnessed in a wide assortment of ecosystems (Groudev& Doycheva, 2001;

MacFarlane & Burchett, 2001).

The introduction of organic-metalloids or ionic forms of heavy metals can have dramatic long

term health implications for human populations. Presently, the most common means of exposure are

through inhalation and ingestion of contaminated water sources or food (Liu et al., 2005). The United

States Environmental Protection Agency (EPA) has consequently ranked mercury, cadmium, copper, lead

2

nickel and zinc on their priority list for hazardous pollutants (Mulligan et al., 2001). A comprehensive

review of the medical implications of heavy metal toxicity has been made by Bridges and Zalups (2005).

A wide variety of species have been studied for their heavy metal decontamination potential. One

mechanism, that is highly conserved amongst taxa are genes encoding metallothionein proteins and

peptides that actively bind heavy metals and reduce the metals’ ability to disrupt cellular function

(Daniels et al., 1998; J. Liu et al., 2000; Okubo et al., 2003; Osborna et al., 1997; Palmiter et al., 1982;

Wang & Dei, 2001). Although metallothionein proteins have been extensively investigated in vascular

plants (Kochian et al., 2002; Masi et al., 2002), their ability to be utilized for metal removal from

contaminated sites are limited by associated costs for propagating and harvesting the vegetation.

Prokaryotic, algal and fungal capacities to cope with metal stress have been extensively investigated

because of their relative biological simplicity, ease of culture, and the molecular similarity of their

decontamination mechanisms to mammalian counterparts (Bannon et al., 2002). These mechanisms are

known to decontaminate a variety of metals including, but not limited to Zn(II), Cu(II), Cd(II), Co(II) and

Pb(II). Additionally, prokaryotic species have been identified to contain a variety of operons containing

genes involved in the process of metal resistance. These are capable of regulating the stress response

associated with ameliorating high concentrations of heavy metals. Thus far, the use of the mer operon

(mercury), and other metal resistance operons, such as zntR (zinc), cueR (copper), cadR (cadmium), are

responsible for increasing the exportation of the metals from the Eubacteria, as well as forestalling the

metals from embedding in the cellular membrane (Nascimento et al., 2003; Osborna et al., 1997; Permina

et al., 2006). Additionally, prokaryotes, algae and fungi have also been demonstrated to have the ability

to biotransform heavy metals into metal-sulfides that have a reduced biological availability due to their

insoluble nature (Clemens et al, 1999; Collard & Matagne, 1990; Lefebvre et al. 2007; Scarano &

Morelli, 2003).

3

Vascular plants and aquatic macrophytes have been identified to possess the ability to bind and

detoxify heavy metals; much of the knowledge derived from organisms have been applied to

understanding the heavy metal tolerance mechanisms in algae (Davis et al., 2003; Steffens, 1990). Many

of the genes associated with heavy metal detoxification and metal transport that have been sequenced in

vascular plants have been used to identify potential homologues in aquatic macrophytes and algae

(Clemens et al., 1999; Schäfer et al., 1997; Vatamaniuk et al., 1999). Various algal species have been

isolated as candidates for heavy metal removal due to their ability to hyper-accumulate heavy metals and

their elevated growth rates by comparison with heavy metal tolerant macrophytes (Davis et al., 2003).

With regards to how species detoxify heavy metals, sulfur remains an essential component in

these processes. Sulfur is contained in a variety of cellular components, most notably the amino acids

cysteine and methionine, and plays necessary roles in biochemical processes such as reduction-oxidation

reactions, heavy metal tolerance and secondary metabolites production.

This review will investigate the processes of heavy metal tolerance and bioconversion within

microorganisms with particular emphasis placed on the response to metals and metal transport

mechanisms employed by algae, cyanobacteria, and other photosynthetic microorganisms. Sulfur is

important in these processes because metal-sulfide formation prevents the metals from becoming

biologically available, and limits reactive oxygen production.

1.2 Membrane transport of heavy metals:

One of the most important challenges for cells is selectively importing essential heavy metals, while

excluding the importation of harmful forms. Due to the large number of heavy metal species and

compounds with which they are associated, identifying the mechanism by which they cross the cellular

membrane can be difficult. In Chlamydomonas reinhardtii, there is believed to be a total of 486 possible

4

transporters, of which there are 69-ATP binding cassette (ABC) complexes and 29 P-type ATPases that

are known to transport heavy metals (Hanikenne et al., 2005; Merchant et al., 2007). The large number

of potential transporters in C. reinhardtii rivals those found in higher plants. The large variety of

membrane transporters found in many algal species makes it very difficult to definitively prove which of

these transporters is responsible for the uptake of particularly harmful heavy metals. More so, most metals

can be taken across the membrane through other ion transporters by molecular mimicry, or they may be

brought across the membrane by absorption when they are bound to secondary metabolites. Copper (II)

and cadmium (II) are capable of entering the cell though divalent ion channels, particularly through Ca(II)

channels (Brautigam et al., 2009; Lamai et al., 2005; Lee et al., 2001).

Presently it has been demonstrated that the red alga, Cyanidium caldarium is tolerant of Al(III) within the

50-100 mM range (Yoshimura et al., 2000). Aluminum (III) has been shown to enter cells by utilizing

iron (Fe III) transporters, suggesting that aluminum is a molecular mimic of iron (Guida et al., 1991).

Surprisingly, the concentrations of aluminum within the cell, remains relatively low in relation to the

external concentrations, indicating that C. caldarium contains a mechanism for the exclusion of

aluminum. Furthermore, as the temperature increases beyond the optimum of C. caldarium growth, the

ratio of absorption between Fe and Al shifts towards the importation of Al(III), suggesting a loss in metal

specificity in Fe transporters with increased temperature(Guida et al., 1991).

Heavy metals can also be bound extracellularly with low and high molecular weight thiols, such

as glutathione. These heavy metal thiol compounds are then be transported across the membranes through

thiol transporters or through endocytosis (Gueldry et al., 2003; Li et al., 1997). One metal that has been

heavily studied with this form of transport is silver. Silver has been shown to be transported readily into

the algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata, when it is bound to

thiosulfate and other sulfate compounds (Hiriart-Baer et al., 2006), apparently crossing the plasma

5

membrane in bound form (Fortin & Campbell, 2000). Once inside the cell, the silver thiosulfates tend to

be accumulated and are energically costly for the cell to metabolize. The breakdown of the thiols, causes

the silver to be converted into its ionic form, increasing its toxicity within the cell. Metals bound with

thiols tend to be more stable and less likely to cause oxidative damage, although high levels can have the

similar deleterious effects to their ionic counterparts. Table 1 outlines a list of transport methods by which

heavy metals can gain access to autotrophic eukaryotes, fungi and prokaryotes.

Heavy metals can also bind with high molecular weight thiols, such was class III

metallothioneins, which are brought into the cell through endocytosis (Waldron & Robinson, 2009). The

importation of metallothioneins may present a source of cysteine, and once the metallothioneins are

broken down, heavy metals are transported out of the cell through ATP dependent transporters (Pinto et

al., 2003). In a reverse process, metal chelators, such as class III metallothioneins, can become a means of

metal excretion through exocytosis. However, one of the deleterious effects to the exportation of

metallothioneins is that under lower pH conditions, or high temperatures, they can dissociate putting the

metal back into its ionic form, increasing the extracellular concentration of heavy metals. Additionally,

induction of UV-B damage on the cell has been determined to increase the permeability of the cellular

membrane by damaging the lipid bi-layer or by damaging the nutrient transporters, allowing for metals to

more readily gain access into the cell (Rai et al., 1998).

6

Table 1Heavy metal mimicry and membrane transport

Metal Toxic mimicry Transporters Species

Fe(III)

Fe(II)

Al(III)

Cu(II),Zn(II),

Mn(II),Co(II)

Fe Transporters

Fet4p

Cyanidium caldarium R-11

Escherichia coli

Saccharomyces cerevisiae

(Yoshimura et al., 2000)

(Guida et al., 1991)

(Van Ho et al., 2002)

Fe(II),

Ca(II)

Pb(II) DMT1

(Fe transporter)

Saccharomyces cerevisiae

S. cerevisiae

(Bannon et al., 2002)

PO43 AsO4

3 Pho84 and Pho87 Pi

Transporter

S. cerevisiae

Holcus lanatus

(Bun-ya et al., 1996)

(Meharg & Macnair, 1990)

SO42-

SeO42−

AgS2O3

Cr(IV)

Sulfate permease

ABC Transporter

SulP

SulT (ABC)

Selenastrum capricornutum

Thalassiosira pseudonana

Chlamydomonas reinhardtii

E. coli

(Williams et al., 1994)

(Riedel et al., 1991)

(Fortin & Campbell, 2000)

(Lindberg & Melis, 2008)

(Kertesz, 2001)

Hg(II) Hg(II) MerT & MerP E. coli

(Chen & Wilson, 1997)

Cu(I) Ag(I)

Type1- P-type

ATPases

(monovalent)

YbaR

Arabidopsis thaliana

E. coli

(Hussain, et al., 2004)

(Hanikenne et al., 2005)

(Permina et al., 2006)

Ca(II),Cu(II)

Zn(II),

Co(II),

Cd(II), Pb(II)

Type 1-P-type

ATPases

(Divalent)

CadA transporter

A. thaliana

E. coli

(Hussain, et al., 2004)

(Hanikenne et al., 2005)

(Permina et al., 2006)

Zn(II)

Cu(II)

W(II) ABC Transporters;

TupA, TupB

Eubacterium acidaminophilum (Andreesen & Makdessi, 2008)

(GS)X* Hg(GS)2

Cd(GS)

Ycf1p S. cerevisiae (Gueldry et al., 2003)

(Li et al., 1997)

*glutathione conjugates. Adapted from (Lefebvre & Edwards, 2010)

7

Ideally heavy metals are prevented from entering cells through mimicry or through potential

channels. In several cyanobacterial and algal species, the production of external cellular membrane

polysaccharides have been determined to bind heavy metals to prevent them from gaining cellular access

(Brautigam et al., 2009). Gloeothece sp. strain PCC 6909 can produce negatively charged extracellular

membrane polysaccharides that interact with positively charged heavy metal ions, immobilizing them on

the outside of the cellular membrane (Micheletti et al., 2008; Philippis et al., 2007). One problem with

this approach, is that the metal ions can be easily displaced when the external environment becomes

acidified.

1.3 Toxicity of heavy metals

The different ionic forms of heavy metals can have variable results on cells. Although there are

several understood means of heavy metal entry, it is still unclear how each cell will react to different

concentrations of heavy metals once they have gained entry. Heavy metals have been demonstrated to

cause acidification of the cytoplasm, disruption of homeostasis, and depolarization of plasma and

organelle membranes (Conner & Schmid, 2003; Waldron & Robinson, 2009). During depolarization, ion

gradients are disrupted, which are required for the proper function of organelles and the plasma

membrane.

Additionally, oxidative stress can be increased with the increasing concentrations of heavy metals

because of their role in the creation of reactive oxygen species, while also suppressing the antioxidation

mechanism (Sies, 1990). There is a direct correlation between the abundance of heavy metals within the

cell in relation with the increase in reaction oxygen species (Winterbourn, 1982). An example of this

occurs in sunflowers, where Fe(II) and Cu(II) in abundance, within the cytosol, causes a decrease in the

activity of antioxidizing enzymes (Gallego et al., 1996). Exposure to Cu(II) and Cd(II) in sub-lethal

concentrations caused a decrease in growth by 60% suggesting an increase in oxidative stress in relation

8

to heavy metals (Collan et al., 2003). Furthermore, in Gracilaria tenuistipitata, copper tends to be

responsible for causing more oxidative stress due to its disruption of catalase, ascorbate peroxidase, and

superoxidase dismutase which are important antioxidating enzymes.

Interesting, cellular maintenance requires essential heavy metals to be integrated into the

antioxidative enzymes used to dispose of reactive oxygen species. This causes a paradox to exist in that

essential heavy metals utilized in the cellular process of removal of oxidative damage may also be

responsible for their promotion (Stauber & Florence, 1987). The presence of copper, zinc or iron in

abundance can cause the production of reactive oxygen species while simultaneously these metals are

cofactors in Cu-, Zn-, and Fe-superoxide dismutases (Pinto et al., 2003). Antioxidant production has

been thoroughly studied in vascular plants (Foyer et al., 2002), the process of antioxidant synthesis at the

molecular level has yet to be investigated to the same extent in photosynthetic microbes (Holovská et al.,

1996; Okamoto et al., 1996). Some species of algae may react to heavy metal stress by inducing the

production of proline, which has been determined in higher plants to be an active response to damage to

membranes and proteins. It is hypothesized that the production of proline reduces reactive oxygen species

through chemically reacting with the hydroxyl radicals and through physical quenching of the free

radicals (Alia et al., 2001; Siripornadulsil et al., 2002)

Presently, mitochondrial and chloroplast function may be disrupted by the presence of heavy metals

in elevated concentrations (Okamoto et al., 2001). The intense electron fluxes within the cellular

compartments of algae elevate oxygen and metal ion concentrations, putting mitochondrial and

chloroplast enzymes at an increased risk of oxidative damage (Pinto et al., 2003). Voigt and colleagues

(2002) have determined that cadmium accumulated in high concentrations in the chloroplasts and

mitochondria in comparison to other cellular organelles (Voigt & Nagel, 2002). Due to the sensitivity of

chloroplasts and mitochondria, the accumulation of cadmium and the increased production of reactive

9

oxygen species can lead to partial or total disruption of their function. Cadmium has been shown to be

particularly damaging to the chloroplast. Cadmium concentration in the chloroplast causes not only the

increase in the synthesis of reactive oxygen species but also disrupt the photosynthetic pigments (Lamai

et al., 2005). Additionally, Pb(II) is capable of binding to the superstructure of the thylakoids and altering

their function and eventually causing total disruption of photosystem II (Heng et al., 2004).

Algal photosynthetic and metabolic activity as well as the overall electrical gradient of the cell can be

severely impeded by oxidative damage. Furthermore, an increase in the abundance of light can further

promote the presence of reactive oxygen species within the cell by causing a proportional increase in the

number of excited molecules, such as triplet state chlorophyll and singlet state oxygen (Arunakumara &

Zhang, 2008). Singlet state oxygen is capable of causing strong oxidation of other molecules in which

they form an interaction (Pinto et al., 2003).

Heavy metals present in substantial concentrations extracellularly and intracellularly have the

potential to inhibit nutrient uptake as well as metabolic activity. The presence of Pb(II) and Cu(II) and

some metalloids (particularly AsO4) at toxic concentrations in cyanobacteria and green algae acts to

competitively inhibit the uptake of NH4+, urea, and PO4

3- into the cell (Rai et al., 1998). Surprisingly,

extracellular concentrations of Zn(II) are thought to cause more damage to the microalgae function by

inhibiting the uptake of Ca(II), which is essential for cellular signaling (Takahashi et al., 1997) , and vital

for Ca-ATPase activity which is required for cellular division (Stauber & Florence, 1990).

1.4 Uptake and assimilation of sulfate

Sulfur it an essential component in a variety of amino acids, particularly cysteine and methionine, and

integral for the proper scaffolding of many proteins. Inorganic sulfate is taken up from the environment

by organisms in the form as a readily available and stable form of sulfur. Inorganic sulfate is actively

transported into the cells by active transport systems in: bacteria (Kertesz, 2001), algae (Melis & Chen,

10

2005; Merchant et al., 2007; Pollock et al., 2005), yeast (Smith et al., 1995), and higher plants

(Hawkesford and De Kok, 2006; Takahashi et al., 1997). Upon transporting the sulfate into algal cells, it

is then immediately sequestered into the chloroplast where it is synthesized into cysteine, or it is

sequestered into the vacuole where it is stored.

The regulation of the sulfate assimilation pathway has been identified to be associated with three

genes in the green alga Chlamydomonas reinhardtii: sac1, sac2, and sac3 (Leustek et al., 2000a). The

sac1 gene encodes an integral protein that has conserved homology with a dicarboxylate transporter

(Davies et al., 1996). sac1 may regulate the sulfur concentration within the cell, and be involved in

activating the sulfate assimilation pathway (Figure 1).

In higher plants and algae, sulfate is first translocated to the chloroplast, while in cyanobacteria this

process is in the cytoplasm (Melis & Chen, 2005; Ravina et al., 2002). For sulfate reduction to occur in

algae and cyanobacteria, it is first converted to adenylylsulfate by ATP sulfurylase (APS).

Adenylylsulfate is then reduced further by APS reductase to produce sulfite. Sulfite reductase then

reduces sulfite into sulfide. This sulfide is then immediately transferred into cysteine by the SAT-OASTL

bi-enzymatic complex that covalently binds the sulfide to serine to produce cysteine (Giordano et al.,

2005; Saito, 2000; Takahashi et al., 1997).

11

Cysteine serves as a cellular pool for reduced sulfur within cells (Giordano et al., 2005) to be

employed in the formation of thiol containing compounds such as glutathione and, along with methionine,

in protein synthesis. A group of proteins and peptides that contain high thiol contents from cysteine are

the metallothioneins involved in metal binding.

Figure 1 Flow diagram of sulfate reduction pathway in C. reinhardtii. Adapted from Melis et al.,

(2005) and Lefebvre & Edwards (2010). Abbreviations: adenylylsulfate reductase (APS reductase),

sulfate permease (SulP).

12

1.4.1 Sulfate transporters:

Present analysis suggests that C. reinhardtii has 486 predicted membrane transporters, which

have high sequence homology as well as the diversity of transporters that is common in vascular plants

(Merchant et al., 2007). Of these transporters 61 ion channels, 124 primary ATP dependent transporters,

293 secondary transporters, have been genetically identified. Eight recognized genetic homologues to

sulfate transporters in C. reinhardtii have been reported but the protein complexes have yet to be purified.

Of eight identified sulfate transporters in C. reinhardtii four are thought to be in the H+/

SO42-

family,

which is characteristic of vascular plants, one is an ABC-type SO42-

similar to bacterial sulfate

transporters, and three are in the Na+/SO4

2- family found in unicellular eukaryotes (Merchant et al., 2007).

Sulfur is an essential element for cell function and plant growth. As a result, photosynthetic

organisms need an effective means of transport for sulfate (Davies et al., 1996; Ha et al., 1999; Smith et

al., 1995). Sultr1, Sultr2, and Sultr3 have several strongly conserved domains shared with the Arabidopsis

sulfate co-transporter ATPases while another gene sequence sulp is very similar the cysT gene found in

cyanobacteria (Irihimovitch & Yehudai-Resheff, 2008; Lindberg & Melis, 2008; Melis & Chen, 2005;

Pollock et al., 2005). Sultr1, sultr2, and sultr3 have been isolated from Arabidopsis and transformed into

yeast lacking sulfate transporters where they were able to rescue their growth. This suggests that the

protein encoded by the cDNA was responsible for the transport of sulfate (Kataoka et al., 2004;

Takahashi et al., 1997). Furthermore, it is suggested that Sultr1, Sultr2, and Sultr3 have variable levels of

affinity for sulfate (Km, 3.6 µM; Km, 0.41 mM,; Km>1.2 mM, respectively).

Sultr1, Sultr2, and Sultr3 are thought to coordinate and form an ABC type transporter responsible

for the absorption of sulfate into the cytosol. From the cytosol, the inorganic sulfate is then transferred

directly into the vacuole or the chloroplast by passing through a SulP holocomplex that transfers the

13

sulfate into these organelles (Figure 2). However, the transport mechanism of the sulfate past the plasma

membrane has yet to be identified in algal species.

SulP in C. reinhardtii is localized to the chloroplast envelope and may be, as its sequence analysis

suggests, responsible for high affinity transport of SO42-

into the chloroplast and vacuole (Lindberg &

Melis, 2008; Melis & Chen, 2005). Recently the identification of another sulfate permease, sulP2 has

been recognized in C. reinhardtii to be a transmembrane protein that may form a heterodimer complex

with SulP, thereby making a transporter complex (Figure 2). Putative membrane folding models for SulP2

suggest that the six transmembrane helices form an inverted structure that corresponds with SulP folding

analysis. Together, SulP and SulP2 are thought to form a holocomplex with a variety of ABC-type

transporter subunits forming a chloroplast pore (Chen et al., 2005; Lindberg & Melis, 2008).

Two other gene products that have been associated with SulP and SulP2 in C. reinhardtii, have

sequence homology with ABC transporter subunit proteins previously isolated in the cyanobacteria S.

leopoliensis (Laudenbach & Grossman, 1991) and the red alga Cyanidioschyzon merolae (Matsuzaki et

al., 2004). These genes are sbp and sabc (Chen et al., 2003; Lindberg & Melis, 2008; Melis & Chen,

2005). Sabc possesses sequence similarities with ATP dependent proteins and it has been suggested that it

is hydrolyzed ATP to mitigate sulfate migration through the SulP-SulP2 pore (Lindberg & Melis, 2008;

White & Melis, 1995). Sbp is thought to be a sulfate binding protein that helps sulfate to enter the SulP1

and SulP2 holocomplex. Western blot analysis has revealed the protein complex of Sbp, SulP1, SulP2 and

Sabc to be 380 kDa in size.

14

Figure 2 Hypothetical structure of the sulfate permease holocomplex fixed in the chloroplast envelope.

SulP and SulP are hypothesized to form a heterodimer complex. Vertical arrow indicated direction of

sulfate transport. Modified from (Melis & Chen, 2005). Abbreviations: sulfate binding protein (sbp),

sulfate permease (SulP), sulfate ATP binding casette (Sabc).

Oddly, it seems that the location of the sulfate transporters genes sulP and sulP2, are not

consistently present in either the nuclear or chloroplast genome. Each species appears to have

transcriptional sequences for sulP, sulP2, sbp, and sabc in either the chloroplast or nuclear genomes, with

some species having the transcript in either location without a distinct pattern (Matsuzaki et al., 2004). An

example of this is C. merolae, in which the sulP and sulP2 are encoded in the chloroplast genome, while

the sbp and sabc are encoded in the nuclear genome (de Koning & Keeling, 2004; de Koning & Keeling,

2006). This further adds to the difficult nature of determining the genes responsible for the production of

sulfate transporters.

1.4.2 Gene regulation during metal and sulfate starvation

In terms of stress response, either through sulfate starvation or sublethal levels of heavy metals

can potentially cause the up-regulation of genes within the pathway. During sulfate starvation there is a

15

rapid response by the cell causing an increase in the mRNA transcript levels of Sabc and Sbc, which are

key components of sulfate transport into chloroplasts(Lindberg & Melis, 2008), these increase the affinity

of the sulfate permease (SulP) ABC transporter complex to sulfate, driving cytosolic and vacuole stored

sulfate into the chloroplast. Interestingly, the gene response in terms of starvation for sulfate, also causes

an up regulation in protein response for phosphurus. Pho4p, the phosphorus deprivation responsive

transcription factor, can actively substitute for the sequence specific DNA-binding protein Cp1p, which is

required for the activation of methionine biosynthetic genes (Pollock et al., 2005). During stress response

to limitation of phosphorus and sulfur, there is evidence that similar stress response genes are activated

leading to the up-regulating of some of the same chaperone and protease proteins that help the cell. This

is thought to be a secondary response or even potentially a cross regulatory measure between the S and P

assimilation pathways (Pollock et al., 2005).

Figure 3 Hypothetical sulfate starvation regulation model in C. reinhardtii.

Modified from the model proposed by Saito (2000) in Arabidopsis.

16

In C. reinhardtii, there is an increase in the sulfur assimilation pathway activation in relation to

exposure to cadmium. Cd(II) caused there to be an increase in SAT and O-acetylserine(thiol)lyase activity

by 110% within a 24 hour period (Domínguez et al., 2003). Furthermore, O-acetylserine(thiol)lyase

mRNA transcripts have been shown to dramatically increase in A. thaliana in relation to the presence of

heavy metals, suggesting that it is essential in the pathway for cysteine production (Barroso et al., 1999).

This increase may be directly related to the demand for the bioconversion of metals to metal-sulfides, the

increased production of cysteine-rich phytochelatins needed to detoxify the cadmium.

Domínguez et al. (2003), determined that C. reinhardtii exposure to Cd(II) showed significant

increases in glutamate dehydrogenase activity as well as NAD+ and NADP

+-isocitrate dehydrogenases.

Particularly, there was a 75% increase in the activity of glutamate dehydrogenase in relation to the

controls that is consistent with findings from maize (Gouia et al., 2000) and tobacco (Restivo, 2004). The

process of glutamate production however, required the nitrogen assimilation pathway to be functional in

order for ammonium to be incorporated into 2-oxoglutarate. It is proposed that this may be another

potential pool of hydrogen sulfide for metal-sulfide formation. Furthermore, the glutamate has been

identified as an important precursor to in phytochelatin within higher plants and microalgae (Rauser,

1999).

1.5 Metallothioneins

Metallothioneins are peptides and relatively small proteins, rich in cysteine and glycine amino acid

residues, that are produced when metals such as zinc, copper and cadmium are in excess. Metal-sulfide

formation may be facilitated by metallothioneins (Rauser, 1999; Scarano & Morelli, 2003).

A strong cellular response may be activated when eukaryotes are exposed to metals, inducing the

synthesis of class II metallothioneins (MtII). Mt(II)s have been identified to occur in cyanobacteria, algae

17

and higher plants while class III metallothioneins (MtIII) are found mostly algal species, higher plants and

fungi. Class III metallothioneins are also commonly referred to as phytochelatins (Perales-Vela et al.,

2006). The classes of these metal binders differ in their positions and numbers of cysteines and by how

they are produced. Class I and II Mts are encoded by their respective genes whereas class III Mts are

enzymatically synthesized. There will be particular emphasis on Class II and III metallothioneins,

1.5.1 Class II metallothioneins

The regulatory response to how organisms employ class II metallothioneins has yet to be fully

elucidated. The age of the organism, enzyme sensitivities to specific metals, the essential or non-essential

nature of the metals can all play a factor in which MtIIs are synthesized (Schäfer et al., 1997). MtII are

thought to be induced by specific metals. Zinc ions are preferentially bound by MtIIs (Cavet et al., 2003).

The cyanobacterium, Synechococcus PCC 7942, synthesizes a 56 amino acid cysteine-rich

protein, SmtA. SmtA is strongly activated during exposure to cadmium, copper, and zinc (Cavet et al.,

2003). Similar cysteine-rich proteins have been determined to be induced by metals in the algae Chlorella

and Euglena (Gekeler, et al., 1988). In Synechococcus, Zn(II) is known to maximally induce the

transcription smtA while copper and cadmium produce less strong gene responses (Cavet et al., 2003).

SmtB has also been expressed, which is a strong trans-acting repressor of smtA (Cavet et al., 2003; Turner

et al., 1993). Mutants that lack functional smtA exhibit a several fold decrease in zinc tolerance (Turner et

al., 1993). Other prokaryotic species (Blindauer et al., 2003) as well as algae (Gekeler, et al., 1988) have

similar gene sequences to smtA.

1.5.2 Class III metallothioneins

Several algal species have adapted to live in heavy metal rich environments. Ten divisions and 24

genera of algae possess MtIII complexes as their primary means of heavy metal stabilization (Perales-

Vela et al., 2006). MtIII production appears to play a major role in the adaptive ability of these species to

18

cope with the heavy metals. MtIII synthesis appears to be ubiquitous to algae and is preferentially

induced by high concentrations of Cd(II) and Cu(II) (Gekeler et al., 1988). These metallothioneins, also

known as phytochelatins, are enzymatically synthesized and composed of short chain polypeptides rich in

cysteine. The most potent activator of phytochelatin production is Cd(II), followed by Pb(II), Zn(II), and

Cu(II) (Perales-Vela et al., 2006). The promotion of phytochelatin production has been linked to the

presence of extracellular metals and metalloids (Steffens, 1990b).

The gamma bond present between glutamate and cysteine in phytochelatins cannot be formed during

protein translation, and thus needs to be facilitated enzymatically. The gamma bond is made by

phytochelatin synthase, an amylcysteine dipeptidyl transpeptidase (Grill et al., 1989; Vatamaniuk et al.,

2004). This enzyme has the general mechanism of [γGlu-Cys]n-Gly→ [γGlu-Cys]n+1-Gly +Gly 136

(Cobbett & Goldsbrough, 2002; Gekeler, et al., 1988). Respective genes for phytochelatin synthase have

been isolated from yeast, higher plants, and algae (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et

al., 1999). Thus far, however, regulatory mechanisms governing the induction of phytochelatins remain

to be fully understood. Mutants of Arabidopsis thaliana lacking the ability to enzymatically synthesize

phytochelatins show increased sensitivity to Cd(II) (Howden et al., 1995). Mt(III) complexes may play

an essential role in stabilizing heavy metals.

1.5.3 Labile and non-labile phases of metals

Cd(II), Pb(II), Zn(II), Cu(II) and Co(II) within cells are known to form labile and non-labile phases

(Lee et al., 1996). Labile Cd(II), bound to phytochelatins, is capable of being mobilized and exported.

Non-labile phase metals that are bound to cytoplasmic components are relatively unavailable for export.

In the marine diatom, Thalassiosira weissflogii, the efflux of phytochelatins results in a physiological

removal of Cd(II) from the cells ( Lee et al., 1996). Heavy metals may have variable times for each metal

to occupy labile and non-labile in different species. In Synechococcus, there is a delay prior to growth in

19

media supplemented with Cd(II), indicating that the cells are amplifying metallothionein genes in

response to the stress (Cavet et al., 2003; Turner et al., 1993).

Cytosolic fractions taken from species of cyanobacteria and algae after exposure to Cd(II) and Zn(II)

show 30% of these metals bound with metallothioneins (Bierkens et al., 1998; El-Enany & Issa, 2000;

Torres et al., 1997). Class III metallothioneins can also exist as low and high molecular weight variants.

In low molecular weight forms the metal is bound to thiol groups, whereas in the high molecular weight

forms, additional inorganic sulfur is incorporated into the MtIII complexes. Scarano and Morelli (2003)

determined that this sulfur forms in the range of nanometres in diameter. The addition of sulfur to MtIII

complexes appears to stabilize and improve their detoxification capabilities.

1.5.4 Sequestration and compartmentalization of phytochelatins

Microalgal species have been known to sequester metal-MtIII complexes into their vacuoles

(Heuillet et al., 1986; Ortiz et al., 1995). In green algae heavy metals have also been shown to accumulate

in the cell wall and within organelles, with precipitation of Cd(II), Ca(II) and S(II) being detected in the

vacuole (Ballan-Dufrançais et al., 1991). A variety of other species have been characterized to precipitate

Cd(II), Cu(II), Hg(II) and Cr(II) on the cell membrane (El-Enany & Issa, 2000; Hammouda et al., 1995;

Nassiri et al., 1997; Turner et al., 1993). The accumulation of metals can also occur in the mitochondria

and chloroplasts of species devoid of vacuoles (Mendoza-Cozatl et al., 2004; Nagel et al., 1996).

There are three possibilities for the partitioning of heavy metals metallothioneins into organelles,

particularly the mitochondria and chloroplast. Firstly, the MtIIIs may be synthesized in the cytosol where

they bind to heavy metals, then imported into an organelle for storage. Secondly, MtIIIs may be

synthesized inside the organelle, where they then sequester metals, forming heavy molecular weight

complexes with inorganic sulfur. Thirdly, both of these pathways may co-exist and MtIII may be

synthesized in the cytosol as well as in organelles. In Chlamydomonas reinhardtii, 60% of Cd(II) was

20

found within the chloroplast (Nagel et al., 1996). It is worth noting, that the chloroplast in C. reinhardtii

is a predominant structure in the cell.

1.5.5 Cellular exportation of phytochelatins

Phytochelatin-metal complexes can be exported from the cell via exocytosis. However, the

exportation of these complexes do not appear to remain stable once outside the cell. Cd(II) and Pb(II)

MtIII complexes readily disassociate to their free ionic forms in the media (Lee et al., 1996; Pawlik-

Skowronska, 2000). Although heavy metal MtIII complexes are adequate to detoxify and sequester heavy

metals intracellularly, these may not be ideal for bioremediation purposes. MtIII metal complexes can be

exported and dissociate when cytosolic heavy metal concentrations become elevated or if the media is

low in pH (Lee et al., 1996; Pawlik-Skowronska, 2000). Unfortunately, under these conditions the metal

ions become once again biologically available.

1.5.6 Anaerobic metal-sulfide production

Sulfate reducing bacteria possess the ability to form hydrogen sulfide in turn, can act to precipitate

metal ions into insoluble metal-sulfides (Groudeva et al., 2001). Anaerobic bacteria can form sulfides

with Fe(III), U(VI), Cr(VI), Te(VII), Mo(VI) and Pd(II) (Lloyd et al., 2001). Sulfur reducing bacteria

have already been incorporated into bioremediation efforts using up-flow anaerobic packed bed reactors

and other industrial decontamination procedures (Craggs et al., 1996; Jong & Parry, 2003). These

procedures utilize the metal-sulfide synthesis of sulfur reducing bacteria to detoxify heavy metal

contaminated waste. Unfortunately, sulfur reducing bacteria efficiency of metal-sulfide production is

inhibited as the concentration of metal-sulfides increase. Metal-sulfides sterically hinder sulfur

metabolism by preventing sulfate and organic compounds from coming into contact with relevant

enzymes (Utgikar et al., 2002).

21

One drawback to the use of these bacteria in bioremediation is that they have a low tolerance for free

metals, such as Cd(II), Zn(II) or Ni(II) (Valls & de Lorenzo, 2002); heavy metal concentrations as low as

20 µM can be toxic to most species of sulfur reducing bacteria. A drawback to bioremediation

applications using sulfur reducing bacteria is that they require anoxic environments in order to function

properly. Maintaining these conditions in an open system can be problematic.

1.5.7 Aerobic metal-sulfide biotransformation

Metal-sulfide biotransformation has been characterized for mercury in algae, cyanobacteria, and fungi

(Kelly et al., 2006; Lefebvre et al., 2007). The spread of mercury has resulted from industrial processes.

The industrial activity releases Hg(0) which can eventually accumulate in watershed ecosystems. The

widespread assumption that Hg(II) is bound to thiol chelates such as the metallothioneins does not appear

to be universal. Several algae, cyanobacteria and fungi can synthesize mercury sulfide, while releasing a

negligible amount of Hg(0), in response to Hg(II) exposure (Kelly et al., 2006; Kelly et al., 2007a;

Lefebvre et al., 2007). However, the pathway for mercury sulfide conversion remains to be elucidated

(Kelly et al., 2006).

Under pH stable conditions, the cyanobacterial species Limnothrix planctonica, Synechococcus

leopoliensis and Phormidium limnetica biotransformed Hg(II) into β-HgS while producing limited

concentrations of volatile Hg(0) (Lefebvre et al., 2007). Importantly, highly toxic methyl-mercury was

not produced in these species under these conditions.

Similar mercury sulfide biotransformation has been revealed for eukaryotic algae. Selenastrum

minutum, Chlorella fusca var. fusca, Galdieria sulphuraria and Navicula pellicosa were capable of

biotransforming mercury into meta-cinnabar, however the rates at which the transformations occurred

was dramatically different among the species (Kelly et al., 2006). G. sulphuraria completed the

22

transformation within a matter of minutes while S. minutum, C. fusca and N. pellicosa took hours detoxify

the mercury (Kelly, et al., 2007a).

G. sulphuraria was capable of transforming 100 ppb Hg(II) into 90% into β-HgS within 20 minutes

(Kelly et al., 2007b). G. sulphuraria is adapted to flourishing in volcanic and low acidity watersheds

around the world, which may account for the species ability to biotransform mercury at such a rapid rate

(Gross & Oesterhelt, 1999; Gross et al., 1998). Volcanic activity can release high amounts of mercury

(Gross et al., 1998), and extremophiles such as G. sulphuraria would thus have to be capable of tolerating

high levels of Hg(II). G. sulphuraria’s coping mechanisms for the low pH associated with high metal

concentrations has been linked to type III ascorbate peroxidases (APX), that buffer the extra cellular

matrix pH, by using hydrogen peroxide as an electron acceptor (Oesterhelt et al., 2008). De Gara et al.

(2004) determined that class III ascorbate peroxidase activity is associated with cellular wall modification

which may act as a biotic defence against pathogens and pH fluctuations. Ascorbate peroxidase is thought

to limit lipid peroxidation by detoxifying reactive oxygen species that can cause cell membrane damage

(Hirooka et al., 2009).

The aerobic production of metal-sulfides is thought to occur by two phases (Kelly et al., 2006). When

first exposed to the metal ions, metal-sulfide formation occurs rapidly, this is known as the rapid phase.

The rapid phase is dependent upon a readily available intercellular pool of sulfur present within the cell

(Kelly et al., 2007a). Following this rapid phase, the production of metal-sulfides slows considerably; the

rate may be limited by the speed at which the pool can be synthesized by the organism. It can be

speculated that the sulfur in the metal-sulfide is facilitated by the formation of high molecular weight

phytochelatins (Scarano & Morelli, 2003).

23

1.6 Objectives

Aerobic biotransformation has yet to be fully utilized as a method for bioremediation of heavy

metal contaminated sites. Further research is to be proposed complimenting the work previously

spearheaded by Kelly et al. (2006) and Lefebvre et al. (2007). Cadmium, copper and zinc, have been

chosen as toxicants due to their high toxicity and relative abundance (Gadd, 2004).

Thus far the pathway has not been determined for sulfur used in metal-sulfide synthesis nor has

there been an investigation into the role of thiols in the process of biotransformation. Kelly et al. (2006)

proposed that the synthesis of metal-sulfides follows two phases: a rapid phase, where the algae

synthesize metal-sulfides using a pool of sulfur, possibly from the cysteine pool, and the slow phase,

where the cell has depleted its cysteine reserves and the rate of metal-sulfide synthesis is dependent upon

the rate of de novo cysteine formation.

Sulfur from thiols are suspected to be transferred from cysteine, and thus the ability to synthesize

metal-sulfides could be dependent upon the thiol pool present within the cell. We seek to provide

evidence that the abundance of sulfate, sulfite or cysteine, provided to the cells will enhance the

biotransformation rate for Chlaymdomonas reinhardtii, Synechoccocus leopoliensis and C. merolae; i.e.

increase the efficiency in the synthesis of metal-sulfides. By understanding the phases and rates of

biotransformatiom, critical steps in the relevant biosynthetic pathway could be identified.

Serine acetyltransferase (SAT), O-acetylserine (thiol) lyase (OASTL) and cysteine desulfhydrase

are thought to play primary roles in heavy metal tolerance (Nozaki et al., 2001; Wang et al., 2000). By

determining the enzyme activity in response to sulfate, sulfite, and cysteine supplementation as well as

under cadmium, copper and zinc stress, their role in the sulfur assimilation may be elucidated.

24

Chapter 2

Materials and Methods

2.1 Culture sources and growth conditions

The eukaryotic alga Chlamydomonas reinhardtii (UTEX 90) was obtained from the Culture

Collection of Algae, University of Texas at Austin. Cultures were grown in liquid High Salt Medium

(HSM) (Sueoka, 1960) composed of 10.6 mM KH2PO4, 9.5 mM NH4Cl, 4.25 mM K2HPO4, 2.02 mM

MgSO4∙7H2O, 0.09 mM CaCl2, 6 µM FeCl3∙6H2O, 3 µM H3BO3, 2.1 µM MnCl2∙4H2O, 0.025 µM ZnCl2,

1 µM Na2EDTA∙2H2O, 0.3 µM NaMoO4∙2H2O, 0.11 µM CoCl2∙6H2O, 0.07 nM CuCl2∙2H2O in double

deionized water.

Synechococcus leopoliensis (UTEX 2434), a cyanobacteria species, was obtained from the

Culture Collection of Algae, University of Texas at Austin. Cells were grown in 50x Cyanobacteria BG-

11 Freshwater Solution (Sigma Aldrich, catalogue # C3061) (Rippka et al., 1974). The BG-11 fresh

water solution was diluted in double deionized water to the concentrations of: 353 µM NaNO3, 6.1 µM

MgSO4∙7H2O, 4.9 µM CaCl2∙2H2O, 4.6 µM K2HPO4, 0.9 µM H3BO3, 624.6 nM citric acid, 428.5 nM

ferric ammonium citrate, 183 nM MnCl2∙4H2O, 55.8 nM EDTA disodium magnesium, 35.6 nM

NaMoO4∙2H2O, 15.4 nM ZnSO4∙7H2O, 6.3 nM CuSO4∙5H2O, 3.4 nM Co(NO3)2∙6H2O.

Cyanidioschyzon merolae 10D was acquired from the Microbial Culture Collection of the

National Institute for Environmental Studies (Tsukuba, Japan). C. merolae cultures were plated and

grown in a Cyanidium medium (Allen, 1959). Cyanidium medium is composed of 10 µM (NH4)2SO4, 2

µM K2HPO4, 1 µM MgSO4∙7H2O, 0.5 µM CaCl2, 7.16 nM Fe-Na-EDTA∙3H2O, 4.67 nM H3BO3, 0.949

nM MnCl2 4H2O, 0.105 nM (NH4)6Mo7O24 4H2O, 0.0765 nM ZnSO4 7H2O, 0.316 nM CuSO4 5H2O, in

double deionized water. The medium was adjusted to pH 3.5 with HCl.

25

All chemicals were obtained from Sigma-Aldrich (Oakville, CA) or Fisher Scientific (Ottawa,

Canada).

S. leopoliensis, and C. reinhardtii were grown in 1.5 L pyrex glass bioreactors (Figure 4) under

fluorescent lighting of 150 µEinsteins/m2/s at 28°C for 18 hour photoperiods. Cells were kept suspended

by aerating at a 1L per min flow rate. C. merolae was grown similarly except that the temperature was

maintained at 45°C. (Gross et al., 1998).

2.2 Sulfate, sulfite, and L-cysteine treatments

Supplemental sulfate and sulfite were added to create a ten times increase in the level of sulfur

from the original media. All L-cysteine treatments were supplemented with 2x the amount of sulfur, due

Figure 4 Model of scale bioreactor used for the culture of S. leopoliensis, C. reinhardtii, and C.

merolae.

26

to the toxicity of L-cysteine at higher concentrations. Stock concentrations for sulfate, sulfite and cysteine

were composed as follows:

(i) Sulfate. K2SO4 (Fisher Scientific) was composed of 48.374 grams per liter and

suspended in deionized water to give a stock concentration of 277.53 mM.

(ii) Sulfite. K2SO3 (Sigma Aldrich) was composed of 43.932 grams per liter and

suspended in deionized water to give a stock concentration of 277.53 mM.

(iii) Cysteine. L-cysteine (Sigma Aldrich) was composed of 33.63 grams per litre in

deionized water to give a stock concentration of 277.53 mM.

Table 2 The concentration of sulfur in the base algae growth media

Medium Sulfur (g/L)

BG-11 0.010085813

High Salt 0.002596665

Allen's 0.314817618

2.3 Supplemental treatment groups

Each species was exposed to a variety of supplemental treatments to determine the effect of sulfur

nutrition on heavy metal resistance and biotransformation. The treatment groups are as follows:

(i) Control: The cultures were grown in unmodified algal growth media.

(ii) 10X sulfate: Cultures were supplemented with K2SO4 to yield a ten times increase in

the amount of sulfur at the time of metal exposure.

(iii) 10X sulfite: Cultures were grown in media supplemented with K2SO3 to ten times

normal sulfur content at the time of metal exposure.

27

(iv) 2X L-cysteine: Cultures were supplemented with L-cysteine at the time of metal

exposure to yield a 2 fold increase in sulfur in the media.

Pretreated cultures were grown in bioreactors under supplemented conditions for 240 hrs prior to

exposure to heavy metals. All treatments were performed in 100 mL of media in glass cell culture jars

with translucent magenta caps. Illumination was 300µEinsteins/m2/s with 120 rpm rotary shaking.

Temperatures were 27°C for S. leopoliensis and C. reinhardtii and 45°C for C.merolae. All cultures

started at a cell density of O.D.665 equal to 0.1. They were given the following treatments with metals:

(v) 10X pretreated and supplemented with sulfate: Cultures were resuspended in fresh

10x sulfate media at the time of heavy metal exposure.

(vi) 10X pretreated and supplemented with sulfite: Cultures were diluted and

resuspended in fresh 10X sulfite media at the time of heavy metal exposure.

(vii) 2X pretreated and supplemented with L-cysteine: Cultures were then diluted and

resuspended in fresh 2X L-cysteine media at the time of heavy metal exposure.

2.4 Heavy metal treatments:

The cells were exposed to divalent metal ions added to the media as CdCl2, CuCl2 or ZnCl2.

Stocks containing 5 g/L were stored at 4°C until used.

2.5 Metal toxicity

Cell cultures were grown for 240 hrs supplemented with 10x sulfate, sulfite and 2X L-cysteine

media, from a cell density of O.D.665 equals 0.1 to a late log phase of growth (O.D.665 ≈ 1.0). Aliquots

from the cultures were then diluted in fresh media back to an optical density of 0.1 under sterile

conditions with their respective media. One hundred mL cell cultures from each of the trial groups

28

(section 3.3) were transferred into 150 mL glass cell culture jars, to which either Cd(II), Zn(II) or Cu(II)

was added from metal chloride stock solutions. These culture jars were then vigorously swirled using an

orbital shaker (VWR) at 120 rpm for 1 min under constant light, before 200 µL aliquots from each jar

were distributed into the wells of sterile 96 well spectrophotometer plates (Costar 9017). The 96 well

plates were incubated under (300 𝜇𝐸−1 𝑚2) at a photo period of 18 hrs. The temperatures were as

described for the bioreactors. Cultures were continuously shaken on an orbital shaker (VWR) at 120rpm.

Cellular growth was measured three times daily for 10 days using a Spectra Max Plus Spectrophotometer

(Molecular Devices, Sunnyvale, CA).

2.6 Bradford protein assays

Bradford assays were determined by following the protein microplate bioassay procedure

supplied by Bio-Rad (Mississauga, Canada). Protein Assay Dye Reagent concentrate was diluted 5 times

in distilled water. Samples were homogenized using a BulletBlender (Next Advance, Averill Park, NY)

for 5 minutes on its maximum speed. The homogenized cells were then transferred into fresh 1.5 mL

microcentrifuge tubes and centrifuged at 1000 g for 5 min to pellet. Samples were taken from the

supernatant. Aliquots of 80 µL of sample were taken from each of the cell lysates and diluted with 720 µL

of distilled water. To this 200 µL of dye reagent was added to each tube, vortexed and the samples

incubated at room temperature for 5 minutes. A 200 µL aliquot was then transferred into a 96 well

spectrophotometer plate (Costar 9017), and then read at 595nm in a Spectra Max Plus Spectrophotometer.

2.7 Enzyme assay sample preparation

One hundred mL aliquots of the cell culture were transferred into 100 mL plant cell culture jars

(Sigma Aldrich). Metal treatments for use in the enzyme analysis experiments were determined from the

growth and resistance experiments as outlined in Chapter 3, 4 and 5. Sublethal concentrations were used

to measure the enzyme activity across all supplemental nutrition samples.

29

Table 3 Heavy metal concentrations created for enzyme analysis.

From these 100 mL plant cell culture jars, 10 mL aliquots were taken for 0 hr, 6 hr, 12 hr, 24 and

48 hr intervals, were added into 15 mL ready centrifuge tubes (Fisher, 05-539-12) and then stored at -

80°C. Ten milliliters aliquots of cells from each of the treatment groups were collected and placed into 15

mL centrifuge tubes (VWR 21008-089) during the following time points: 0, 3, 6, 12, 24, 48 hr after the

initial addition of heavy metals. Samples were immediately centrifuged at 3,000 g for 10 minutes at 4°C.

The supernatant was removed and 1 mL of 10 mM potassium phosphate buffer (pH 7.5) was added (Chu

et al, 1997). The pelleted cells were then gently vortexed to suspend in cultures. Then, 0.04-0.06 grams of

0.1 mm glass beads (Next Advance) were then placed into properly labeled 1.5 mL microcentrifuge tubes.

The samples from the 15 mL Falcon tubes were then transferred into 1.5 mL microcentrifuge tubes.

Samples were homogenized for 5 minutes at maximum speed using a BulletBlender. Homogenized

samples were then stored at -80°C until required.

2.8 Acid labile sulfide analysis

Acid labile sulfide analysis followed the protocol developed by Siegel, (1967) with several small

modifications. One hundred µL samples described above were used for the determination of sulfide

Growth medium Heavy metal Concentration (µM)

BG-11 freshwater cyanobacteria medium Cd(II) 1, 2, 2.5

Cu(II) 1, 2.5, 5

Zn(II) 1, 2, 2.5

High Salt Medium (Sueoka et al., 1967) Cd(II) 25, 50, 100

Cu(II) 1, 2.5, 5

Zn(II) 50, 100, 300

Cyanidium media (Doemel & Brock, 1971) Cd(II) 25, 50, 100

Cu(II) 1, 2.5, 5

Zn(II) 50, 100, 300

30

content. These were transferred into 1.5 mL microcentrifuge tubes. To this was added 100 µL 0.02M N,N-

dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 0.1 mL of 0.3 M FeCl3 in 1.2 N HCl. Parafilm

was used to seal the microcentrifuge cap, followed by an incubation of 20 min at room temperature, in the

dark. Any precipitate that formed was removed by centrifugation at 10,000 g, at room temperature for 10

minutes. Two hundred microliters of the remaining supernatant was then transferred into the wells of a 96

well plate and optical density was measured at 670nm. Total sulfide concentration was determined by

comparison with a Na2S standard curve (Appendix G). Data analysis required transfer from SoftMaxPro

4.8 to Microsoft Excel.

2.9 Cysteine desulfhydrase activity

Cysteine desulfhydrase activity was determined by following a modified protocol from Chu et al.

(1997). One hundred µL of samples (≈ 0.00035-0.00068 mg protein) in 10mM phosphate buffer solution

(PBS) were transferred into 1.5 mL microcentrifuge tubes. The reactions were started by the addition of

900 µL 0.11 mM L-cysteine. The microcentrifuge tubes were then mixed and incubated at 37°C for 1 hr.

Hydrogen sulfide production was quantified by following the protocol described in section 2.8 and

measuring the product on a Spectra Max Plus Spectrophotometer.

2.10 Serine acetyltransferase and O-acetylserine(thiol)lyase activity

The serine acetyl-transferase (SAT) and O-acetylserine(thiol)lyase (OASTL) combined enzyme

assay was modified from Dominguez et al. (2003). One hundred µL of cellular lysate (≈ 0.00035-

0.00068 mg protein) was added to a 1.5 mL microcentrifuge tube, along with 20 µL of 100 mM

potassium phosphate buffer (pH 7.3). Then, 9.5 µL of 400 mM L-serine was added to the reaction tube

followed by the addition of 6.75 µL of 401.56 mM acetyl coenzyme A. Ten µL of NaS2 (100 mM) was

then added into the reaction tube along with 72 µL of de-ionized water. The microcentrifuge tubes were

31

vortexed and incubated for 20 minutes at 30ºC and the reaction was then terminated with the addition of

25 µL of 25% trichloroacetic acid.

The L-cysteine produced was then measured by transferring 200 µL of the supernatant from the

1.5 microcentrifuge tubes into 5 mL test tubes containing 0.2 mL of 99.5% acetic acid ninhydrin reagent.

The ninhydrin reagent was composed of 250 mg ninhydrin, 6 mL glacial acetic acid and4 mL

concentrated HCl made daily. This was mixed for 30 minutes in the dark at room temperature before use.

The test tubes were then placed into a 100ºC water bath for 10 minutes followed by rapid cooling in a wet

ice bath. The ninhydrin reaction was terminated by the addition of 1.4 mL of 99% ethanol. Two hundred

microlitre aliquots were then read on a Spectra Max Plus Spectrophotometer at 560nm. Enzymatic

measurements are presented in a per protein basis. A standard curve was prepared using predetermined

linear concentrations of L-cysteine Appendix I.

2.11 Statistics

Two way analysis of variance (ANOVAS) and Tukey-Kramer post hoc tests were performed

using JMP 8.0 software (SAS Incorporated). Where appropriate, two tailed T-tests assuming unequal

variance were analyzed using Microsoft Excel 2007. All experiments include representative standard

error (SE). Experiments were performed at least in triplicate and the results are indicative of n=3 for

enzymatic assays. SE is presented in all figures by the error bars, where not visible SE is smaller than the

character at that point.

32

Chapter 3

Determining the detoxification capabilities of photosynthetic microorganisms

to cadmium

3.1 Introduction

Cadmium toxicity is a prevalent environmental contaminant, causing adverse effects to a wide

variety of ecosystems. As a result, human-cadmium interaction has become more common, posing

undesirable health effects in humans. Cadmium is a known carcinogen, and has been linked to renal

failure, cellular senescence, and inhibition of essential enzymes responsible for proper cellular function

(Elinder et al., 1985; Garcia-Morales et al., 1994; Sataruga et al., 2000). Cadmium acts by displacing

Ca(II) and Zn(II) as cofactors in numerous enzymes, while also disruption membrane potentials (Heng et

al., 2004). In plants and algae high concentrations of cadmium can negatively affect nitrate, phosphate

and sulfate assimilation (Domínguez, et al., 2003; Gouia et al., 2000; Mosulén et al., 2003; Rai et al.,

1998), photosynthesis (Voigt & Nagel, 2002), carbohydrate metabolism (Permina et al., 2006) and plant-

water interactions (Domínguez-Solís et al., 2004).

Previous research has determined that photosynthetic microorganisms and fungi have the

capability to biotransform heavy metals into metal-sulfides; metal-sulfides are less toxic, biologically

unavailable, and have lower solubilities in relation to their ionic metal counterparts (Kelly et al., 2007a;

Kelly et al., 2006; Lefebvre et al., 2007). Metal biotransformation holds potential for effective

bioremediation of heavy metal contaminated aquifers. Biotransformation may offer a cost effective

alternative to conventional chelation remediation approaches, and may hold promise to remove detoxify

heavy metals below the US Environmental Protection Agency’s water quality standards. The exposure

of 200 ppb of Hg(II) to Galdieria sulphuraria led to the transformation of 90% of the Hg(II) into β-

33

cinnabar within 20 minutes (Kelly et al. 2006). The availability of cysteine and intermediate compounds

of sulfate have been demonstrated to increase the resistance and accumulation of Cd(II) in Arabidopsis

thaliana and C. reinhardtii (Domínguez-Solís et al., 2004; Mendoza-Cozatl et al., 2004). By testing three

candidate autotrophic microorganisms, C. reinhardtii, S. leopoliensis, and C. merolae to Cd(II), the

process of cadmium sulfide production was characterized. Because of the vital nature of sulfide in

detoxifying Cd(II) the supplementation of cultures with additional cysteine, sulfite and the sulfate was

investigated. This may yield an increase in the heavy metal biotransforming capabilities. Key enzymes of

the sulfur assimilation pathway were also studied to further validate this pathway’s role in cadmium

detoxification in cyanobacteria and freshwater algae.

Materials and methods are presented in Chapter 2.

3.2 Results

3.2.1 Cadmium tolerance

When C. reinhardtii, S. leopoliensis and C. merolae were exposed to various concentrations of

Cd(II), between 0 µM and 500 µM, the species demonstrated a wide range of tolerances (Figures 5a, b, c).

C. merolae was capable of tolerating 5 and 250 fold higher concentrations than C. reinhardtii and S.

leopoliensis, respectively. Additionally, there was a significant increase in biomass in the 10x pretreated

sulfate trials for each of the candidate species over a 10 day period (ANOVA, p < 0.05) and in each trial,

the pretreated sulfate group did not significantly diverge in biomass from the respective control treatments

(ANOVA, p > 0.05). Sulfite and cysteine trials did not yield a significant increase in biomass (p > 0.05).

3.2.2 Sulfide production

Acid labile sulfide production was measured at three time intervals (0, 24 and 48 hours) to assess the

ability of C. reinhardtii, S. leopoliensis and C. merolae to detoxify 100 µM, 2 µM, and 100 µM of Cd(II),

34

respectfully (Figures 6a,b,c). There was a significant increase in the amount of sulfide produced by C.

merolae than by the other candidate species following 24 hrs in the 10x sulfate pretreatment and the

sulfate treatment groups (p < 0.05). Additionally, there was a significant increase in the production of

sulfide by C. merolae in both the 10x sulfate pretreatment and 10x sulfate supplemented groups between

0 and 24 hours (p < 0.05). In both C. reinhardtii and the C. merolae the pretreatment with sulfate had

significantly greater sulfide production in relation to S. leopoliensis at 48 hrs (p < 0.05). In all three

species there was a significant increase in the amount of sulfide produced, except in the sulfite and

cysteine pre-treatments, over 48 hrs (p < 0.05). Interestingly, although pre-treatment sulfate provided no

significant difference in biomass to the control in the growth trials, there was a strong increase in labile

sulfide over 48 hours in relation to the other treatment groups (p < 0.05).

3.2.3 Cysteine desulfhydrase

There was a significant difference in cysteine desulfhydrase after 48 hrs between the 10x pretreatment

sulfate, in C. merolae, C. reinhardtii, and S. leopoliensis (p< 0.05) (Figure 7). C. merolae had the highest

cysteine desulfhydrase activity at 48 hrs followed by C. reinhardtii, and S. leopoliensis. There was a

significant increase in cysteine desulfhydrase activity in all the supplemental treatments for C. merolae

(p < 0.05). Contrastingly, immediate down regulation in cysteine desulfhydrase activity occurred for all S.

leopoliensis cultures (Figure 7b) with a slow recovery to the 0 hr starting activity. There was also

significantly lower cysteine desulfhydrase activity (p < 0.05) in the BG-11 S.leopoliensis control

treatments in relation to the other nutritionally enhanced cultures. For C. reinhardtii, only the control and

sulfate enhanced treatments showed any significant increase in cysteine desulfhydrase activity between

the time of heavy metal exposure and 48-hours after (p < 0.05). The 10x sulfate pre-treatment also

showed significantly greater cysteine desulfhydrase activity over the course of the 48 hr time period than

all other treatments (p < 0.05).

35

3.2.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity

There was a gradual increase in SAT and OASTL combined activity over 48 hrs for each of the

three candidates (Figures 8a, b, c), with a plateau observed for S. leopoliensis after 24 hrs. Interestingly,

each of the three species had significantly different initial SAT/OASTL activities (ANOVA, p < 0.05). In

C. reinhardtii and C. merolae there was a significant increase in activity after 48 hrs by comparison to the

initial activity (p < 0.05). Over 48 hrs, there was significantly higher SAT and OASTL activity in the

control C. merolae culture in relation to the controls for S. leopoliensis and C. reinhardtii

(ANOVA, p < 0.05). Interestingly, the pre-cysteine treatments had significantly lower activities than the

other C. reinhardtii and S. leopoliensis treatments for the duration of the 48 hrs of measurement

(ANOVA, p < 0.05).

36

Figure 5 Cadmium tolerance growth analyses for C. reinhardtii 150 µM Cd(II)(A), S. leopoliensis 2.5

µM Cd(II) (B), and C. merolae 500 µM Cd(II) (C). The supplemental nutritional trials are as follows:

control 0 µM Cd(II)( ), pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3 ( ),cysteine (

), pre-cysteine ( ), and control at respective Cd(II) upper limit( ).The 0 hr OD665

measurement was 0.1. SE represented by error bars (n = 4). Please note, y-axis scales are different heights

for A, B and C.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0hr 18hr 28hr 36hr 56hr 74hr 96hr 120hr 168hr 216hr

OD

665

Time (hours)0 24 48 72 96 120 144 168 192 216

0

0.2

0.4

0.6

0.8

1

1.2


OD

665

Time (hours)

0 24 48 72 96 120 144 168 192 216

0

0.5

1

1.5

2


OD

665

Time (hours)0 24 48 72 96 120 144 168 192 216

A

B

C

37

Figure 6 Cadmium induced sulfide formation at 0 hr ( ), 24 hr ( ), and 48 hr ( ) for C. reinhardtii

100 µM Cd(II) (A), S. leopoliensis 2 µM Cd(II) (B) and C. merolae 100 µM Cd(II) (C). SE represented

by error bars (n=3). Please note the y-axis scales are different amongst the graphs.

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine HSM

Sulf

ide

pro

du

ctio

n

µM

ole

s/m

g p

rote

in

0

0.1

0.2

0.3

0.4

0.5

0.6

pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine BG-11

Sulf

ide

pro

du

ctio

n

µM

ole

s/m

g p

rote

in

0

0.5

1

1.5

2

2.5

3

3.5

4

pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine Allen's media

Sulf

ide

pro

du

ctio

n

µM

ole

s/m

g p

rote

in

A

B

C

38

Figure 7 The effect of cadmium on cysteine desulfhydrase activity of C. reinhardtii 100 µM Cd(II) (A),

S. leopoliensis 2 µM Cd(II) (B) and C. merolae 100 µM Cd(II) (C) . Treatments included: pre SO4

( ), pre SO3 ( ), SO4 ( ), SO3 ( ), cysteine, ( ), pre-cysteine (

), and control ( ). SE included as error bars (n=3), standard error smaller than markers

in some points. Please note, y-axis is not the same scale for each graph.

0

1

2

3

4

5

6

7

8

0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs

Sulf

ide

pro

du

ctio

n

(U/m

g p

roe

tein

)

0

0.5

1

1.5

2

2.5

3


Sulf

ide

pro

du

ctio

n(

nm

ole

s/m

g p

rote

in)

0

5

10

15

20

25


Sulf

ide

pro

du

ctio

n

(U/m

g p

rote

in)

A

B

C

39

Figure 8 The effect of cadmium on serine acetyl-transferase and O-acetylserine(thiol)lyase activity for

C. reinhardtii 100 µM Cd(II) (A), S. leopoliensis 2 µM Cd(II)(B), and C. merolae 100 µM Cd(II) (C).

Treatments symbols are as follows: pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3

( ), cysteine, ( ), pre-cysteine ( ), and control media ( ). SE included as

error bars (n=3). Please note, the y-axis is not the same scale throughout the figure.

0

2

4

6

8

10

12

14


Cys

tein

e p

rod

uct

ion

( U

/mg

pro

tein

)

0

0.5

1

1.5

2

2.5

3


Cys

tein

e p

rod

uct

ion

(

U/m

g p

rote

in)

0

5

10

15

20


Cys

tein

e p

rod

uct

ion

(

U/m

g p

rote

in)

A

B

C

40

3.3 Discussion

3.3.1 Cd(II) tolerance and supplemental sulfate, sulfite and cysteine.

C. merolae has been demonstrated to have extremely high tolerance to Cd(II) in comparison to

the other two tested candidates. In relation to C. reinhardtii, and S. leopoliensis, C. merolae was capable

of surviving 5 and 250 times the upper toxicity limits of the other species, respectively (Figure 5). C.

merolae is an extremophile capable of growing even under pH conditions below 2.0, high concentrations

of ionic heavy metals, and temperatures ranging up to and beyond 60ºC (Gross & Oesterhelt, 1999).

Cd(II) in high concentrations can induce similar negative consequences to the cells to those of low pH.

Cd(II) is known to induce lipid peroxidation, which can cause extensive damage to the cell membrane and

organelles; lipid peroxidation and reactive oxygen species (ROS), occur at higher frequency under acidic

conditions. Chlamydomonas, exposed to as little as 50 µM cadmium, showed an increase in lipid

peroxidation activity and a decrease in cellular growth rates (Siripornadulsil et al., 2002).

Galdieria sulphuraria, a close relative to C. merolae, expresses 4 unique class III ascorbate

peroxidase enzymes, and 2 class I peroxidases (Barbier et al., 2005; Oesterhelt et al., 2008). Ascorbate

peroxidases have been strongly correlated with higher tolerance to acidity and extreme temperature by

preventing reactive oxidative damage (Hirooka et al., 2009). Analysis of Galdieria’s cell wall fractions

and extracellular space yielded high concentrations of the four types of class III peroxidases which were

responsible for cell wall modification. Similar modifications may occur in C. merolae, preventing Cd(II)

induced lipid peroxidation and reactive oxygen accumulative damage (Barbier et al., 2005). C. reinhardtii

and S. leopoliensis may not have the same capacity to modify their cellular barriers and are known to

have less gene duplications for ascorbate peroxidase classes compared to C. merolae (Matsuzaki et al.,

2004; Shrager et al., 2003). S. leopoliensis and other cyanobacteria react to increased concentrations of

41

heavy metals by increasing their number of efflux ATPases (Outten et al., 2000). However high

extracellular Cd(II) concentrations can still pose a threat of lipid peroxidation of the cell membrane. Even

though S. leopoliensis may be capable of excluding Cd(II) from its internal cellular matrix, ROS damage

may cause considerable stress on cell membrane transporters as well as increase the possibility of lipid

peroxidation of the cellular membrane.

Additionally, differences in metal transporters may be account for variance in Cd(II) tolerance.

Hanikenne et al. (2005) analyzed gene metal transporter genome sequences between C. merolae and C.

reinhardtii and found there is limited homology in metal transporters. C. merolae is naturally exposed to

environments condusive of high heavy metal content (Misumi et al., 2008), and thus requires to have very

specific metal transporter activity capable of limiting the uptake of toxic mimics (Yoshimura et al., 2000).

Cyanidium caldarium was able to exclude Al(III) in the growth medium even when concentrations

exceeded 1 mM. Al(III) is a molecular mimic for Fe(III) and can be taken up by iron transporters.


It is worth noting that the addition of a pretreatment of sulfate to all of the candidate species provided

higher tolerance to Cd(II) and the cultures were only marginally lower in biomass after 240 hrs compared

to the controls. Sulfate pretreatments also yielded significantly more biomass in comparison to sulfite and

cysteine treatments. Cd(II) stress has been known to induce sulfate limiting conditions (Mosulén et al.,

2003; Perales-Vela et al., 2006) which may give these pretreated lines a competitive advantage. With an

ability for cells to assimilate sulfate over 10 days into thiols, this may create a viable thiol pool that can be

readily employed to biotransform Cd(II) that enters the cell. Sulfate assimilation is a costly process

requiring 732 kJ mol-1

of energy to reduce one sulfate into sulfide. Cellular growth and metabolism is

limited by their ability to produce energy. By temporally displacing the energy allotments over 10 days

may allow the pretreatment groups to focus their energy towards combating oxidative stress caused by

42

Cd(II) at the time of exposure without the added energy stress of sulfate reduction and cysteine exclusion.

In each species, the sulfate pretreatment trial group produced higher growth and more labile sulfide in

comparison to their respective sulfite and cysteine treatments (Figure 6). Sulfite and cysteine have the

potential to damage cells by binding with NAD+, or by disrupting the disulfide interactions in protein

scaffolds (Baker et al., 1983; Kopriva et al., 2008). This may have accounted for the poor production of

labile sulfide compared to the pretreatment with sulfate. The algae under sulfite and cysteine stress

probably had to expend additional energy to cope with the added stress from bisulfite and cysteine.

C. merolae has 30 times the concentration of sulfate in its media in relation to S. leopoliensis and

nearly 60 times that of high salt media. Sulfate permease transporters for C. merolae may have higher

interactions with sulfate and in response to heavy metals may mediate higher sulfate uptake compared to

the other candidates. C. merolae is naturally found in sulfur rich hot springs (Gross et al., 1998). Due to

sulfur’s abundance C. merolae may be capable of desensitizing SAT more readily than C. reinhardtii and

S. leopoliensis and utilize the sulfur assimilation pathway for detoxification without having the threat of

depleting sulfate. C. merolae possesses one additional SAT and two additional OASTL homologues

(Nozaki et al., 2001), which supports this hypothethesis. C. merolae SAT and cysteine desulfhydrase

activity increased 20 fold over a 48 hr period (Figures 7c, 8c) and elicited a significant increase in activity

in relation to C. reinhardtii and S. leopoliensis (p < 0.05). Of considerable interest, in this respect, SAT

and OASTL are known to have regulatory roles in controlling the expression levels of the enzymes and

transporters in sulfate assimilation (Saito, 2004). Therefore, additional SAT and OASTL genes may

enhance the sulfate gene expression under metal stress.

43

Chapter 4

The examination of zinc biotransformation by photosynthetic microbes

4.1 Introduction

Zinc is an essential heavy metal and is used for a wide variety of cellular functions, including but

not limited to, zinc finger motifs used to bind to DNA, and detoxification of reactive oxygen species

(ROS) (MacDiarmid et al., 2000; Maret, 2003). Zinc has become more prevalent as an environmental

contaminant due to the wide number of industrial and commercial products that utilize it (Groudeva et al.,

2001; Jarup, 2003). Its toxicity has been heavily investigated in industrialized areas around the Rhine

river where it has been deposited from heavy metal smelting and refinement (Behra et al., 2002). Zinc in

high extracellular concentrations can be lethal to cells by preventing the uptake of Ca(II), which is vital to

algal species as a secondary messenger, but also as a co-factor for several calcium dependent protein

kinases (Hrabak et al., 2003; Mikolajczyk et al., 2000; Saito, 2004). Zinc is also a strong reducing agent,

which in high cellular concentrations can disrupt reduction-oxidation reactions (Maret, 2003; Rijstenbil et

al., 1994). Additionally, a range of 10-1500 µM of zinc is capable of causing a 50% reduction in the

photosynthetic rate (Maret, 2003; Takamura et al., 1989) as well as the production of reactive oxygen

species (Lyons et al., 2000; MacDiarmid et al., 2000).

Previous biotransformation studies have indicated that photosynthetic microalgae and

cyanobacteria are capable of biotransforming heavy metals into insoluble metal-sulfides (Lefebvre et al.,

2007). These metal-sulfides are biologically unavailable, precipitate out of solution, and may be removed

from contaminated water sources (Lefebvre & Edwards, 2010). Three species will be examined as

candidates for bioremediation of Zn(II); C. reinhardtii, S. leopoliensis, and C. merolae. Each species can

be easily cultured, have fully sequenced genomes (Matsuzaki et al., 2004; Merchant et al., 2007; Miller et

44

al., 1999), and are abundant in a multitude of fresh water environments. Sulfate limitation has been

determined to cause lower tolerance to Cd(II) and Cu(II)

(Mosulén et al., 2003) and even though Zn(II) contamination is prevalent, little emphasis has been placed

on determining the role of sulfur on detoxifying it. Assessing the role of the sulfur assimilation pathway

in photoautotrophic microbial tolerance and resistance to zinc is examined through determining the

effects the supplemental sulfur feeding of C. reinhardtii, C. merolae and S. leopoliensis on sulfide

production and the activities of relevant enzymes.


4.2 Results

4.2.1 Zinc resistance

In all three species pretreatment with sulfate was shown to promote the best performance of all

the metal stressed lines (Figures 9a, b, c). C. reinhardtii was able to cope with up to 600 µM of Zn(II),

however the sulfate pretreatment resulted in the most significant growth in relation to the other species

stressed at the same concentration (p < 0.05). S. leopoliensis had a much lower overall tolerance to zinc

in relation to C. reinhardtii and C. merolae, however, it demonstrated an ability to grow in all treatments

over 240 hr, which was not seen in the two other species tested. C. merolae pre-treated with sulfate had a

significant increase in biomass over the 240 hrs of exposure (p < 0.05) by comparison to this species’

other treatments (ANOVA; p < 0.05). Moreover, the pretreatment with sulfate proved to grow at the same

rate as the control with no metal exposure (ANOVA, p > 0.05).

45

Figure 9 Growth analyses for C. reinhardtii 600 µM Zn(II)(A), S. leopoliensis 10 µM Zn(II) (B), and C.

merolae 1000 µM Zn(II) (C). The supplemental nutritional trials are illustrated as follows: control 0 µM

Zn(II)( ), pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3 ( ), cysteine ( ), pre-

cysteine ( ), and control at respective Zn(II) upper limit( ).The 0 hr OD665 measurement was 0.1.

SE represented by error bars (n= 4). Please note, y-axis scales are different for A, B and C.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


OD

665

Time (hours)0 24 48 72 96 120 144 168 192 216

0

0.2

0.4

0.6

0.8

1

1.2


OD

665

Time (hours)

0 24 48 72 96 120 144 168 192 216

0

0.5

1

1.5

2


OD

665

Time (hours)

0 24 48 72 96 120 144 168 192 216

A

B

C

46

4.2.2 Acid labile sulfide analysis

In nearly all of the treatments there was an increase in acid labile sulfide from the original 0 to 48

hr (Figure 10a, b, c). In C. reinhardtii and C. merolae, only the sulfate treatments had a significantly

higher amount of sulfide produced (p < 0.05) in relation to the control treatment. S. leopoliensis had no

significant change in sulfide production in the sulfite treatments and cysteine treatments, and these did

significantly poorer at producing labile sulfides than the BG-11 control trial (p < 0.05). All of the C.

merolae treatments had a significant increase in the amount of sulfide over the 48 hr duration of the

experiment (p < 0.05).


Patterns of cysteine desulfhydrase activity over a 48 hr exposure to Zn(II) differed between the

candidate species (Figure 11). Cysteine desulfhydrase activity was higher in the pre-cysteine treatments

for all species prior to exposure to Zn(II), and, in the case of C. reinhardtii ,it was significantly higher

than all the other treatments (p < 0.05). The sulfite, cysteine, and sulfate treatments had significantly

higher activity over the entire 48 hr by camparison to the control in S. leopoliensis (ANOVA, p < 0.05).

All of the C. merolae treatments had an increase in sulfide production over the 48 hr period with the

exception of the cysteine treatments that declined.

4.2.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity

SAT/OASTL activity measured by cysteine production is outlined in Figure 12. HSM had

significantly higher activity at 48 hrs in relation to the other sulfur treatments (p < 0.05). S. leopoliensis

showed no increase in the final activity of SAT/OASTL between the beginning and the end of 48 hr

47

(p > 0.05). There was a significant increase in the control for C. merolae in comparison to the other

treatments at the end of the trial (p < 0.05).

48

Figure 10 Zinc induction of sulfide formation at 0 hr ( ), 24 hr ( ), and 48 hr ( ) for C. reinhardtii

100 µM Zn(II) (A), S. leopoliensis 2 µM Zn(II) (B) and C. merolae 100 µM Zn(II) (C). SE represented by

error bars (n=3). Please note, y-axis scales are different for A, B and C.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


Sulf

ide

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ole

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0.1

0.2

0.3

0.4

0.5

0.6


Sulf

ide

pro

du

ctio

n

µM

ole

s/m

g p

rote

in

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine Allen's Media

Sulf

ide

pro

du

ctio

n

µM

ole

s/m

g p

rote

in

A

B

C

49

Figure 11 Zinc effects on cysteine desulfhydrase activity. Zinc concentrations are as follows: C.

reinhardtii 100 µM Zn(II) (A), S. leopoliensis 2 µM Zn(II) (B), and C. merolae 100 µM Zn(II) (C).

Treatments are as follows pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3 ( ),

cysteine, ( ), pre-cysteine ( ), and HSM ( ). SE included as error bars (n=3).

Please note, y-axis scales are different for A, B and C.

0

0.5

1

1.5

2

2.5

3


Sulf

ide

pro

du

ctio

n

( U

/mg

pro

tein

)

0

0.5

1

1.5

2

2.5


Sulf

ide

pro

du

ctio

n(U

/mg

pro

tein

)

0

5

10

15

20


Sulf

ide

pro

du

ctio

n

( U

/mg

pro

tein

)

A

B

C

50

Figure 12 Zinc effects on serine acetyltransferase and O-acetylserine(thiol)lyase activity. C. reinhardtii

100 µM Zn(II) (A), S. leopoliensis 2 µM Zn(II) (B), and C. merolae 100 µM Zn(II) (C). Treatments are as

follows pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3 ( ), cysteine, ( ),

pre-cysteine ( ), and control ( ). SE included as error bars (n=3). Please note, y-axis

scales are different for A, B and C.

0

1

2

3

4

5

6

7


Cys

tein

e p

rod

uct

ion

(

U/m

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in)

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1.5

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Cys

tein

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(

U/m

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16


Cys

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C

A

B

51

4.3 Discussion

4.3.1 Zinc tolerance

Zinc plays an essential role in many gene expression and metabolic processes. However, at high

concentrations Zn(II) can induce ROS formation, inhibit the uptake of Ca(II), and disrupt photosynthesis

(Rijstenbil et al., 1994). Zn(II) induced ROS production can also encourage lipid peroxidation with

similar negative effects to Cd(II). This can be inhibited by the production of cell membrane embedded

metallothioneins, proteins with high thiol content, (Thomas et al., 1986) exhibited by some marine algal

species under zinc stress.

Due to Zn(II)’s ability to induce ROS when it is in high concentration, similar cellular responses

may be exhibited to these under Cd(II) stress. As outlined in Chapter 3, C. merolae has a much larger

gene family of ascorbate peroxidases that prevents oxidative damage and increase cellular membrane

tolerance to low pH and high temperature. This similar principle may be employed to explain the variance

in candidate tolerance to Zn(II). C. merolae was able to survive double the concentration that C.

reinhardtii could tolerate and 10 times the Zn(II) concentration of S. leopoliensis (Figure 9). As

observed during Cd(II) exposure (chapter 3: Figure 5), sulfite and cysteine treatments did not produce

significant increases in biomass for C. merolae, S. leopoliensis, and C. reinhardtii (ANOVA, p > 0.05).

Sulfite and cysteine are toxic even under sulfur limiting conditions (Baker et al., 1983; Hawkesford & De

Kok, 2006; Kopriva et al., 2008). It is likely, that since there was not an increase in growth that sulfite

and cysteine may not be intermediates utilized by algae and cyanobacteria for the sequestration and

detoxification of heavy metals. As an alternative intermediate dimethylsulfoniopropionate (DMSP) has

been shown to be a compatible solute involved in osmoprotection in bacteria and algae as well as a

cryoprotection agent in algae (Kiene et al., 2000). Furthermore, more than 70% of organic sulfur is stored

as DMSP in marine algal cells (Keller et al., 1999). However this sulfur intermediate is limited to marine

52

algae and bacteria and has not been shown to form in significant concentrations in freshwater species.

Another intermediate may exist in the form of Fe(S), but this remains to be investigated.

Interestingly, S. leopoliensis had favorable growth increases in sulfate and control treatments

during Zn(II) exposure, while C. merolae only had a significant increase in biomass in the sulfate

pretreated cells (Figure 9a, c). S. leopoliensis’s genome contains ZntR operons that are transcribed when

zinc is in toxic concentrations (Iohara et al., 2001; Osborna et al., 1997; Wu & Rosen, 1993). ZnTR codes

for zinc efflux ATPase transporters that can readily export zinc and lower intercellular concentrations

(Helmann et al., 1989). Zinc sulfide production, cysteine desulfhydrase and SAT/OASTL activity for S.

leopoliensis over 48 hrs was minimal in comparison to C. reinhardtii and C. merolae (Figures 10, 11, 12).

Due to the limited enzyme activity in S. leopoliensis, biotransformation may be a secondary mechanism

for detoxification and additive to ZntR activity.

4.3.2 C. reinhardtii and C. merolae sulfur assimilation

Eukaryotes, such as C. reinhardtii and C. merolae, may utilize the sulfur assimilation pathway as

their main defense against heavy metal contamination. Both species showed a significant and gradual

increase in SAT and cysteine desulfhydrase activity after 48 hrs in the control groups (Figures 11, 12a, c).

There was also lower activity in the pretreated cells, indicating that ample thiol and sulfide intermediates

may be stored intracellularly. Although there was an induction in cysteine desulfhydrase and SAT

activity in Zn(II) the activity was considerably lower by comparison with Cd(II) (Chapter 3: Figures 7, 8).

Perales-Vela and colleagues (2006) determined that the induction of class II and III metallothioneins were

preferentially induced by Cd(II) followed by Zn(II) and Cu(II), respectively. Metallothionein assimilation

is correlated to be regulated by the sulfur assimilation pathways regulatory mechanisms (Fraser et al.,

2002; Ravina et al., 2002). Zinc’s limited ability to induce the sulfur assimilation pathway, may explain

some of the discrepancies in the lower labile sulfide found in all of the zinc trials in comparison to

53

cadmium. Kelly et al. (2007) determined that a fast and a slow phase took place during the

biotransformation period. It was proposed that initially a readily available thiol pool was synthesized into

metal-sulfides, followed by a limitation in the production of sulfides by the sulfur assimilation pathway,

with a quelling of sulfide production in 24 hrs to 48 hrs. The sulfate pretreatment sulfate and the control

treatment showed significant increases in labile sulfide produced over 24 hrs (Figure 10) (p < 0.05). This

may shed light on a similar fast and slow phase biotransformation process occuring in C. reinhardtii, C.

merolae, and S. leopoliensis for ZnS.

High extracellular zinc concentrations can inhibit the uptake of calcium (Rijstenbil et al., 1994),

which has been found to be essential in triggering the sulfate assimilation pathway (Mosulén et al., 2003;

Pollock et al., 2005). Due to calcium’s role in activating cysteine sensitive and SAT/OASTL,

extracellular Zn(II) concentrations may have prevented SAT desensitization in S. leopoliensis (Figure

12b) and thus allowed for a transcriptional upregulation in the sulfate assimilation pathway. Interestingly,

C. merolae has multiple copies of SAT and OASTL genes, and even under calcium limited conditions

may be capable of up-regulating cysteine desensitized SAT/OASTL gene expression.

These factors may all be responsible for the large range in SAT, cysteine desulfhydrase and

sulfide production between treatments in these photosynthetic microorganisms.

54

Chapter 5

Copper biotransformation and the role of the sulfur assimilation pathway in

photoautotrophic microbes

5.1 Introduction

Copper is one of the most abundant heavy metals on earth and is sought after for a large number

of industrial and commercial applications. Industrial effluent, agricultural and urban runoff, mining

operations and fossil fuel refineries are responsible for comprising the main sources of copper pollution in

the environment (Malik, 2004). When essential heavy metals, such as copper are in abundance, cells are

challenged with having to selectively import and maintain proper concentrations of these ions while at the

same time preventing their sequestration above desired concentrations that would disrupt metabolic

activity. Although copper is an essential heavy metal for proper cellular function, copper can cause

increased oxidative damage through generation of reactive oxygen species (Blindauer et al., 2003; Paperi

et al., 2006). Paradoxically, in many organisms copper super-oxidase dismutase proteins are used by the

organisms to remove reactive oxygen species. So at high concentrations copper is both helpful and a

hindrance to proper cellular function. Furthermore, copper is capable of binding to cellular membrane

proteins and bacterial sheaths which can disrupt membrane potentials and facilitate the reactive oxygen

species (ROS) formation (Collan et al., 2003; Micheletti et al., 2008; Philippis et al., 2007). Additionally,

high ionic concentrations of copper disrupt the function of photosystem II and can also inhibit cellular

division (Stauber & Florence, 1987).

Mosulén and colleagues (2003) determined that C. reinhardtii exposed to high concentrations of

Cu(II) and Cd(II) stimulated the uptake of sulfate. Sulfate assimilation has also been linked to various

other heavy metals including: lead, gold, colbalt, silver, and mercury (Kelly et al., 2006; Mandal et al.,

55

2006). Previous studies from our laboratory indicated that aerobic algae and cyanobacteria have the

potential to biotransform mercury (II) into meta-cinnabar (β-HgS) when they were exposed to

concentrations as high as 200 ppb (Kelly et al., 2007b; Kelly et al., 2006; Lefebvre et al., 2007). This

study investigates the role of supplemental sulfur nutrition on the ability to increase the tolerance to

Cu(II) in C. merolae, S. leopoliensis, and C. reinhardtii. It also characterizes sulfide production and the

activities of serine acetyltransferase and cysteine desulfhydrase in response to copper.


5.2 Results:

5.2.1 Copper tolerance and supplemental sulfur nutrition

The addition of copper in concentrations as high as 30 µM was determined to inhibit the growth of C.

reinhardtii, S. leopoliensis and C. merolae (Figure 13a, b, c) significantly in relation to controls

(ANOVA, p < 0.05). For C. reinhardtii, exposure to 5µM Cu(II) lead to only a limited growth response

when pretreated with sulfate, this was not significantly different from the control exposed to the same

copper concentration (ANOVA p < 0.05). Interestingly, copper exhibited the highest inhibition of

cellular growth at lower concentrations by comparison to the Cd(II) and Zn(II) in all three species (See

Chapters 3 and 4). Oddly, in C. merolae, additional sulfate supplementation at the time of Cu(II) exposure

did not produce a change in the biomass production.


Total sulfide production was determined using the techniques outlined by Seigel (1967) and were

analyzed for each of the three candidates at 0, 24 and 48 hrs (Figure 14). When exposed to 2.5 µM Cu(II)

there was not a significant increase or decrease, between 0 hr and 48 hrs, in sulfide production in each of

56

the candidates (p > 0.05). However, there was a large divergence between the labile sulfide found

between C. reinhardtii, S. leopoliensis, and C. merolae.

57

Figure 13 Copper tolerance analyse. C. reinhardtii 5 µM Cu(II)(A), S. leopoliensis 5 µM Cu(II) (B), and

C. merolae 30 µM Cu(II) (C). The supplemental nutritional trials are illustrated as follows: control 0 µM

Cu(II)( ), pre SO4 ( ), pre SO3 ( ), SO4 ( ), SO3 ( ), cysteine ( ), pre

cysteine ( ), and control at respective Cu(II) upper limit( ). The 0 hr OD 665 measurement was

0.1. SE represented by error bars (n= 4). Please note, y-axis scales are different heights for A, B and C.

0

0.2

0.4

0.6

0.8

1

1.2

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OD

665

Time (hours)

0 24 48 72 96 120 144 168 192 216

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OD

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Time (hours)

0 24 48 72 96 120 144 168 192 216

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Time (hours)

0 24 48 72 96 120 144 168 192 216

C

A

B

58


Both C. reinhardtii and C. merolae had gradual increases in cysteine desulfhydrase activity over 48

hrs, while all treatments for S. leopoliensis showed a decrease in activity during 6-24 hrs with a recovery

to baseline activity by 48 hrs (Figure 15a, b, c). There was also significantly higher enzyme activity for

the pre-sulfate, pre-cysteine and cysteine treatments for C. merolae between 0 and 48 hrs (p < 0.05). C.

reinhardtii showed a significant increase in cysteine desulfhydrase for the sulfate pretreatment and

cysteine pretreated cells from the time of copper exposure to 48 hrs (p < 0.05). Interestingly, there was

no significant change in activity of S. leopoliensis during 48 hr exposure to Cu(II) even amongst the

pretreatment cells.

5.2.3 Serine acetyltransferase activity and O-acetylserine(thiol)lyase activity

Figure 16 outlines the dynamic shifts in SAT /OASTL activity for the candidate microbes. The C.

reinhardtii control without copper had significantly higher activity and cysteine production in relation to

the rest of the trials (p < 0.05). The other trials for C. reinhardtii did not vary significantly from the time

of initial exposure to Cu(II) through 48 hrs (p> 0.05). Surprisingly, there was no increase in enzymatic

cysteine production for S. leopoliensis in all of the lines after 3 hrs (p > 0.05). S. leopoliensis showed an

increase pre-sulfate enzyme activity only after three hours of exposure. All C. merolae supplemented

trials had a gradual increase in SAT activity over 48 hrs. The control had a 22 fold increase in activity in

comparison to the initial activity and 48 hrs (p < 0.05). Pretreatment with cysteine had the lowest

induction of SAT activity and was significantly lower than the C. merolae control and sulfate

pretreatment trials (p < 0.05).

59

Figure 14 Copper induction of sulfide formation at 0 hr ( ), 24 hr ( ), and 48 hr ( ) for C. reinhardtii

(A), S. leopoliensis (B) and C. merolae (C). Copper concentration was 2.5 µM. SE represented by error

bars (n=3). Please note, y-axis is not the same scale for A, B and C.

0

0.05

0.1

0.15

0.2

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0.3

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0.4


Sulf

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µM

/mg

pro

tein

0

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0.02

0.03

0.04

0.05

0.06

0.07

pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine Allen's media

Sulf

ide

pro

du

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n

µM

/mg

pro

tein

A

B

C

60

Figure 15 Copper effects on cysteine desulfhydrase activity. C. reinhardtii (A), S.leopoliensis (B), and C.

merolae (C) were exposed to 2.5 µM Cu(II), . Treatments are as follows: pre SO4 ( ), pre SO3 (

), SO4 ( ), SO3 ( ), cysteine, ( ), pre-cysteine ( ), and control (

). SE included as error bars (n=3). Please note, y-axis is not the same scale for A, B and C.

0

0.2

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16


Sulf

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(U/m

g p

rote

in)

A

B

C

61

Figure 16 Serine acetyltransferase and O-acetylserine(thiol)lyase activity induced by 2.5 µM Cu(II) for

C. reinhardtii (A), S. leopoliensis (B), and C. merolae (C). Treatments are as follows pre SO4 ( ),

pre SO3 ( ), SO4 ( ), SO3 ( ), cysteine, ( ), pre-cysteine ( ), and

control ( ). SE included as error bars (n=3). Please note, y-axis scale is not the same throughout

the figure.

0

2

4

6

8

10

12


Cys

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U/m

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)

A

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62

5.3 Discussion

5.3.1 Cu(II) tolerance

Our upper level tolerance analysis for Cu(II) provides evidence that by supplementing cells with

sulfate prior to exposure to heavy metals can provide an increase in tolerance to higher levels of Cu(II)

(Figure 13), as there was a significant increase in biomass for all sulfate pretreated candidates after 240

hrs (p < 0.05). There was no significant difference in the growth of C. merolae pretreated with sulfate in

relation to the non-metal treated control (p < 0.05). Similar to the upper level of tolerance to Zn(II) and

Cd(II) when supplemented with sulfite and cysteine (see Chapters 3 and 4). There is also no evidence of

increased tolerance to Cu(II). In high concentrations, sulfite is interchangeable with bisulfite, which is a

known disrupter of NAD+ and FAD and can interfere with disulfide bonds vital to protein tertiary

structure (Baker et al., 1983). With the addition of Cu(II) and other heavy metals, there is the potential for

acidification, which increases the lethality of sulfite by pushing the reaction towards bisulfite. Although

the energy requirement for the production of sulfide is lower from sulfite in relation to the reduction of

sulfate (732 kJ mol-1

), the stress from the disruption of NAD+ and protein scaffolding appears to offset the

benefits. Cysteine in even small concentrations can be lethal by inhibiting favorable oxidation reactions

and by causing DNA damage through the Fenton reaction (Awano et al., 2003; Park & Imlay, 2003).

These stresses can be enhanced with the presence of heavy metals.

Interestingly, all species had lower tolerance to Cu(II) than to Cd(II) or Zn(II), (see Chapters 3

and 4). Copper is known to inhibit cellular growth and division in fresh water and marine diatoms at

concentrations of as low as 1 µM (Stauber & Florence, 1987). Although the mechanisms for how copper

inhibits cellular division is yet to be fully understood, it is postulated that excessive concentrations of

copper limit the formation of methionine, which is required for proper cellular division in algal species

(Davies et al., 1996b). Additionally, copper is thought to bind to thiol groups on tubulin; tubulin is

63

required for spindle fibre formation during mitosis (Jensen et al., 1991). Due to the low ability for C.

merolae, C. reinhardtii, and S. leopoliensis to accumulate biomass it is postulated that copper may be

strongly inhibiting cellular division either by limiting the availability of methionine or by binding with

tubulin thiols.

5.3.2 Sulfide production under Cu(II) stress and the sulfur assimilation pathway

Surprisingly, acid labile sulfide analysis yielded no significant changes in sulfide production in all

treatments and species over 48 hrs (Figure 14). Copper (II) sulfide has a very low solubility product (Ksp

8.5 x 10-45

) and in even miniscule concentrations are insoluble in water (Shea & Helz, 1989). This

solubility constant is 18-20 orders of magnitude lower than CdS (Ksp 8.0 x 10-27

) and ZnS (Ksp 2 x 10-25

).

Due to the low solubility product, the acid labile sulfide analysis may not be able to dissociate S2-

from

CuS. CuS is only soluble in high concentrations of acid, and the dilution of 100 mL with 7.2N HCl

outlined by Seigel (1965) may not have been effective at effecting solubilization. The sulfide present in

the analysis may be iron sulfide, which appears to be produced by cells as a storage pool. Iron sulfide is

used in a variety of enzymes as a cofactor and forms in clusters surrounding SAT/OASTL as an

immediate pool of sulfide for the formation of cysteine (Saito, 2000; Saito, 2004). Iron sulfide also has a

relatively high solubility product (Ksp 1.14 x 10-3

) and can easily dissociate freeing the sulfide to OASTL

(Morse et al., 1987; Pinto et al., 2003). It is suggested that the sulfide determined during the acid labile

sulfide analysis may have been iron sulfide instead of CuS.

However, there is a strong indication that Cu(II) is capable of being biotransformed in C.

reinhardtii and C. merolae. SAT/OASTL and cysteine desulfhydrase activity for C. merolae increased by

2-3 fold in all treatments over 48 hrs, indicating that under Cu(II) stress these cells are inducing the

sulfate assimilation pathway and thereby, reducing cysteine. These findings are consistent with similar

results from Stauber and Florence (1989) that showed significant depletion of the intracellular thiol pools

64

of Nitzschia closterium, AsterioneIla glacialis, and Chlorella vulgaris after exposure of each to copper in

excess.

Similar to when S. leopoliensis was exposed to Cd(II) and Zn(II) (Chapter 3 and 4), there was a

deactivation in cysteine desulfhydrase activity and only limited fluctuations in SAT activity at the 3 hr

increment (Figures 15b, 16b). Synechococcus PCC7942 is known to contain two unlinked genes, copA

and copB (copper uptake and efflux ATPases respectively), that are regulated independently (Silver &

Phung, 1996). The increase in copB expression may be favoured over metallothionein production due to

the inability of the cells to transport or sequester Mt(II) bound Cu(II) (Kanamaru et al., 1993). The efflux

of Cu(II) may be the primary detoxification mechanism employed by S. leopoliensis.

These trends will be more thoroughly discussed in Chapter 6.

65

Chapter 6

General discussion

6.1 Growth and heavy metal tolerance

Throughout the Cd(II), Cu(II), and Zn(II) heavy metal tolerance analyse, pretreatment with

sulfate demonstrated the ability to infer higher resistance to each of the metal ions (Figures 4, 9, 13) than

the sulfite, cysteine and control treatment groups. The reduction of sulfate into sulfide is an energy costly

process for organisms, having to consume 732 kJ mol-1

per reduction (Leustek et al., 2000). The sulfate

reduction to sulfide is approximately twice as energy costly as the reduction of nitrate into ammonia at

347 kJ mol-1

. As a result, the assimilation of sulfate into sulfide adds stress to the organism. Allowing

each species to incorporate and reduce sulfate for 10 days prior to exposure, limits the combined stress of

sulfate assimilation and heavy metal ROS formation. The addition of sulfate, and the reduction time of

10 days, allows the species to acclimatize to the higher concentrations of sulfate, also transporting, storing

and converting sulfate into thiols and perhaps sulfide such as FeS. Sulfate is a relatively stable compound,

capable of being stored, and has limited toxicity in relation to more reduced forms of sulfur. Due to the

abundance of sulfate, each species would be capable of producing optimal pools of thiols, which may aid

in the production of metallothioneins or be used for the biotransformation of the ionic metal into metal-

sulfides during stress. By temporally separating the stress of the reduction of sulfate and the exposure to

heavy metals, each species may be capable of maintaining their energy stores, while also having ample

thiol or sulfide pools in order to detoxify the heavy metals. Additionally, added sulfate may allow for

more sulfate permease- sulfate interaction, preventing the cells from entering into a state of sulfate

starvation during metal-sulfide production.

66

Contrastingly, supplemental nutrition with sulfite and cysteine proved to be detrimental and

produced only limited resistance. Interesting, C. merolae, the most tolerant candidate to Cd(II), Zn(II),

and Cu(II) (Figures 4, 9, 13), was very sensitive to sulfite treatment and was unable to grow in 10x

sulfite. Although sulfite is more reduced than sulfate, and would require less energy to produce cysteine

(Giordano et al., 2005), sulfite and bisulfate can be toxic in high concentrations to algal and

cyanobacterial species (Baker et al., 1983; Rabinowitch et al., 1989). In solution, bisulfite and sulfite are

interchangeable. Baker and his colleagues (1983) determined that the toxicity of sulfite and bisulfate were

directly linked to the pH within which microorganisms were grown; as the pH was lowered the bisulfite

and sulfite pool became more lethal as the equilibrium shifted to bisulfite.

Allen’s media was optimized to a pH of 3.5 which would have converted nearly 100% of the 10x

supplemented sulfite into bisulfite while the HSM, which had a pH of 6.0, may have only converted 80%

of the sulfite to bisulfite. Bisulfite and sulfite can cause cellular and biochemical damage on a variety of

levels. One of the most frequent interactions with bisulfites is that they can react with aldehyde and

ketone structures of 5 and 6 carbon sugars, disrupt the disulfide linkages of proteins distorting protein

scaffolding, and negatively interact with cofactors such as NAD+, FMN, and FAD (Babich & Stotzky,

1978). NAD+ is required in the light reaction of photosynthesis. These disruptions may have added

additional stress to C. merolae, C. reinhardtii and S. leopoliensis during the pretreatment as well as

during exposure to heavy metals. This may explain why there was no significant increase in biomass for

either C. reinhardtii or S. leopoliensis treated with sulfite (Figures 4a b, 9a b, 13a b) at the upper tolerance

limits for Cu(II), Zn(II), and Cd(II).

The cysteine supplementation also did not confer higher resistance to Cd(II), Cu(II), or Zn(II)

(Figures 4, 9, 13). Cysteine is highly reduced and in limited amounts can be toxic to organisms by

inhibiting oxidation or by inducing deoxyribose degradation. In E. coli, high levels of intracellular

67

cysteine are capable of causing DNA degradation through the Fenton reaction (Park & Imlay, 2003).

Similarly when the concentration of cysteine is more abundant than sulfur, cells gain a higher

susceptibility to damage from hydrogen peroxide formation. Furthermore, unused cysteine residues can

form disulfide bonds with functional proteins, disrupting their conformation, ultimately lowering their

efficiency (Denk & Bock, 1987; Leustek et al., 2000). Even 2x supplementation with cysteine, in relation

to the sulfur levels, may have caused additional stress and DNA damage. Additionally, in nearly all of the

cysteine pretreatments there was significantly higher SAT activity at the time of metal exposure in

relation to the other treatment groups (p < 0.05). This may indicate that the levels of intercellular cysteine

may have exceeded optimal conditions, triggering the cells to break down unwanted cysteine into

pyruvate, sulfide, and ammonia (Figures 7a, b, 11 a, b, c, 15a, b, c). Increased initial cysteine

desulfhydrase activity in some of the treatments may have been an active means to prevent DNA damage

from excessive intercellular cysteine concentrations.

C. merolae was capable of surviving and flourishing in higher concentrations of Cd(II), Zn(II)

and Cu(II) than S. leopoliensis or C. reinhardtii. C. merolae is an extremophile, capable of surviving at

low pH, high temperatures, and high concentration metal conditions (Matsuzaki et al., 2004; Misumi et

al., 2008; Ohta et al., 2003). Cyanidioschyzon merolae has already been identified to tolerate high

concentrations of metals that produce reactive oxygen species, such as Cd(II), Zn(II) and Cu(II) (Hirooka

et al., 2009; Misumi et al., 2008). It also has extreme tolerance for trivalent metal ions and is capable of

growing in 10 mM of Al(III) (Misumi et al., 2008). As outlined in Chapters 3-5, the C. merolae has a

diverse number of ascorbate peroxidase enzymes that are embedded in the cell membrane as well as

excreted into the extracellular matrix (Hirooka et al., 2009; Oesterhelt et al., 2008). Ascorbate peroxidase

activity is known to increase the tolerance to pH and high temperatures by limited lipid peroxidation (De

Gara, 2004; Hirooka et al., 2009). The metal tolerance divergence between species may be hypothesized

68

to be due to the cellular membrane transporters. Genetic comparisons between C. merolae and C.

reinhardtii have very little sequence homology between heavy metal transporter families (Hanikenne et

al., 2005). Cyanidioschyzon and its relatives contain very specific metal transporters that can select

specific metal ions while excluding toxic mimics. Yoshimura et al. (2000) determined that Cyanidium

caldarium could exclude Al(III) from entering through Fe(III) transporters.

By a simple supplementation of algal media with 10x sulfate and allowing the species to

acclimatize to the higher concentrations, it is possible to increase their tolerance to cadmium, zinc, and

copper. Our study indicates that the pretreatment of C. merolae with sulfate resulted with 5 fold more

resistance to Cd(II) in relation to the selected resistant 10D strains developed by Shirabe et al. (2010).

Although our lines were capable of increasing their biomass at 5 times the concentration of cadmium,

their rate of growth was slower when compared to these other resistant lines (Shirabe et al., 2010). This

difference in rate may have been due simple variance in the light and dark cycles that can enhance the cell

division of C. merolae (Kuroiwa, 1998). C. merolae was grown under relatively high intensity lights

during the metal trials, which may have been responsible for impeding growth. As a result,

supplementation with sulfate prior to heavy metal exposure exhibits the similar results to the growth times

of selected resistant lines.

6.2 Heavy metal-sulfides

The ability for organisms to detoxify non-essential metals within the cell is vital to survival. Even

essential heavy metals, in above desired concentrations, can be harmful or fatal to the organism. In

response, some microbes have developed useful strategies to remove heavy metals by biotransforming the

ionic form into metal-sulfides. A variety of studies have characterized that algae and vascular plants

produces cadmium sulfide in response to cadmium stress (Mendoza-Cazatl & Moreno-Sanchez, 2005;

69

Reese et al., 1992; Scarano & Morelli, 2003). A variety of other metal-sulfides can also be synthesized by

bacterial, algal and fungal species. These metals include: gold, silver, lead, zinc, and cobalt (Mandal et

al., 2006).

For both the zinc and the cadmium exposure treatments, most of the production of metal-sulfides

occurred in the first 24 hrs (Figures 7, 11, 15). This was especially apparent for C. merolae and C.

reinhardtii sulfate pretreatments. Kelly et al. (2006a) hypothesized that the production of acid labile

sulfide followed two phases; a fast and a slow phase. The fast phase was associated with the immediate

exposure to the heavy metal, where it is hypothesized that there is an immediate use of the thiol pool to

detoxify the intercellular heavy metals. The slow phase follows after the depletion of the rapidly available

thiol pool because biotransformation then limited by the sulfate assimilation pathways’ ability to produce

sulfide; i.e. the slow phase due to the depletion of the intercellular thiol pool is restrained by the rate at

which the cell can assimilate sulfate. The sulfate pretreatments would have had the potential to

accumulate optimal intercellular concentrations of thiols, to biotransform a higher amount of ionic metals

into metal-sulfides than the control, sulfite, and cysteine treatments. Furthermore, the additional sulfate in

the media would have allowed for the cellular rate of assimilation during the slow phase to not be

impeded by the limitation in sulfate, while cadmium stress may have caused sulfate to be a limiting

nutrient in the other treatments. Additional sulfate may allow for the thiol depletion to be more gradual,

allowing for the upregulation of rate limiting enzymes in the sulfur assimilation pathway. This is

reinforced by the notion that during cadmium stress, sulfate limitation has been linked to lower metal

tolerance (Mosulén et al., 2003; Wang et al., 2000).

C. merolae produced 3 times the cadmium sulfide in relation C. reinhardtii exposed to the same

concentrations (p < 0.05). By comparison to S. leopoliensis, C. merolae was capable of producing nearly

6 times the CdS (p < 0.05). Additionally, Cd(II) exhibited nearly double the sulfide production in

70

comparison to Zn(II) even though these were present in the media at the same molar concentration.

Dominguez et al. (2003) determined that cadmium concentrations are capable of causing a strong up

regulation in cysteine synthesis and sulfate assimilation in sulfate starved C. reinhardtii. Cu(II) and

Zn(II) are essential heavy metals when these cells would have to cope with the high levels of these

metals. They may be actively exported through specific Cu(II) and Zn(II) ATPase transporters to prevent

the over accumulation (Blindauer et al., 2003; Collan et al., 2003; Micheletti et al., 2008). Bacterial

species contain a series of metal specific operons responsible for the rapid exportation of Cu(II) and

Zn(II) from the cell when there is an excess in the environment (Hobman et al., 2005). Synechococcus

PCC7942 is known to contain two unlinked genes, copA and copB (copper uptake and efflux ATPases

respectively), that are regulated independently (Silver & Phung, 1996). The increase in copB expression

may be favoured over metallothionein production due to the inability of the cells to transport or sequester

Mt(II) bound Cu(II) (Kanamaru et al., 1993). A similar mechanism if Zn(II) efflux has been attributed to

czc and czr chemiosmotic antiporters (Nies et al., 1989; Nies, 1999).

Furthermore, algae and cyanobacteria have very specified responses to metals which typically

involve the formation of class II or III metallothioneins (MtII and III respectively) (Pinto et al., 2003) and

may explain the variance in CdS, ZnS, and CuS levels. MtIII and II production appears to play a major

role in the adaptive ability of these species to cope with heavy metals. MtIII is ubiquitous in algae, and

are preferentially induced by Cd(II), followed by Pb(II), Zn(II) and Cu(II), respectively (Perales-Vela et

al., 2006). MtII’s and MtIIIs are cysteine-glycine rich polypeptides that stabilize, isolate and

facilitate the exportation of heavy metals. Scarano and Morelli (2003) indicated that MtIII metal

ion complexes are stabilized by S2-

. Ravina et al (2002) proposed that algae cysteine synthesis

and reduction is sensitive to sac1 sac2, and sac3 post trancriptional control. Stress from Cd(II)

may cause a strong gene response from sac1, leading to the formation of metallothioneins while

71

simultaneously activating cysteine desulfhydrase. The formation of MtII and MtIII complexes

may aid in the synthesis of metal-sulfides and the divergence in CdS, ZnS, and CuS could be due

to the different potentials for the metals to induce a strong sac1 response.

Surprisingly, there was no increase in sulfide production in all three species after the exposure to

copper (Figure 14). Copper sulfide has a very low dissociation constant, (Ksp 8.5 x 10-45

) which may have

prevented the extraction of the sulfide for analysis using the method outlined by Siegel (1965). The

solubility product for copper sulfide is 18-20 orders of magnitude lower than CdS (Ksp 8.0 x 10-27

) and

ZnS (Ksp 2 x 10-25

). The variance between each of the species in terms of their baseline sulfide in the

copper study may be due to varying concentrations of iron sulfides present within the cells as co-factors

for a variety of enzymes (Horner et al., 2002). Iron sulfide, for example, plays an critical role in the sulfur

assimilation pathway by forming clusters around sulfite reductase in order to stabilize free sulfide that has

been reduced for SAT (Saito, 2004). The FeS allows for SAT to have a readily available pool of sulfide to

produce cysteine without the delay of reduction of sulfate. The concentration and size of these FeS

clusters may be variable between each of the species and may be dependent on the efficiency of sulfite

reductase, SAT or even associated with the pool of available Fe(II). FeS solubility product is very high

(Ksp 1.14 x 10-3

) allowing for sulfide to be sequestered by SAT activity (Morse et al., 1987).

Furthermore, it may be possible that MtIII production is high and synthesis of CuS is minimal in relation

to CdS and ZnS and in combination with the low solubility product difficult to characterize.

6.3 Cysteine desulfhydrase

One millimole of Cd(II) was capable of inducing a 4 fold increase in the level of cysteine

desulfhydrase activity in Clostridium thermoaceticum (Cunningham & Lundie, 1993), this produced a 1-

1.3 ratio: cadmium to sulfide. Our study indicates very similar trends, with an approximate 4 fold increase

in C. reinhardtii treatments over 48 hrs and a 5-20 fold increase in the treatments for C. merolae when

72

Cd(II) 100 µM. In terms of metal remediation, sulfide forms a strong bond with cadmium, zinc, and

copper causing the metal to precipitate. The reduction of cysteine to sulfide, pyruvate and ammonia has

limited energy input (293 kJ mol−1

) which is a small portion of the energy required to reduce sulfate

(732 kJ mol-1

) into sulfide (Domínguez-Solís et al., 2004; Hawkesford and De Kok, 2006; Kopriva et al.,

2008). The increase in cysteine desulfhydrase activity in C. reinhardtii and C. merolae may be an

indicator to the fast phase biotransformation process observed by Kelly et al. (2006). In this fast phase the

cells would be reacting to the stress of heavy metals by reducing available thiols to form biologically

unavailable metal-sulfides. Furthermore, the increase in activity of cysteine desulfhydrase activity was

directly linked to the SAT/OASTL activity and increased production of cysteine (Figures 8a, c, 12a, b,

16a, b). The binding of CdS and other metals tends to be site specific and tends to accumulate on the cell

wall, indicating that cysteine desulfhydrase activity may be located on the cell wall, and a present first

line of defense against metals (Cunningham & Lundie, 1993; Hall, 2002; Micheletti et al., 2008).

Interestingly, there was a decrease in the activity of cysteine desulfhydrase observed in S.

leopoliensis for all treatments with a gradual recovery to the initial activity (Figures 7b, 11b, 15b).

Cysteine activity was initially high, indicating that the activity of the enzyme is tied directly to the

concentration of available cysteine for reduction. Furthermore, cysteine activity increased in tandem with

the increase in SAT/OASTL activity (Figures 8b, 12b, 16b). Synechococcus PCC7942 has the capability

to activate copper, zinc, mercury and cadmium efflux ATPase transporters (Nies et al., 1989). High

concentrations may exhibit a quick activation of cysteine desulfhydrase followed by production of copper

and divalent efflux transporters to remove zinc and copper. This may be a more energetically conservative

method to keep adequate concentrations of zinc and copper, without having to expend the energy to

activate the sulfur assimilation pathway. Over expression of cysteine desulfhydrase has been found to

have negative effects on bacterial growth and was detrimental to the cells (Chu et al., 1997; Park &

73

Imlay, 2003), but limited activation may be beneficial. However, the addition of cysteine to the growth

medium alleviated the negative effects of the cysteine desulfhydrase activity in E. coli (Wang et al.,

2000). Thus higher production of cysteine may offset the negative consequences of the cysteine

desulfhydrase activity in S. leopoliensis.

6.4 Serine acetyltransferase and O-acetylserine(thiol)lyase activity

SAT and OASTL form a dimer complex (Sirko et al., 2004) that synthesizes cysteine from serine

and sulfide. Serine acetyltransferase has been characterized as the allosteric enzyme in the regulation of

the pathway for sulfate assimilation. Saito (2000) determined that SAT sensitivity to cysteine controlled

translation of the gene products in Arabidopsis’ sulfur assimilation pathway. During sulfur limiting

conditions SAT becomes desensitized and activates a strong signaling cascade to express APS, APR and

sulfite reductase (Sirko et al., 2004). C. merolae may have gained tolerance due to extra gene copies of

SAT and OASTL that C. reinhardtii and S. leopoliensis do not possess (Nozaki et al., 2001). The

increased production of cysteine and sulfide by cysteine desulfhydrase may actually be because of

enhanced transcription of SAT and OASTL.

Oxidative stress from heavy metals is known to strongly induce the production of cysteine and

glutathione (Ravina et al., 2002). This induction potential has been similarly demonstrated to occur in C.

reinhardtii, S. leopoliensis, and C. merolae. C. merolae was capable of increasing SAT/OASTL activity

by over 20 fold over 48 hrs for the Cd(II) and Cu(II), while Zn(II) only increases this activity by 5 fold.

Zn(II), in high extracellular concentrations may reduce the uptake rate of calcium into the cell (Hussain et

al., 2004; Stauber & Florence, 1990), which is required to activate SAT activity (Saito, 2000; Saito, 2004)

and turn on sulfate assimilating genes. SAT/OASTL activity is directly linked to the availability of O-

acetyl-l-serine (Leon et al., 2001) and is triggered by calcium signaling (Saito, 2004). Due to

74

SAT/OASTL’s role in activating strong gene responses, the enzyme may very well be the limiting factor

in the sulfur assimilation pathway (Sirko et al., 2004).

The addition of pretreated sulfate limits the activation of SAT/OASTL activity significantly

between in C. merolae, C. reinhardtii, and S. leopoliensis (p < 0.05). The ability for the cells to assimilate

sulfate into cysteine over 10 days, would provide these treatments with an adequate pool of thiols that

could be readily reduced to sulfide under metal stress conditions. This pool may have allowed for there to

be a gradual increase in SAT/OASTL activity, which would have not been as energy costly to the cell

lines as would immediate activation in many genes. Each of the control treatments had significantly

higher activation of SAT during cadmium exposure in comparison to the sulfite, cysteine, and sulfate

treatments (p < 0.05), indicating that higher levels of sulfur compounds exhibits less pressure on the SAT

pathway.

6.5 Future directions

Presently, a move towards determining the flux in the thiol pools during exposure to Cd(II),

Zn(II) and Cu(II) would be of interest to determine how thiol pools change in relation to heavy metal

stress. This could be useful in determining supplementation requirements during particularly stressful

times in the detoxification process of heavy metals. This analysis could be completed using the HPLC

protocols outlined by Masi et al. (2003).

Due to the high tolerance of C. merolae to Cd(II), Cu(II) and Zn(II), further research should be

devoted to understanding the enzyme kinetics associated with C. merolae’s sulfur assimilation pathway. It

may hold promise for large scale bioremediation applications. By determining rate limiting enzymes in

the sulfur assimilation pathway this could lead to more targeted feeding strategies or the generation of

metal resistant mutants that have increased enzyme efficiency.

75

Sulfite and cysteine proved to be ineffective at increasing metal tolerance and seem unlikely

sources of the sulfide utilized during metal-sulfide formation. SAT/OASTL are known to be surrounded

by a pool of FeS (Saito, 2004). Iron sulfide may be the sulfur donor used during the heavy metal

biotransformation process. Supplemental feeding with iron sulfide should be conducted to determine its

role in the rapid phase of biotransformation.

76

Summary

1. Chlaymdomonas reinhardtii, Synechoccocus leopoliensis and Cyanidioschyzon merolaes’ ability

to tolerate Cd(II), Cu(II) and Zn(II) were assessed over 10 days of exposure.

2. C. reinhardtii, S. leopoliensis and C. merolases’ growth media were enriched with sulfate, sulfite,

and cysteine to determine the effect of sulfur metabolites on metal sulfide biotransformation.

Sulfite and cysteine treatments were found to be detrimental to cell growth under metal stress.

3. C. merolae, S. leopoliensis, and C. reinhardtii had significantly higher tolerance to Cd(II), Cu(II),

and Zn(II) when the growth medium was enhanced with sulfate ten days prior and at the time of

heavy metal stress. This may allow for the microbes to build up a pool of thiols that can help

detoxify heavy metals.

4. Sulfate pretreatment for C. merolae, S. leopoliensis and C. reinhardtii had significantly higher

sulfide formation than the sulfite and cysteine treatments when exposed to Cd(II) and Zn(II);

there was no change in sulfide levels to Cu(II) for all treatments. Cd(II) and Zn(II) are known to

induce metallothionein production which may facilitate metal sulfide formation.

5. There was no significant induction of sulfide associated with Cu(II) exposure. Cu(II) has a very

low solubility product and the acid labile sulfide analysis employed may not have been adequate

to dissociate CuS.

6. C. merolae exhibited a 15-20 fold increase in cysteine desulfhydrase and serine acetyltransferase

activity to Cd(II), Cu(II) and Zn(II). This suggests that these enzymes play an important role in

metal detoxification in C. merolae.

7. There was a 5-8 fold increase cysteine desulfhydrase and serine acetyltransferase for C.

reinhardtii in response to each metal.

77

8. There was limited enzyme activity in S. leopoliensis under Cd(II), Cu(II) and Zn(II) stress. It is

suggested that S. leopoliensis primarily exports heavy metals when they are in excess while

biotransformation may only have a secondary role.

78

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Appendix A: Microorganism quantification and unit conversions

Table 4 S. leopoliensis quantification unit conversions

Units Equivalence

Culture turbidity (OD665) 1.0

Protein (mg/L) 680.4 ± 35.3

Chlorophyll (mg/L) 18.4 ± 0.07

Cell weight (wet) (mg/L) 4733.3 ± 11.5

Cell weight (dry) (mg/L) 866.6 ± 20

Cell count (cells/L) 176,737,500 ± 6,523

Table 5 C. reinhardtii quantification unit conversions

Units Equivalence


Protein (mg/L) 540.7 ± 30.9


Cell weight (wet) (mg/L) 7666.6 ± 110.15

Cell weight (dry) (mg/L) 666.6 ± 23.09


Table 6 C. merolae quantification unit conversions

Units Equivalence


Protein (mg/L) 357.7 ± 34.1


Cell weight (wet) (mg/L) 8400 ± 28.3

Cell weight (dry) (mg/L) 765 ± 14.1


A

C

100

Appendix B: Trace metal concentrations present in algal growth media.

Table 7 Trace metal concentrations present in algal growth media.

mg/500 mL

Stock concentration

(µmoles)

% metal in

compound

concentration in final

medium (µmoles)

High salt medium:

Fe 79.89 591.1 0.207 0.1221

Cu 0.006 0.070 0.373 2.6E-05

Co 1.3 10.93 0.248 0.0027

Zn 1.64 24.1 0.480 0.0115

Cyanobacteria BG11 medium:

Fe 0.003 21.4 0.199 0.0042

Cu 0.0395 316.4 0.255 0.08052

Co 0.0247 207.6 0.248 0.05142

Zn 0.111 772.1 0.227 0.1756

Modified Allen's medium:

Fe 97 717.7 0.2074 0.14828

Co 2 16.8 0.248 0.00416

Zn 5 73.36 0.4796 0.03518

101

Appendix C: Metal concentrations in media employed for growth analysis

Table 8 Metal concentrations employed in media for growth analysis

Final concentration in 100 mL cell

media metal aliquot in 100 mL (μL) Stock concentration of metal (M)

Cd(II) (μM)

500.00 1905.40 0.03

450.00 1711.66 0.03

400.00 1518.65 0.03

375.00 1422.41 0.03

350.00 1326.35 0.03

325.00 1230.47 0.03

300.00 1134.76 0.03

200.00 753.71 0.03

175.00 658.89 0.03

150.00 564.24 0.03

100.00 375.47 0.03

50.00 187.39 0.03

25.00 93.61 0.03

12.50 46.78 0.03

6.25 23.39 0.03

1.50 5.61 0.03

Zn(II) (μM)

1000.00 1409.63 0.07

900.00 1266.91 0.07

800.00 1124.59 0.07

700.00 982.67 0.07

600.00 841.13 0.07

500.00 699.98 0.07

400.00 559.21 0.07

300.00 418.84 0.07

250.00 348.79 0.07

200.00 278.84 0.07

150.00 208.99 0.07

100.00 139.23 0.07

50.00 69.57 0.07

Cu(II) (μM)

100.00 137.32 0.07

50.00 68.62 0.07

35.00 48.02 0.07

30.00 41.16 0.07

27.50 37.73 0.07

25.00 34.30 0.07

22.50 30.87 0.07

20.00 27.44 0.07

15.00 20.57 0.07

10.00 13.72 0.07

5.00 6.86 0.07

1.00 1.37 0.07

102

Appendix D: Cellular counts vs optical density

0

0.2

0.4

0.6

0.8

1

1.2

0 40000 80000 120000 160000 200000

OD

66

5

0

0.2

0.4

0.6

0.8

1

1.2

0 2000 4000 6000 8000 10000

OD

66

5

0

0.2

0.4

0.6

0.8

1

1.2

0 10000 20000 30000 40000 50000 60000 70000

OD

66

5

Cell Count

Figure 4Cellular count of S. leopoliensis (A), C. reinhardtii (B), and C. merolae (C)

compared with optical density 665nm. SE is represented with error bars (n=3).

A

B

C

103

Appendix E: Chlorophyll extractions

0

24

68

10

12141618

20

0 1 2 3 4 5 6

tota

l ch

loro

ph

yll µ

g/m

l

OD665

0

2

4

6

8

10

12

14

0 0.5 1 1.5 2 2.5 3

tota

l ch

loro

ph

yll µ

g/m

L

OD665

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2 2.5 3

tota

l ch

loro

ph

yll µ

g/m

L

OD665

Figure 5 Total chlorophyll extracted from S. leopoliensis (A), C. merolae (B), and

C. reinhardtii (C). SE is represented by the error bars (n=4).

A

B

C

104

Appendix F: Bradford BSA assay standard curves

Figure 6 Bradford assay standard curve for protein concentration (biorad reagent kit). SE indicated by

error bars (n=3).

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.1 0.2 0.3 0.4 0.5 0.6

OD

59

5

Protein concentrations µg/ mL

105

Appendix G: Acid labile sulfide analysis standard curve

0

20

40

60

80

100

120

0 0.02 0.04 0.06 0.08 0.1 0.12

Sulf

ide

Co

nce

ntr

atio

n (

µM

Na

2S)

OD670

Figure 7 Standard curve calculated using the acid labile sulfide analysis with Na2S (Seigel, 1967). SE

indicated by error bars (n=3).

106

Appendix H: Cysteine desulfhydrase enzyme activity standard curve

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100 120

OD

67

0

Sulfide concentration (µM)

Figure 8 Standard curve of cysteine dyseulhydrase activity outlined by Wang et al. (2000). SE

presented as error bars (n=3).

107

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.5 1 1.5 2 2.5

OD

56

0

µM Cysteine

Appendix I: Serine acetyltransferase and Serine acetyl-transferase and O-

acetylserine(thiol)lyase activity

Figure 9 Serine acetyltransferase standard curve using D-cysteine. Protocol was derived from

Dominguez et al. (2003). Standard error is represented by the vertical error bars (n=3).

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