THE CHARACTERIZATION OF METAL BIOTRANSFORMATION ...
Transcript of 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
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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.
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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.
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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
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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
3.3.2 Sulfide production ...................................................................................................................... 41
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
4.2.3 Cysteine desulfhydrase .............................................................................................................. 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
5.2.1 Sulfide production ...................................................................................................................... 55
5.2.2 Cysteine desulfhydrase .............................................................................................................. 58
5.2.3 Serine acetyltransferase activity and O-acetylserine(thiol)lyase activity................................... 58
5.3 Discussion ......................................................................................................................................... 62
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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
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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
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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
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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)
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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
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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).
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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
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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
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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).
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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)
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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
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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
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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,
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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).
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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).
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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
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
0.5
1
1.5
2
0hr 18hr 28hr 36hr 56hr 74hr 96hr 120hr 168hr 216hr
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
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n(
nm
ole
s/m
g p
rote
in)
0
5
10
15
20
25
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
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
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
( U
/mg
pro
tein
)
0
0.5
1
1.5
2
2.5
3
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
0
5
10
15
20
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
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.
3.3.2 Sulfide production
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.
Materials and methods are presented in Chapter 2.
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
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
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
0.5
1
1.5
2
0hr 18hr 28hr 36hr 56hr 74hr 96hr 120hr 168hr 216hr
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).
4.2.3 Cysteine desulfhydrase
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
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
0.2
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
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n
( U
/mg
pro
tein
)
0
0.5
1
1.5
2
2.5
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n(U
/mg
pro
tein
)
0
5
10
15
20
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
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
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
0
0.5
1
1.5
2
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
0
2
4
6
8
10
12
14
16
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
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.
Materials and methods are presented in Chapter 2.
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.
5.2.1 Sulfide 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
1.4
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
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
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
C
A
B
58
5.2.2 Cysteine desulfhydrase
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
0.25
0.3
0.35
0.4
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
0.05
0.1
0.15
0.2
0.25
pre-sulfate pre-sulfite sulfate sulfite cysteine pre-cysteine BG-11
Sulf
ide
pro
du
ctio
n
µM
/mg
pro
tein
0
0.01
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
ctio
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
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n
(U/m
g p
rote
in)
0
0.5
1
1.5
2
2.5
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n(U
/mg
pro
tein
)
0
2
4
6
8
10
12
14
16
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Sulf
ide
pro
du
ctio
n
(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
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
(
U/m
g p
rote
in)
0
5
10
15
20
25
0 hrs 3 hrs 6 hrs 12 hrs 24 hrs 48 hrs
Cys
tein
e p
rod
uct
ion
( U
/mg
pro
tein
)
A
B
C
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
Culture turbidity (OD665) 1.0
Protein (mg/L) 540.7 ± 30.9
Chlorophyll (mg/L) 13.3 ± 0.04
Cell weight (wet) (mg/L) 7666.6 ± 110.15
Cell weight (dry) (mg/L) 666.6 ± 23.09
Cell count (cells/L) 9,136,250 ± 6,285
Table 6 C. merolae quantification unit conversions
Units Equivalence
Culture turbidity (OD665) 1.0
Protein (mg/L) 357.7 ± 34.1
Chlorophyll (mg/L) 2.2 ± 0.02
Cell weight (wet) (mg/L) 8400 ± 28.3
Cell weight (dry) (mg/L) 765 ± 14.1
Cell count (cells/L) 55,406,250 ± 5,465
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).