bioremoval of copper and nickel on living and non-living euglena gracilis a thesis submitted in
TRANSCRIPT
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Bioremoval of copper and nickel on living and non-living Euglena gracilis 1
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A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master 5
of Science in the Faculty of Arts and Sciences 6
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TRENT UNIVERSITY 13
Peterborough, Ontario, Canada 14
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Environmental and Life Sciences MSc. Graduate Program 21
April 2016 22
© Cameron Winters 2016 23
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Abstract 25
Bioremoval of Copper and Nickel on living and non-living Euglena gracilis 26
Cam Winters 2016 27
This study assesses the ability of a unicellular protist, Euglena gracilis, to remove 28
Cu and Ni from solution in mono- and bi-metallic systems. Living Euglena cells and 29
non-living Euglena biomass were examined for their capacity to sorb metal ions. 30
Adsorption isotherms were used in batch systems to describe the kinetic and equilibrium 31
characteristics of metal removal. In living systems results indicate that the sorption 32
reaction occurs quickly (<30 min) in both Cu (II) and Ni (II) mono-metallic systems and 33
adsorption follows a pseudo-second order kinetics model for both metals. Sorption 34
capacity and intensity was greater for Cu than Ni (p < 0.05) and were described by the 35
Freundlich model. In bi-metallic systems sorption of both metals appears equivalent. In 36
non-living systems sorption occurred quickly (10-30 min) and both Cu and Ni 37
equilibrium uptake increased with a concurrent increase of initial metal concentrations. 38
The pseudo-first-order model was applied to the kinetic data and the Langmuir and 39
Freundlich models effectively described single-metal systems. The biosorption capacity 40
of Cu (II) and) was 3x times greater than that of Ni (II). Sorption of one metal in the 41
presence of relatively high concentrations of the other metal was supressed. Generally, 42
it was found that living Euglena remove Cu and Ni more efficiently than non-living 43
Euglena biomass in both mono- and bi-metallic systems. It is anticipated that this work 44
should contribute to the identification of baseline uptake parameters and capacities for Cu 45
and Ni by Euglena as well as to the increasing amount of research investigating 46
sustainable bioremediation. 47
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Keywords: biosorption, kinetics, Cu, Ni, accumulation, bioremediation, Euglena gracilis 48
Acknowledgements 49
I would like to offer my sincere thanks to my supervisor Dr. Céline Guéguen for 50
her guidance and support of this work. It has been my pleasure to work in her laboratory 51
and I am thankful for her clear perspective and valuable counsel. My thanks go also to 52
supervisory committee members Dr. Eric Sager and Dr. Neil Emery both for their time 53
and advice regarding the work. Additionally, I would like to thank Dr. Sager for his 54
friendship and guidance throughout my undergraduate and graduate studies. This work 55
would not have been possible without the assistance of Antoine Perroud whom I thank 56
for his ICPMS analysis and unfailingly good humour. I would also like to acknowledge 57
Jean- François Koprivnjak and the Water Quality Centre at Trent University for 58
assistance and expertise. My thanks also go to Noble Purification Inc. for their support 59
and encouragement of this project. Financial support from the Ontario Centre for 60
Excellence, NSERC, and the Canada Research Chair program is greatly appreciated. I 61
must also thank all my colleagues in the lab, especially Vaughn Mangal, Yong Xiang Shi, 62
and Chad Cuss for their valuable input and friendship. Finally, I thank my parents for 63
their abiding patience, love, and support over the course of this endeavour. It means 64
more than words can say, and I dedicate this work to you both. 65
Authors’ contributions 66
CW and CG developed study design and CG contributed revisions for all chapters. CW 67
collected and analyzed the data and wrote the first draft of all manuscripts. AN provided 68
technical and financial support for Chapter 2. All authors read and approved final drafts. 69
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Table of Contents 70
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Chapter 1: General Introduction 72
1 Metal pollution ............................................................................................................ v 73
2 Ecological and Human Health Effects of Metals ........................................................ 2 74
3 Conventional Methods of Metal Remediation ............................................................. 2 75
4 Bioremediation: an alternative method for metal removal .......................................... 5 76
5 Euglena gracilis: a potential biosorbent for metal remediation ................................ 13 77
6 Thesis objectives........................................................................................................ 14 78
References ......................................................................................................................... 18 79
Chapter 2: Equilibrium and kinetic studies of Cu (II) and Ni (II) sorption and 80
accumulation on living Euglena gracilis 81
Abstract ..................................................................................................................... 26 82
1 Introduction ............................................................................................................... 27 83
2 Materials and Methods .............................................................................................. 28 84
3 Results and Discussion .............................................................................................. 34 85
4 Conclusions ............................................................................................................... 39 86
References ......................................................................................................................... 41 87
Chapter 3: Equilibrium and kinetic studies of Cu (II) and Ni (II) biosorption on non-88
living Euglena gracilis 89
Abstract ..................................................................................................................... 51 90
1 Introduction ............................................................................................................... 52 91
2 Materials and Methods .............................................................................................. 56 92
3 Results and Discussion .............................................................................................. 60 93
4 Conclusion ................................................................................................................. 64 94
Chapter 4: General Conclusion ......................................................................................... 78 95
1 Euglena biomass characterization and toxicity ......................................................... 78 96
2 Sorption kinetics ....................................................................................................... 79 97
3 Sorption Isotherms .................................................................................................... 80 98
4 Significance of the work ........................................................................................... 83 99
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List of Figures 101
Chapter 1: 102
Figure 1: Euglena gracilis at 400x magnification 103
Figure 2: Growth curve for Euglena gracilis 104
Chapter 2: 105
Figure 1: Experimental data for EC50 toxicity assay on living E. gracilis 106
for (a) Cu2+
and (b) Ni2+
107
Figure 2: Experimental data for Cu2+
sorption kinetics on living E. gracilis 108
at initial concentrations of (a) 20 ug L-1
and (b) 50 ug L-1
. Curves 109
represent pseudo-second-order kinetic model. 110
Figure 3: Experimental data for Ni2+
sorption kinetics on living E. gracilis 111
at initial concentrations of (a) 3 mg L-1
and (b) 100 mg L-1
. Curves 112
represent pseudo-second-order kinetic model. 113
Figure 4: Experimental data for a) Cu2+
and b) Ni2+
sorption equilibrium 114
on living E. gracilis. Curve represents the Freundlich model. 115
Figure 5: Experimental data for Cu2+
and Ni2+
sorption equilibrium from 116
binary-metal solution on living E. gracilis. Error bars represent SE. 117
Chapter 3: 118
Figure 1: FTIR spectra of dried euglena biomass 119
Figure 2: Experimental data for Cu2+
sorption kinetics on non-living E. 120
gracilis at initial concentrations of (a) 20 ug L-1
and (b) 50 ug L-1
(c) 1 mg 121
L-1
(d) 25 mg L-1
. Curves represent PFO (a, c) and PSO (b, d) kinetic 122
models. 123
Figure 3: Experimental data for Ni2+
sorption kinetics on non-living E. 124
gracilis at initial concentrations of (a) 1 mg L-1
and (b) 2 mg L-1
(c) 4 mg 125
L-1
(d) 20 mg L-1
. Curves represent PFO (b, d) and PSO (a, c) kinetic 126
models. 127
Figure 4: Experimental data for Cu2+
sorption equilibrium on non-living E. 128
gracilis. Curves represent the Freundlich and Langmuir models. 129
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Figure 5: Experimental data for Ni2+
sorption equilibrium on non-living E. 130
gracilis. Curves represent the Freundlich and Langmuir models. 131
Figure 6: Experimental data for Cu2+
and Ni2+
sorption equilibrium from 132
binary-metal solution on living E. gracilis. Error bars represent SE. 133
Chapter 4: 134
Figure 1: Percentage metal removal of Cu and Ni in mono-metallic and bi-135
metallic solutions by living and non-living E. gracilis. 136
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List of Tables 149
Chapter 2: 150
Table 1: Pseudo-second-order kinetics parameters for the sorption of 151
Cu2+
and Ni2+
on living Euglena gracilis cells (±SE). 152
Table 2: Freundlich adsorption isotherm parameters for the sorption of 153
Cu2+
and Ni2+
on living Euglena gracilis cells at pH 5. 154
Chapter 3: 155
Table 1: Kinetic model parameters for the biosorption of Cu on non-living 156
Euglena gracilis cells – pseudo-first-order model. 157
Table 2: Kinetic model parameters for the biosorption of Ni on non-living 158
Euglena gracilis cells – pseudo-first-order model. 159
Table 3: Langmuir adsorption isotherm parameters for the biosorption of 160
Cu and Ni on non-living Euglena gracilis cells at pH 5. 161
Table 4: Freundlich adsorption isotherm parameters for the biosorption of 162
Cu and Ni on non-living Euglena gracilis cells at pH 5. 163
Chapter 4: 164
Table 1: Kinetic parameters of biosorbents for Cu and Ni in mono-metallic 165
systems. 166
Table 2: Equilibrium parameters of biosorbents for Cu and Ni in mono-167
metallic systems. 168
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1 Metal pollution 173
Globally, freshwater resources are subjected to steadily increasing demands for 174
withdrawal which are occurring concurrently with rising rates of development, 175
industrialization, and population growth. This demand has been projected to increase by 176
a factor of 55% by 2050 and the strain which this requirement will induce on existent 177
reserves of freshwater could result in greater than 40% of the global population living in 178
areas of acute water stress by the middle of the 21st century (Miletto, 2015). 179
Additionally, that water which is put to anthropogenic uses, domestically, agriculturally 180
or industrially, may not return to natural reserves, or if so, may be contaminated or 181
degraded to an extent which serves to preclude human consumption and poses risks to 182
ecosystem health. Metal pollution is a global concern in terms of freshwater 183
contamination (ie. further reducing available stock) and has serious ramifications in both 184
the developed and developing world (Akpor and Muchie, 2010; Kumar et al., 2015; 185
Vijayaraghavan and Balasubramanian, 2015). The chemical properties of metals are 186
such that even at dilute concentrations, levels of contaminants are persistent in the 187
environment to an extent that bioaccumulation and/or biomagnification can promulgate 188
toxic effects throughout the food web including the human body (Kumar et al., 2015). It 189
is evident therefore, that a need exists to not only judiciously manage water usage, but 190
also to develop methods for the remediation of contaminated water if the expected 191
demand for this resource is to be met. 192
193
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2 Ecological and Human Health Effects of Metals 194
This demand for water is driven in large part by the needs of the global 195
manufacturing sector, which, by 2050, are expected to rise by 400% (Miletto, 2015). The 196
effluent produced by these industries (ie. electroplating, milling, circuit board printing, 197
petroleum refining, wood processing, mining) has been identified as a key source of 198
pollutants that enter water bodies and include metals such as arsenic, cadmium, 199
chromium, copper, cobalt, iron, lead, mercury, nickel and zinc (Barakat, 2011). Metals 200
are commonly divided into two general categories; trace elements which are required for 201
biological function but are toxic at higher concentrations (e.g. Cu, Ni, Co, Zn) and those 202
which have no known nutritional significance yet remain highly toxic (e.g. Pb, Cd, Hg, 203
Cr) (Herrera-Estrella et al., 2009). Metals which are essential to physiological function 204
are controlled via a network of intracellular biochemical processes that serve to maintain 205
an appropriate osmotic balance (Albergoni et al., 1980; Piccinni, 1989). Those metals 206
which are not involved in homeostatic maintenance (e.g. non-essential) and tend to be 207
compartmentalized within cell vesicles and/or organelles and therefore provide a pathway 208
for toxic species to bio-accumulate (Devars et al., 2000). This persistence and 209
circulation of metals in the environment pose a threat to the well-being of plant and 210
animal life and consequently, via food-web connections, to human health. 211
3 Conventional Methods of Metal Remediation 212
Rising population growth and continued extraction of natural resources ensure 213
that the discharge of metal effluent to ecosystems will remain an environmental priority 214
both in Canada and across the globe. Conventional methods of treatment of industrial 215
3
effluent have proven to be effective at removing or eliminating metals but remain 216
problematic in terms of application due to a number of substantial limitations. Many of 217
these technologies require an extensive degree of infrastructure (e.g. storage and 218
conveyance) and energy requirements, thus necessitating large capital investment and 219
operational costs at the industrial scale (Lesmana et al., 2009). In addition to potentially 220
prohibitive expenses, these methods may be limited generally in terms of their treatment 221
effectiveness at relatively lower concentrations (e.g. 1 – 100 mg/L) as well as the 222
production of secondary waste streams (e.g. toxic sludge) containing high concentrations 223
of metal ions (Ahalya et al., 2003; Wang and Chen, 2009). An effective cleaning 224
process must not only possess the capacity to manage variation in terms of both effluent 225
quality and quantity, but also the variability associated with the physio-chemical 226
parameters (e.g. pH, inorganic/organic contaminants, dissolved/colloidal/volatile species) 227
of the effluent within a cost framework that is applicable at the commercial scale (Eccles 228
1995; Vijayaraghavan and Balasubramanian 2015). 229
Chemical precipitation is a commonly utilized method of removal which has 230
traditionally been a primary process for the treatment of metal-laden effluents 231
(Kurniawan et al., 2006). Ion exchange processes, as compared to chemical 232
precipitation, are also a commonly used technique for the removal of metal ions from 233
aqueous solutions (Papadopoulos et al., 2004). Adsorption, the physical and/or chemical 234
bonding of ions on to another molecule, is also the primary process that drives the use of 235
organically and/or inorganically based adsorbents for water purification. These 236
manufactured sorbents can take a wide range of chemical forms and surface structures, 237
which, in general, may be carbon, mineral, or synthetically based (Dabrowski, 2001). 238
4
Membrane filtration is a physio-chemical process in which substances are separated via a 239
semi-permeable membrane across which a driving force (e.g. pressure) is applied to 240
compel molecules of a defined size through the membrane while those exceeding this 241
constraint are rejected and remain in solution. As a component of wastewater treatment, 242
common membrane filtration techniques include ultrafiltration, nanofiltration, reverse 243
osmosis, and electro-dialysis (Fu and Wang 2011). 244
It is apparent that the selection of the most appropriate method of physio-chemical 245
metal remediation of wastewaters is dependent on chemical, economic, and 246
environmental factors which are specific to the industrial context in which the pollution is 247
generated. The initial concentrations of target metals and the ionic components of the 248
wastewater, the necessary capital investments and operational costs, the flexibility and 249
reliability of infrastructure, and finally the environmental impact must be taken into 250
consideration (Kurniawan et al., 2006). Potentially prohibitive costs due to chemical and 251
energy requirements and secondary pollution generation may bar techniques which are 252
decidedly effective and consequently, these physio-chemical methods remain limited in 253
their use and industrial application despite the common benefits of rapid processing, 254
relative ease of control, and operational flexibility. As an alternative, the bioremediation 255
of wastewater offers several advantages which include the minimization of chemical and 256
or biological sludge, operation over a broad range of physio-chemical conditions, 257
relatively low capital investment and low operational cost, and an increased efficiency in 258
ameliorating the toxicity of dilute effluents (Abbas et al., 2014; Flouty and Estephane 259
2012). 260
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4 Bioremediation: an alternative method for metal removal 261
Bioremediation may be defined as the use of biological organisms for removing 262
pollutants from contaminated environmental systems, a subset of which is 263
phycoremediation; the use of macro- or microalgae to remove, transform, or otherwise 264
render inert the toxic effects of pollutants. Plants have ability to accumulate high levels 265
of metals however, they tend to grow at slower rates, generate smaller amounts of 266
biomass and show lower fitness in comparison to those which are not exposed, therefore 267
identifying algal species which show an enhanced resistance to metals accompanied by 268
metal accumulation and fast-growing biomass offers potential for bioremediation 269
applications (Rodriguez-Zavala et al., 2007). Microorganisms have a proven capability 270
to take up metals from aqueous solutions, especially when concentrations in the effluent 271
range from less than 1 to about 20 mg/L (Brierley 1990). A key factor in this use of 272
(living) algal biomass for remediation is the ability of microalgae to discriminate between 273
those metals which are essential to growth and survival (e.g. Cu, Fe) and those which are 274
not (e.g. Pb, Cd). At the cellular scale, metals which do not play a physiological role or 275
essential metals at excessive concentrations, can accumulate and cause disorders in 276
important metabolic pathways (Clijsters and Van Assche 1985). Microalgae, and other 277
related eukaryotic organisms, produce peptides (e.g. metallothioneins and phytochelatins) 278
which possess the capacity to bind metals into organo-metallic complexes and transport 279
them to cell vacuoles and/or organelles in order to maintain cytosol concentrations of 280
metals at appropriate and non-toxic levels (Perales-Vela et al., 2006). The use of algae 281
as a system for metal bioremediation has several benefits when compared to conventional 282
methods. These include: 1) a rapid metal uptake capacity (He and Chen 2014), 2) less 283
6
energy requirements (Razzak et al., 2013), 3) a capacity for biomass re-use and metal 284
recovery (Volesky 2003), 4) a large surface to volume ratio (Khoshmanesh et al., 1997), 285
and 5) applicable to both high and low concentrations (Monteiro et al., 2012). 286
4.1 Biosorption and bioaccumulation 287
Bioremediation may be understood in terms of two relatively broad categories; 288
biosorption and bioaccumulation. Biosorption refers most generally to the characteristic 289
displayed by non-living biological substances to bind and accrue metal ions from aqueous 290
solutions; an interaction between the biosorbent and metal ions which is a physical and 291
chemical process that occurs independently of any metabolic activity (Gupta et al., 2015). 292
Biosorption is a broad term which refers to the variety of mechanisms of metal-biomass 293
sorption, the type of biological material used, biotic and abiotic environmental factors, in 294
addition to the presence or absence of metabolic influences (Fomina and Gadd 2014). 295
More precisely, the removal of metals by living microalgae has been termed 296
bioaccumulation; an active process which includes both adsorption and absorption and is 297
dependent on the metabolic activity of the organism (Gupta et al., 2015). 298
Bioaccumulation typically encompasses a bi-phasic process: 1) a rapid, reversible, and 299
non-metabolic (e.g. passive) adsorption of metal ions to functional groups on the cell 300
surface, and 2) a slower, metabolically-driven transport of metal ions across the cell 301
membrane which can be subsequently bound inside the cell (Kumar et al., 2015). In 302
living organisms, biosorption and bioaccumulation are not mutually exclusive and both 303
may serve to remove metals from solution. Both biosorption and bioaccumulation are 304
relatively complex actions in which several mechanisms may be operating 305
simultaneously. Several potential mechanisms which have been identified include: 1) 306
physical adsorption (e.g. ion exchange, Van der Waals forces), 2) micro-precipitation of 307
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insoluble metal complexes on the cell surface, 3) complexation of metal ions by organic 308
exudates, 4) metabolically-driven efflux pumps which maintain osmotic balance within 309
the cell, 5) modifying of oxidation state to render an ion less or non-toxic, 6) 310
volatilization of metals, 7) binding of metal ions to proteins and polypeptides (e.g. 311
metallothioneins and phytochelatins) (Malik 2004; Monteiro et al., 2012; Perales-Vela et 312
al., 2006, Volesky 2003). Removal by means of ion exchange (e.g. biomass protons 313
exchange with metals) has been suggested as the mechanism which most contributes to 314
metal uptake by microalgae, however micro-precipitation and complexation have been 315
proposed as being the most efficient, and some reports have claimed that metabolic 316
uptake by living organisms may account for a major fraction of total metal removal (Crist 317
et al., 1988; Kadukova and Vircikova 2005; Kratchovil and Volesky 1998; Mehta and 318
Gaur 2005). 319
4.2 Factors affecting biosorption/bioaccumulation 320
4.2.1 Abiotic factors 321
4.2.1.1 pH 322
One of the most important variables affecting metal ion sorption by microalgae is 323
the pH of the system in which it occurs (Al-Rub et al., 2006; Chojnacka et al., 2005; 324
Mehta et al., 2002). The dependence of sorption processes on pH arises from its effects 325
on metal speciation, bioavailability, toxicity, as well as the surface characteristics of the 326
biosorbent. Industrial wastewaters can contain metals in a number of different chemical 327
forms which can include the free aqueous ion, metal ions complexed with organic and 328
inorganic ligands, and ions adsorbed to particulate matter in solution (Mehta and Gaur 329
2005). Decreases in pH will result in an increase in the amount of free ions in solution 330
(e.g. greater solubility), a higher bioavailability of metals to algal cells (in living 331
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organisms) and, in turn, the potential for toxic effects to the biota will also increase 332
(Starodub et al., 1987). Volesky and Holan (1995) have described metal sorption as a 333
function of the free ion concentration of the metals in solution. Inorganic ligands which 334
are present in wastewater (e.g. CO32-
, Cl-), as well as organic molecules (e.g. amino 335
acids) can reduce bioavailability of the metal through the formation of complexes which 336
may be insoluble and/or reduced in affinity with the algal material (e.g. less positively 337
charged) (Gadd and Griffiths 1977; Starodub et al., 1987). 338
In addition to its effects on the chemical behaviour of metals pH also determines 339
in large part the chemical properties of the algal surface. At a low pH the negatively 340
charged binding sites of functional groups on the cell surface are occupied with H+ ions, 341
(e.g. sites are protonated) which decreases the electrostatic attraction of cations to the 342
functional sites. As pH increases, the functional groups are deprotonated, become more 343
negatively charged, and therefore metal binding may occur to a greater extent (Donmez et 344
al., 1999; Monteiro et al., 2012). In general, different functional groups become 345
available to bind metal ions as pH levels increase: at pH > 2-5 carboxyl groups are 346
dominant, from pH > 5-9 phosphate groups are also available, and from pH > 9-12 347
carboxyl, phosphate, and hydroxyl groups are potentially able to bind metals (Chojnacka 348
et al., 2005). However, increasing pH also increases the possibility for precipitation of 349
metals as hydroxides thus causing a decrease in the efficiency of removal as less metal is 350
left in solution (Monteiro et al., 2012). Therefore, optimizing pH in any prospective 351
system utilizing microalgae for bioremediation must take into account both the 352
organismal species of the sorbent and the chemical species of the target metal(s) (Mehta 353
and Gaur 2005). 354
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4.2.1.2 Ionic strength and initial metal concentration 355
Industrial effluents are typically composed of a suite of contaminants and 356
impurities that can include metals, both cationic and anionic, light metals, high salinity 357
and total dissolved solids (Ho and McKay 2000). At a lower ionic strength, the 358
concentration of ions in solution is lower, and as a result, more sites on functional groups 359
are available to participate in metal binding (Chen et al., 1997; Dwivedi 2012). 360
Conversely, an increase in ionic strength results in a decrease of available binding sites 361
(e.g. sites are fixed at a given pH), as they are occupied by non-metal ions which can 362
bring about a reduction in total metal removed. Competition between metals for binding 363
sites has typically been found to inhibit metal sorption (Chong and Volesky 1995; Mehta 364
et al., 2002; Vijayaraghavan and Balasubramanian 2015). However, it has been found 365
that some metals in binary mixtures show an increase in metal removed as compared to 366
single metal sorption (Chong et al., 2000). Additionally, binding site competition 367
between major (e.g. Ca2+
, Mg2+
, Al3+
) and trace metal ions or between anions (e.g. PO42-
, 368
CO32-
) and metal ions can reduce metal uptake (Gadd 2009; Lee and Volesky 1997; Lee 369
et al., 2004). Finally, the initial concentration of the metal(s) targeted for removal has 370
important effects for removal efficiency. It is not only a driving force for overcoming 371
mass transfer resistance between the liquid (e.g. high metal concentration) and solid (e.g. 372
low metal concentration) phases, but also affects the quantity of metal sorbed per mass 373
unit of biosorbent (e.g. causing an increase in metal/biomass but reducing overall 374
efficiency) (Fomina and Gadd 2014; Mehta and Gaur 2001; Mehta and Gaur 2005; 375
Zouboulis 1997). In terms of bioremediation with algae and ionic strength it is important 376
to consider not only the solution chemistry as determined by pH and effluent 377
composition, but also the characteristics of the sorbent and metal chemistry. Each 378
10
functional group present has an affinity with certain metal ions dependent on ionic radii, 379
electronegativity, and atomic mass which is reflected in removal efficiency (Matos and 380
Arruda 2003; Tarley and Arruda 2004). 381
4.2.1.3 Temperature 382
Temperature affects several factors which are involved in the process of metal 383
sorption in a number of different ways which include: the stability of ion species, ligands 384
and ligand complexes, as well as the solubility of metal ions in solution (Ruiz-Manriquez 385
et al., 1998). This could account for the inconsistency of results between studies which 386
have found both an enhancement and reduction in metal uptake by algae at increased 387
temperatures while some have found no effect (Aksu 2001; Lau et al., 1999; Mehta et al., 388
2002). Generally, higher temperatures would favour greater solubility of ions in and 389
thus could weaken sorption as metals remain dissolved in solution, whereas removal 390
could be increased via an increasing amount of kinetic energy and binding activity at the 391
cell surface (Lau et al., 1999; Park et al., 2010). In these terms, it becomes clear that 392
these factors are highly interrelated and must be taken into consideration in order to 393
evaluate the suitability for bioremediation of any particular sorbent. 394
4.2.2 Biotic Factors 395
4.2.2.1 Algal Species 396
Based on morphological and physiological differences algal affinities for and 397
tolerance of metals can vary between both species and genus of organisms. The 398
capability of an organism to ameliorate toxic effects of a metal can be measured through 399
the determination of its EC50 value (Nyholm 1990). EC50 values have been found to 400
differ among microorganism genera (Guanzon 1994) as well as among species and these 401
differences are thought to be based not only on the type of metal but also on differences 402
11
in the form and function of the algal cell (Wong et al., 2000). Higher tolerances may be 403
the result of not only environmental drivers, but also may be related to algal size, lipid 404
content and composition, and cell wall structure. Unicellular algae have generally 405
shown a greater sorption capacity due to a relatively higher surface to volume (or mass) 406
ratio (Mehta and Gaur 2005). 407
4.2.2.2 Biomass concentration 408
Metal sorption is not only affected by the size of the algal cell but also by the 409
concentration of the biomass in solution. A higher concentration of biomass may 410
increase removal simply due to an increased number of available binding sites (Fraile et 411
al., 2005). For example, Mehta and Gaur have found Cu and Ni sorption per unit of 412
biomass to be maximal at lower biomass concentrations (2001). Contrarily, increasing 413
biomass concentration has also been reported to reduce sorption as a result of an 414
accretion of cells that reduces the effective surface area available for binding as well as 415
decreasing the distance (e.g. increasing interference) between adsorption sites (Malkoc 416
and Nuhoglu 2005). Therefore, increasing biomass concentration will only improve 417
metal removal to a certain threshold beyond which a decrease is most likely due to a 418
reduction in sorption per unit of algal volume/weight (Gadd 2009). 419
4.2.2.3 Living vs inert biomass 420
Both living and dead biomass have been successfully utilized for metal removal 421
and have been studied extensively although the use of inactivated biomass appears to be 422
the preferred alternative for the majority of metal removal studies (Fomina and Gadd 423
2009; Doshi et al., 2007; Kizilkaya et al., 2012; Kumar et al., 2015; Malik 2004). Each 424
method confers different advantages or limitations which are not only determined by the 425
metabolic statues of the algal material, but also by the environmental parameters and 426
12
target pollutants in the treatment system. For instance, living cells can utilize both 427
passive and active forms of metal uptake wherein metal ions are removed via sorption to 428
the cell surface and through intercellular accumulation (Malik 2004). Live cells are also 429
constantly replenished as cellular reproduction proceeds and the potential for metal 430
toxicity impediments to cellular function can be overcome through the isolation of metal-431
tolerant mutant strains (Kumar et al., 2015; Mailk 2004). Factors such as growth rate, 432
nutrition, and the growing stage of algae can have effects on metal sorption and therefore 433
can be experimentally manipulated for potential optimization of the process (Gadd 2009). 434
Metal sorption (e.g. Ni) has been found to be higher using algal cultures which are in the 435
stationary and decline phases of growth as compared to those in the exponential growth 436
phase potentially due to the creation of more binding sites or improved exposure of those 437
sites (Mehta et al., 2002). Living algae also have the capacity for actively degrading 438
organic pollutants and/or altering valence states of target inorganic pollutants to reduce 439
toxicity whereas this is not possible for metabolically inert biomass (Asku and Tezer 440
2005; Mehta and Gaur 2005). Dead algal biomass however, is advantageous in that 441
there are no requirements for nutrients or continual maintenance of culture conditions 442
once harvesting has taken place, nor is inactivated biomass susceptible to toxic levels of 443
pollutants (Donmez et al., 1999). Additionally, dead biomass can be recharged through 444
desorption methods which separate metals from the biological material, effectively 445
regenerating it for further use and the potential recovery of rare or valuable metals 446
(Kadukova and Vircikova 2005). Conversely, live material cannot be utilized for metal 447
recovery via desorption due to intercellular accumulation, and metabolic exudates 448
13
secreted by living organisms can form organo-metallic complexes which are retained in 449
solution as opposed to removal (Mehta and Gaur 2005). 450
5 Euglena gracilis: a potential biosorbent for metal remediation 451
Euglena gracilis (Figure 1) is a eukaryotic, free floating and flagellated, 452
unicellular organism which has been used as biological model in a myriad of different 453
investigations due to its metabolic plasticity, environmental prevalence, and general 454
adaptability (Einicker-Lamas et al., 2002). Euglena sp., a photosynthetic protist, has the 455
metabolic capability to grow in the presence of high concentrations of metals which 456
include, Cd2+
(Navarro et al. 1997; Devars et al. 1998; Mendoza-Cozatl et al. 2006), Hg2+
457
(Devars et al. 2000); Zn2+
(Mendoza-Cozatl et al. 2006), Pb2+
(Navarro et al. 1997; 458
Mendoza-Cozatl et al. 2006) and Cr6+
(Cervantes et al. 2001). Euglena also have the 459
capacity to tolerate a broad range of environmental circumstances including the extreme 460
conditions (e.g. low pH, high metal concentrations) typically found in acid mine drainage 461
systems (Nakatsu and Hutchinson 1988). Euglena gracilis have been found to tolerate 462
pH levels 2.5-7 with no significant impediments to growth (Olaveson and Nalewajko 463
2000). Euglena grown in wastewater have shown faster growth rates as compared to 464
other algal species while concurrently removing substantial amounts of C, N, and P 465
(Mahaptra et al., 2013). Additionally, E. gracilis is able to grow under photosynthetic, 466
heterotrophic, and photo-heterotrophic conditions, utilizing carbon from several different 467
sources including glucose, ethanol and organic acids such as lactate, acetate, and malate 468
(Rodriguez-Zavala et al., 2007; Santiago-Martinez et al., 2015). 469
14
Euglena gracilis have developed a number of mechanisms for ameliorating metal 470
toxicity through biochemical responses that bring about the safe transport and storage of 471
metal ions in the cell (Rodriguez-Zavala et al., 2007). One mechanism of toxicity in 472
aquatic biota involves the generation of ROS (reactive oxygen species) through exposure 473
to metals and other inorganic and organic pollutants (Livingstone 2001). ROS can 474
oxidize crucial cellular components such as proteins, lipids, and nucleic acids and 475
consequently bring about changes in cell structure as well as genetic mutations (Pinto et 476
al., 2003). In response to metal and oxidative stress E. gracilis has been reported to cope 477
with potential toxicity with a number of different mechanisms which include: 1) the 478
synthesis of metal chelating molecules (e.g. thiols and carboxylate groups), 2) sub-479
cellular compartmentalization of metals, 3) secretion of extracellular chelating molecules 480
(e.g. malate), and 4) chemical reduction of metals (Devars et al., 2000; Lira-Silva et al., 481
2011; Mendoza-Cozatl and Moreno-Sanchez 2005). The increased synthesis of metal 482
chelators such as cysteine, glutathione and phytochelatins and the effective 483
compartmentalization of metals into sub-cellular organelles such as chloroplasts and 484
mitochondria have been reported as the main mechanisms of resistance to metals (e.g. 485
Cd, Hg, Cr) although other mechanisms have been proposed for certain metals (e.g. Zn) 486
which may operate independently and thus be applicable to simultaneous multi-metal 487
remediation of industrial effluent (Mendoza-Coaztl et al., 2005; Lira-Silva et al., 2011). 488
6 Thesis objectives 489
The overall objective of this study is to evaluate Euglena gracilis as a potential 490
biosorbent for metal removal from industrial effluent. The first chapter of this work 491
15
provides background information and context regarding metal pollution, conventional 492
methods of treatment and alternatives for the remediation of industrial wastewaters with 493
biological material such as the protist Euglena gracilis. In chapter 2 we study the 494
performance of living E. gracilis in sequestering Cu and Ni in single- and binary-metal 495
systems at two different pH values to determine the amounts of metal removed by 496
Euglena cells. In chapter 3, we study metal acquisition by non-living Euglena in single- 497
and binary-metal solutions at a single pH to assess the quantities of Cu and Ni removed. 498
Finally, in chapter 4, we discuss key findings, conclusions and directions for future work 499
and an assessment of which moiety (living or non-living) may be more effective in a 500
bioremediative context. This work will contribute an assessment of Euglena gracilis’ 501
capability to remove Cu and Ni from mono- and bi-metallic solutions and a starting point 502
for a wider investigation of the potential application of this organism for bioremediation 503
of industrial effluent. 504
505
506
507
508
509
510
511
512
513
514
16
515
Figure 1: Euglena gracilis at 400x magnification 516
17
517
Figure 2: Growth curve for Euglena gracilis 518
519
520
521
522
523
524
525
526
527
hours
0 20 40 60 80 100 120 140 160 180
ce
lls m
L-1
0
1e+6
2e+6
3e+6
4e+6
5e+6
18
References 528
Abbas, S.H., Ismail, I.M., Mostafa, T.M., and Sulaymon, A.H. 2014. Biosorption of 529
heavy metals: a review. Journal of Chemical Science and Technology 3(4): 74-102. 530 531 Ahalya, N., Ramachandra, T.V., and Kanamadi, R.D. 2003. Biosorption of heavy metals. 532 Res. J. Chem. Environ 7(4): 71-79. 533 534
Akpor, O.B., and Muchie, M. 2010. Remediation of heavy metals in drinking water and 535 wastewater treatment systems: Processes and applications. International Journal of 536 Physical Sciences 5(12): 1807-1817. 537 538 Aksu, Z. 2001. Equilibrium and kinetic modelling of cadmium (II) biosorption by C. 539
vulgaris in a batch system: effect of temperature. Separation and purification technology 540
21(3): 285-294. 541
542
Aksu, Z., and Tezer, S. 2005. Biosorption of reactive dyes on the green alga Chlorella 543 vulgaris. Process Biochemistry 40(3): 1347-1361. 544 545
Albergoni, V., Piccinni, E., and Coppellotti, O. 1980. Response to heavy metals in 546 organisms: I. Excretion and accumulation of physiological and non physiological metals 547
in Euglena gracilis. Comparative Biochemistry and Physiology Part C: Comparative 548 Pharmacology 67(2): 121-127. 549 550
Al-Rub, F.A.A., El-Naas, M.H., Ashour, I., and Al-Marzouqi, M. 2006. Biosorption of 551 copper on Chlorella vulgaris from single, binary and ternary metal aqueous solutions. 552
Process Biochemistry 41(2): 457-464. 553 554
Barakat, M.A. 2011. New trends in removing heavy metals from industrial wastewater. 555 Arabian Journal of Chemistry 4(4): 361-377. 556 557
Brierley, C.L. 1990. Bioremediation of metal-contaminated surface and groundwaters. 558 Geomicrobiology Journal 8(3-4): 201-223. 559
560 Cervantes, C., Campos-Garcia, J., Devars, S., Gutierrez-Corona, F., Loza-Tavera, H., 561 Torres-Guzman, J.C., and Moreno-Sanchez, R. 2001. Interactions of chromium with 562
microorganisms and plants. FEMS Microbiology Reviews 25(3): 335-347. 563 564 Chen, J., Tendeyong, F., and Yiacoumi, S. 1997. Equilibrium and kinetic studies of 565
copper ion uptake by calcium alginate. Environmental Science & Technology 31(5): 566
1433-1439. 567 568 Cho, D.Y., Lee, S.T., Park, S.W., and Chung, A.S. 1994. Studies on the biosorption of 569 heavy metals onto Chlorella vulgaris. Journal of Environmental Science & Health Part A 570 29(2): 389-409. 571 572 Chojnacka, K., Chojnacki, A., and Gorecka, H. 2005. Biosorption of Cr 3+, Cd 2+ and 573
19
Cu 2+ ions by blue-green algae Spirulina sp.: kinetics, equilibrium and the mechanism of 574
the process. Chemosphere 59(1): 75-84. 575 576 Chong, A.M.Y., Wong, Y.S., and Tam, N.F.Y. 2000. Performance of different microalgal 577
species in removing nickel and zinc from industrial wastewater. Chemosphere 41(1): 578 251-257. 579 580 Clijsters, H., and Van Assche, F. 1985. Inhibition of photosynthesis by heavy metals. 581 Photosynthesis Research 7(1): 31-40. 582
583 Crist, R.H., Oberholser, K., Schwartz, D., Marzoff, J., Ryder, D., and Crist, D.R. 1988. 584 Interactions of metals and protons with algae. Environmental Science & Technology 585 22(7): 755-760. 586
587 Dabrowski, A. 2001. Adsorption: from theory to practice. Advances in colloid and 588
interface science 93(1): 135-224. 589 590
Devars, S., Aviles, C., Cervantes, C., and Moreno-Sanchez, R. 2000. Mercury uptake and 591 removal by Euglena gracilis. Archives of microbiology 174(3): 175-180. 592 593
Devars, S., Hernandez, R., and Moreno-Sanchez, R. 1998. Enhanced heavy metal 594 tolerance in two strains of photosynthetic Euglena gracilis by preexposure to mercury or 595
cadmium. Archives of environmental contamination and toxicology 34(2): 128-135. 596 597 Dias, J.M., Alvim-Ferraz, M.C.M., Almeida, M.F., Rivera-Utrilla, J., and Sánchez-Polo, 598
M. 2007. Waste materials for activated carbon preparation and its use in aqueous-phase 599
treatment: a review. Journal of Environmental Management 85(4): 833-846. 600 601 Donmez, G., Aksu, Z., Ozturk, A., and Kutsal, T. 1999. A comparative study on heavy 602
metal biosorption characteristics of some algae. Process Biochemistry 34(9): 885-892. 603 604
Doshi, H., Ray, A., and Kothari, I.L. 2007. Bioremediation potential of live and dead 605 Spirulina: spectroscopic, kinetics and SEM studies. Biotechnology and Bioengineering 606
96(6): 1051-1063. 607 608 Dwivedi, S. 2012. Bioremediation of heavy metal by algae: current and future 609 perspective. Journal of Advance Laboratory Research in Biology 3(3): 229-233. 610 611
Eccles, H. 1995. Removal of heavy metals from effluent streams: why select a biological 612 process? International Biodeterioration & Biodegradation 35(1): 5-16. 613
614 Einicker-Lamas, M., Mezian, G.A., Fernandes, T.B., Silva, F.L.S., Guerra, F., Miranda, 615 K., Attias, M., and Oliveira, M.M. 2002. Euglena gracilis as a model for the study of Cu 616 2+ and Zn 2+ toxicity and accumulation in eukaryotic cells. Environmental Pollution 617 120(3): 779-786. 618 619
20
Flouty, R., and Estephane, G. 2012. Bioaccumulation and biosorption of copper and lead 620
by a unicellular algae Chlamydomonas reinhardtii in single and binary metal systems: a 621 comparative study. Journal of Environmental Management 111: 106-114. 622 623
Fomina, M., and Gadd, G.M. 2014. Biosorption: current perspectives on concept, 624 definition and application. Bioresource Technology 160: 3-14. 625 626 Fraile, A., Penche, S., Gonzalez, F., Blazquez, M.L., Munoz, J.A., and Ballester, A. 2005. 627 Biosorption of copper, zinc, cadmium and nickel by Chlorella vulgaris. Chemistry and 628
Ecology 21(1): 61-75. 629 630 Fu, F., and Wang, Q. 2011. Removal of heavy metal ions from wastewaters: a review. 631 Journal of Environmental Management 92(3): 407-418. 632
633 Gadd, G.M. 2009. Biosorption: critical review of scientific rationale, environmental 634
importance and significance for pollution treatment. Journal of Chemical Technology and 635 Biotechnology 84(1): 13-28. 636
637 Gadd, G.M., and Griffiths, A.J. 1977. Microorganisms and heavy metal toxicity. 638 Microbial ecology 4(4): 303-317. 639
640 Guanzon, N.G., Nakahara, H., and Yoshida, Y. 1994. Inhibitory Effects of Heavy Metals 641
on Growth and Photosynthesis of Three Freshwater Microalgae. Fisheries science 60(4): 642 379-384. 643 644
Gupta, V.K., Nayak, A., and Agarwal, S. 2015. Bioadsorbents for remediation of heavy 645
metals: Current status and their future prospects. Environmental Engineering Research 646 20(1): 1-18. 647 648
He, J., and Chen, J.P. 2014. A comprehensive review on biosorption of heavy metals by 649 algal biomass: materials, performances, chemistry, and modeling simulation tools. 650
Bioresource Technology 160: 67-78. 651 652
Herrera-Estrella, L.R., and Guevara-Garcia, A.A. 2009. Heavy Metal Adaptation. John 653 Wiley & Sons Ltd, Chichester, UK. 654 655 Ho, Y.S., and McKay, G. 2000. Correlative biosorption equilibria model for a binary 656 batch system. Chemical Engineering Science 55(4): 817-825. 657
658 Kadukova, J., and Vircikova, E. 2005. Comparison of differences between copper 659
bioaccumulation and biosorption. Environment international 31(2): 227-232. 660 661 Khoshmanesh, A., Lawson, F., and Prince, I.G. 1997. Cell surface area as a major 662 parameter in the uptake of cadmium by unicellular green microalgae. The Chemical 663 Engineering Journal and the Biochemical Engineering Journal 65(1): 13-19. 664 665
21
Kizilkaya, B., Turker, G., Akgul, R., and Dogan, F. 2012. Comparative study of 666
biosorption of heavy metals using living green algae Scenedesmus quadricauda and 667 Neochloris pseudoalveolaris: Equilibrium and kinetics. Journal of Dispersion Science 668 and Technology 33(3): 410-419. 669
670 Kratochvil, D., and Volesky, B. 1998. Advances in the biosorption of heavy metals. 671 Trends in Biotechnology 16(7): 291-300. 672 673 Kumar, K.S., Dahms, H.-U., Won, E.-J., Lee, J.-S., and Shin, K.-H. 2015. Microalgae-A 674
promising tool for heavy metal remediation. Ecotoxicology and environmental safety 675 113: 329-352. 676 677 Kurniawan, T.A., Chan, G.Y.S., Lo, W.-H., and Babel, S. 2006. Physio-chemical 678
treatment techniques for wastewater laden with heavy metals. Chemical Engineering 679 Journal 118(1): 83-98. 680
681 Lau, P.S., Lee, H.Y., Tsang, C.C.K., Tam, N.F.Y., and Wong, Y.S. 1999. Effect of metal 682
interference, pH and temperature on Cu and Ni biosorption by Chlorella vulgaris and 683 Chlorella miniata. Environmental Technology 20(9): 953-961. 684 685
Lee, H.S., Suh, J.H., Kim, I.B., and Yoon, T. 2004. Effect of aluminum in two-metal 686 biosorption by an algal biosorbent. Minerals Engineering 17(4): 487-493. 687
688 Lee, H.S., and Volesky, B. 1997. Interaction of light metals and protons with seaweed 689 biosorbent. Water Research 31(12): 3082-3088. 690
691
Lesmana, S.O., Febriana, N., Soetaredjo, F.E., Sunarso, J., and Ismadji, S. 2009. Studies 692 on potential applications of biomass for the separation of heavy metals from water and 693 wastewater. Biochemical Engineering Journal 44(1): 19-41. 694
695 Lira-Silva, E., Ramirez-Lima, I.S., Olin-Sandoval, V., Garcia-Garcia, J.D., Garcia-696
Contreras, R., Moreno-Sanchez, R., and Jasso-Chavez, R. 2011. Removal, accumulation 697 and resistance to chromium in heterotrophic Euglena gracilis. Journal of Hazardous 698
Materials 193: 216-224. 699 700 Livingstone, D.R. 2001. Contaminant-stimulated Reactive Oxygen Species Production 701 and Oxidative Damage in Aquatic Organisms. Marine Pollution Bulletin 42(8): 656-666. 702 703
Mahapatra, D.M., Chanakya, H.N., and Ramachandra, T.V. 2013. Euglena sp. as a 704 suitable source of lipids for potential use as biofuel and sustainable wastewater treatment. 705
Journal of Applied Phycology 25(3): 855-865. 706 707 Malik, A. 2004. Metal bioremediation through growing cells. Environment International 708 30(2): 261-278. 709 710 Malkoc, E., and Nuhoglu, Y. 2005. Investigations of nickel (II) removal from aqueous 711
22
solutions using tea factory waste. Journal of Hazardous Materials 127(1): 120-128. 712
713 Matos, G.D., and Arruda, M.A.Z. 2003. Vermicompost as natural adsorbent for removing 714 metal ions from laboratory effluents. Process Biochemistry 39(1): 81-88. 715
716 Mehta, S.K., and Gaur, J.P. 2001. Removal of Ni and Cu from single and binary metal 717 solutions by free and immobilized Chlorella vulgaris. European Journal of Protistology 718 37(3): 261-271. 719 720
Mehta, S.K., and Gaur, J.P. 2005. Use of algae for removing heavy metal ions from 721 wastewater: progress and prospects. Critical Reviews in Biotechnology 25(3): 113-152. 722 723 Mehta, S.K., Tripathi, B.N., and Gaur, J.P. 2002. Enhanced sorption of Cu
2+ and Ni
2+ by 724
acid-pretreated Chlorella vulgaris from single and binary metal solutions. Journal of 725 Applied Phycology 14(4): 267-273. 726
727 Mendoza-Cozatl, D.G., and Moreno-Saqnchez, R. 2005. Cd 2+ transport and storage in 728
the chloroplast of Euglena gracilis. Biochimica et Biophysica Acta (BBA)-Bioenergetics 729 1706(1): 88-97. 730 731
Mendoza-Cozatl, D.G., Rangel-Gonzalez, E., and Moreno-Sanchez, R. 2006. 732 Simultaneous Cd2+, Zn2+, and Pb2+ uptake and accumulation by photosynthetic 733
Euglena gracilis. Archives of environmental contamination and toxicology 51(4): 521-734 528. 735 736
Miletto, M. 2015. Water and Energy nexus: findings of the World Water Development 737
Report 2014. Proceedings of the International Association of Hydrological Sciences 366: 738 93-99. 739 740
Monteiro, C.M., Castro, P.M.L., and Malcata, F.X. 2012. Metal uptake by microalgae: 741 underlying mechanisms and practical applications. Biotechnology progress 28(2): 299-742
311. 743 744
Nakatsu, C., and Hutchinson, T.C. 1988. Extreme metal and acid tolerance of Euglena 745 mutabilis and an associated yeast from Smoking Hills, Northwest Territories, and their 746 apparent mutualism. Microbial ecology 16(2): 213-231. 747 748 Navarro, L., Torres-Marquez, M.E., Gonzalez-Moreno, S., Devars, S., Hernandez, R., 749
and Moreno-Sanchez, R. 1997. Comparison of Physiological Changes in Euglena gracilis 750 During Exposure to Heavy Metals of Heterotrophic and Autotrophic Cells. Comparative 751
Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology 752 116(3): 265-272. 753 754 Nyholm, N. 1990. Expression of results from growth inhibition toxicity tests with algae. 755 Archives of environmental contamination and toxicology 19(4): 518-522. 756 757
23
Olaveson, M.M., and Nalewajko, C. 2000. Effects of acidity on the growth of two 758
Euglena species. Hydrobiologia 433(1-3): 39-56. 759 760 Papadopoulos, A., Fatta, D., Parperis, K., Mentzis, A., Haralambous, K.J., and Loizidou, 761
M. 2004. Nickel uptake from a wastewater stream produced in a metal finishing industry 762 by combination of ion-exchange and precipitation methods. Separation and purification 763 technology 39(3): 181-188. 764 765 Park, D., Yun, Y.-S., and Park, J.M. 2010. The past, present, and future trends of 766
biosorption. Biotechnology and Bioprocess Engineering 15(1): 86-102. 767 768 Perales-Vela, H.V., Pena-Castro, J.M., and Canizares-Villanueva, R.O. 2006. Heavy 769 metal detoxification in eukaryotic microalgae. Chemosphere 64(1): 1-10. 770
771 Piccinni, E. 1989. Response to heavy metals of uni- and multicellular organisms: 772
homologies and analogies. Italian Journal of Zoology 56(3): 265-271. 773 774
Pinto, E., Sigaud-Kutner, T., Leitao, M.A.S., Okamoto, O.K., Morse, D., and Colepicolo, 775 P. 2003. Heavy metal induced oxidative stress in algae 1. Journal of Phycology 39(6): 776 1008-1018. 777
778 Razzak, S.A., Hossain, M.M., Lucky, R.A., Bassi, A.S., and de Lasa, H. 2013. Integrated 779
CO 2 capture, wastewater treatment and biofuel production by microalgae culturing: a 780 review. Renewable and Sustainable Energy Reviews 27: 622-653. 781 782
Rodriguez-Zavala, J.S., Garcia-Garcia, J.D., Ortiz-Cruz, M.A., and Moreno-Sanchez, R. 783
2007. Molecular mechanisms of resistance to heavy metals in the protist Euglena 784 gracilis. Journal of Environmental Science and Health Part A 42(10): 1365-1378. 785 786
Romera, E., Gonzalez, F., Ballester, A., Blazquez, M.L., and Munoz, J.A. 2007. 787 Comparative study of biosorption of heavy metals using different types of algae. 788
Bioresource Technology 98(17): 3344-3353. 789 790
Ruiz-Manriquez, A., Magana, P.I., Lopez, V., and Guzman, R. 1998. Biosorption of Cu 791 by Thiobacillus ferrooxidans. Bioprocess Engineering 18(2): 113-118. 792 793 Santiago-Martinez, M.G., Lira-Silva, E., Encalada, R., Pineda, E., Gallardo-Perez, J.C., 794 Zepeda-Rodriguez, A., Moreno-Sanchez, R., Saavedra, E., and Jasso-Chavez, R. 2015. 795
Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions. 796 Journal of Hazardous Materials 288: 104-112. 797
798 Schiewer, S., and Wong, M.H. 2000. Ionic strength effects in biosorption of metals by 799 marine algae. Chemosphere 41(1): 271-282. 800 801 Starodub, M.E., Wong, P.T.S., Mayfield, C.I., and Chau, Y.K. 1987. Influence of 802 complexation and pH on individual and combined heavy metal toxicity to a freshwater 803
24
green alga. Canadian Journal of Fisheries and Aquatic Sciences 44(6): 1173-1180. 804
805 Tarley, C.s.R.T., and Arruda, M.A.l.Z. 2004. Biosorption of heavy metals using rice 806 milling by-products. Characterisation and application for removal of metals from aqueous 807
effluents. Chemosphere 54(7): 987-995. 808 809 Vijayaraghavan, K., and Balasubramanian, R. 2015. Is biosorption suitable for 810 decontamination of metal-bearing wastewaters? A critical review on the state-of-the-art 811 of biosorption processes and future directions. Journal of Environmental Management 812
160: 283-296. 813 814 Volesky, B. 2003. Sorption and biosorption. BV Sorbex. 815 816
Volesky, B., and Holan, Z.R. 1995. Biosorption of heavy metals. Biotechnology progress 817 11(3): 235-250. 818
819 Wang, J., and Chen, C. 2009. Biosorbents for heavy metals removal and their future. 820
Biotechnology advances 27(2): 195-226. 821 822 Wong, J.P.K., Wong, Y.S., and Tam, N.F.Y. 2000. Nickel biosorption by two chlorella 823
species, C. Vulgaris (a commercial species) and C. Miniata (a local isolate). Bioresource 824 Technology 73(2): 133-137. 825
826 Zouboulis, A.I., Matis, K.A., and Hancock, I.C. 1997. Biosorption of metals from dilute 827 aqueous solutions. Separation and Purification Methods 26(2): 255-295. 828
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Equilibrium and kinetic studies of Cu (II) and Ni (II) sorption and accumulation on 839
living Euglena gracilis 840
Cameron Winters 841
842
Environment and Life Sciences Graduate Program, Trent University, 843
Peterborough, ON, Canada 844
845
846
847
848
849
850
851
26
Abstract 852
Biosorption and bioaccumulation involve the decrease in concentration or removal of 853
metals from aqueous solution through the sequestering of ions by active or metabolically 854
inert biomass. In this study, the potential use of Euglena gracilis, a free-floating, 855
flagellated unicellular species of protist, to remove Cu (II) and Ni (II) ions at 856
environmentally relevant levels from aqueous solutions was investigated. Adsorption 857
isotherms were used in a batch system to describe the kinetic and equilibrium 858
characteristics of metal removal. The effects of pH and initial concentration of metal ions 859
on the adsorption of Cu (II) and Ni (II) ions were examined by fitting experimental data 860
to the Langmuir and Freundlich models. Results indicate that the sorption reaction 861
occurs quickly (<30min) in both Cu (II) and Ni (II) mono-metallic systems and 862
adsorption follows a pseudo-second order kinetics model for both metals. Maximum 863
sorption capacities were found to be greatest at pH 5 for both metals. Removal 864
efficiencies for Cu (II) decreased with higher initial concentrations (3-30 ppb) and 865
conformed to both Langmuir and Freundlich sorption models. Ni (II) removal was found 866
to increase with greater initial concentration values (5-110 ppm) and conformed to the 867
Freundlich isotherm. In bi-metallic systems sorption of both metals appears to be 868
equivalent and results suggest that Euglena may be more appropriate for mining effluent 869
metal removal than comparable organisms due to their ability to simultaneously tolerate, 870
sorb, and accumulate multiple metals in solution in a range of environmental conditions. 871
872
873
27
1 Introduction 874
Industrial wastewaters, generated from metal plating, mining, fertilizer 875
production, tannery operations, battery production, pulp and paper, and pesticide 876
production and application, are sources of heavy metal release into the environment 877
which not only pose an ecological threat, but also may present serious consequences for 878
human health (Borba et al., 2006 Fu and Wang 2011; Kruetzweiser et al., 2013; Paulino 879
et al., 2006). Consequently, there exists a necessity for the efficient removal of toxic 880
metals from industrial effluent before any potential exposure to surface and ground 881
waters. 882
Mining operations have historically contributed to elevated levels of metals in the 883
surrounding environment (Adamo et al., 2012; Keller et al., 2007). Conventional 884
methods of metal removal can include chemical precipitation, resin-based ion exchange, 885
and activated carbons as well as physical methods which utilize filtration, floatation, and 886
coagulation (Fu and Wang 2011). These methods, however, can tend to generate large 887
capital and operational costs, substantial energy requirements, and large volumes of toxic 888
waste materials (Wang and Chen 2009). In addition, these procedures also tend to lack 889
effectiveness at lower (< 100 mg/L) concentrations and can become prohibitively 890
expensive in terms of the volume of wastewater to be treated (Volesky 2001). 891
The need for an alternative process for the removal of heavy metal ions from 892
aqueous solutions has spawned a substantial amount of research regarding the potential 893
and effectiveness of utilizing biological material to bind (and subsequently remove) 894
contaminants from wastewater (Anastopoulos and Kyzas 2015; Gadd 2009; Gupta et al., 895
2015; Kratochvil and Volesky 1998). Living microalgae and other eukaryotic organisms 896
28
have been identified as particularly effective potential biosorbents due to their ability to 897
tolerate high concentrations of metals while growing, low production costs, high surface 898
to area ratios, ubiquity, and ability to remove metals of relatively dilute concentrations 899
(Malik 2004; Monteiro et al., 2012; Perales-Vela et al., 2006). Euglena gracilis is a 900
free-floating, flagellated unicellular species of protist which has been found to tolerate 901
and accumulate heavy metals (Rodriguez-Zavala et al., 2007). Its capacity to tolerate a 902
broad range of pH conditions, in addition to its proven tolerance to heavy metals such as 903
Cd, Cr, and Hg, identifies Euglena as a potential candidate for bioremediation purposes 904
(Mendoza-Cozatl et al., 2006; Olaveson and Nalewajko 2000). 905
The overall goal of this study was to evaluate Euglena gracilis as a potential 906
biosorbent for metal removal from solution. Specifically, we evaluated the performance 907
of live Euglena gracilis in sequestering Cu and Ni in single- and binary-metal systems 908
(which more realistically reflect actual effluent composition) at two different pH values 909
to determine adsorption isotherms and the amounts of Cu and Ni taken up by algal cells. 910
2 Materials and Methods 911
2.1 Test organism, medium and culture conditions 912
Euglena gracilis Klebs were obtained from Boreal Laboratory Supplies Ltd (St. 913
Catharines, ON, Canada). Non-axenic cultures were grown in medium consisting of 914
0.01 g L −1
CaCl 2 (Bishop Canada Ltd), 1.0 g L −1
CH3 COONa.3H2O (Caledon Ltd., 915
Canada), 1.0 g L −1
‘Lab-Lemco’ powder, 2.0 g L −1
tryptone and 2.0 g L −1
yeast extract 916
(Oxoid LDD, Basingstoke, Hampshire, England). Enumeration of Euglena cells was 917
performed with a 0.1 mm Neubauer hemacytometer (Hausser Scientific, USA). All 918
29
media was prepared using Milli-Q water. The pH of the medium was adjusted using 1M 919
HCl or NaOH after autoclaving and maintained between pH 3-5 at 20°C in a Conviron 920
(CMP5090) environmental chamber (Controlled Environments Ltd., Winnipeg, MB, 921
Canada). E. gracilis were grown under a photoperiod of 18:6 (light-to-dark) at an 922
intensity of 210 μmol/ m2/s. Glassware was immersed in 20% HNO3 prior to use for at 923
least 24h and triple-rinsed with Milli-Q water to avoid metal contamination. In addition, 924
any glassware used for culture growth was autoclaved to mitigate bacterial 925
contamination. 926
Toxicity assays (EC50) of these metals to Euglena were performed to establish a 927
50% response to increasing amounts of both Cu and Ni (Bruce and Versteeg 1992). A 4-928
parameter logistic model was utilized to describe the concentration value (Cu2+
64μg L-1
-929
500mg L-1
; Ni2+
0.6mg L-1
-140mg L-1
) at which a 50% mortality response occurs 930
(SigmaPlot Version 12.0). The model is defined as the following equation: 931
932
933
where y is the observed response as dependent variable; x is the test metal concentration 934
as independent variable; c is the inflection point (EC50); a is the limiting response as x 935
approaches zero; d is the effect at infinite x concentration; and b is the Hill-slope 936
coefficient (negative when response decreases with increasing dose (e.g. mortality). 937
938
939
𝑦 = 𝑑 +𝑎 − 𝑑
1 + (𝑥𝑐)
−𝑏 (1)
30
2.2 Experimental procedure 940
2.2.1 Metal solutions 941
Copper (II) and nickel (II) stock solutions (0.01 mol L-1
) were prepared with 942
CuSO4●5H2O (Caledon Laboratory Chemicals) and NiSO4●6H2O (BDH Chemicals) 943
respectively. The pH of working metal solutions was adjusted with 0.1 mol L-1
HCl and 944
0.1 mol L-1
NaOH (Accumet, XL15, USA). Actual metal concentrations were 945
determined utilizing inductively coupled plasma mass spectrometry (ICPMS) (X Series 946
II, ThermoScientific, USA). 947
2.2.2 Cu2+ and Ni2+ Biosorption 948
The amount of Cu2+
and Ni2+
adsorbed at equilibrium, q (μg g-1
) was calculated 949
with the following equation: 950
(2) 951
where Ci is the initial concentration of the metal ion prior to adsorption (μg L-1
) and Ceq is 952
the equilibrium concentration of metal ions in the aqueous phase. V is the volume (L) of 953
the aqueous phase and m is the dry-weight mass of the adsorbent (g). Each experiment 954
was performed in duplicate and the results are presented as averages. All biosorption 955
experiments were performed utilizing the batch technique. 956
2.2.3 Bioaccumulation: sorption kinetics 957
The biosorption kinetics tests were performed at a constant temperature (20°C) in 958
125 ml Erlenmeyer flasks containing E. gracilis (biomass concentration standardized to 1 959
g L-1
) suspended in growth media spiked with metal solutions of either Cu (II) and/or Ni 960
𝑞 =(𝐶𝑖 − 𝐶𝑒𝑞)𝑉
𝑚
31
(II). Kinetic studies were conducted at pH 5.0 and 7.5 and magnetically stirred at 70 rpm 961
for 240 min. Sorption kinetics were evaluated at two different initial concentrations for 962
each metal which reflect environmentally relevant levels: 20 μg L-1
and 50 μg L-1
for Cu 963
and 3 mg L-1
and 100 mg L-1
for Ni in mono-metallic systems only. Aliquots (7 mL) 964
were removed from solution at pre-determined intervals over the time-course of the 965
experiment, 0.7 μm-filtered (GFF, Merck Millipore, Ireland), acidified (ultrapure HNO3) 966
to pH 2.0 and supernatant metal concentrations were measured by ICP-MS. 967
2.2.4 Bioaccumulation: sorption equilibria 968
Sorption of Cu and Ni on living E. gracilis (biomass concentration standardized 969
to g L-1
dry weight) was examined in batch adsorption-equilibrium experiments (120 970
min) at a constant temperature (20°C) in 125 mL Erlenmeyer flasks. Stock solutions of 971
each metal were prepared as described above from which a range of metal concentrations 972
were obtained and added to flasks containing Euglena (biomass standardized to g L-1
). 973
Blank trials without adsorbent and trials without added metal solution were performed for 974
each tested metal concentration. The effect of target metal concentration was studied at 975
pH 5.0 in mono-metallic solutions with concentrations ranging from 3 μg L-1
to 40 μg L-1
976
(for Cu) and from 5 μg L-1
to 110 μg L-1
for Ni. Binary metal solutions were prepared 977
with a range of Cu2+
and Ni2+
concentrations (15-40 μg L-1
Cu; 7-115 μg L-1
Ni) in 125 978
mL Erlenmeyer flasks. Metal concentrations were chosen to reflect levels (e.g. μg L-1
) 979
which have been reported in mining effluent after treatment in a wastewater facility 980
(Hruska and Dube 2004). The pH levels of both mono- and bimetallic solutions were 981
maintained at 5.0 over the duration of the experiments with additions of 0.1M HCl and/or 982
0.1M NaOH. Flasks were magnetically agitated at 70 rpm and temperature was held 983
32
constant at 20°C. Aliquots (7 mL) were taken, filtered, and metal concentrations were 984
determined using ICP-MS. 985
2.3 Data Analyses 986
2.3.1 Sorption kinetic models 987
Adsorption kinetics models were used to evaluate the overall rate of Cu (II) or Ni 988
(II) removal from Euglena–single metal solutions. Kinetic studies were carried out for 989
sorption of Cu2+
and Ni2+
as a function of contact time at two initial concentrations for 990
each metal (Cu2+
= 20 and 50 μg L-1
; Ni2+
= 3 and 100 mg L-1
) at both pH 5 and 7.5 on 991
live Euglena gracilis. Samples were taken at time intervals of 10, 20, 30, 60, 90, 120, 992
and 240 minutes. Two different, commonly used, and easily interpreted models were 993
employed based on their simplicity and their capacity to show the effects of 994
environmental factors (Ho et al., 2000): 995
The pseudo-first-order kinetic equation (PFO; Lagergren 1898): 996
𝑞𝑡 = 𝑞𝑒(1 − 𝑒−𝑘𝑡) (3) 997
where qe is the amount of metal adsorbed (Cu in μg g-1
, Ni in mg g-1
) at equilibrium, qt 998
is the amount of metal adsorbed (Cu in μg g-1
, Ni in mg g-1
) at time t, k1 is the PFO 999
equilibrium rate constant (min-1
) and t is contact time (min). 1000
The pseudo-second-order kinetic equation (PSO; Ho et al., 1996): 1001
(4) 1002
where qe and qt are metal adsorbed at equilibrium and time t, respectively, k2 is the PSO 1003
equilibrium rate constant (Cu in g μg-1
h-1
; Ni in g mg-1
h-1
), and t is contact time. 1004
𝑞𝑡 =𝑞𝑒𝑘2𝑡
1 + 𝑞𝑒𝑘2𝑡
33
2.3.2 Sorption isotherm models 1005
The Langmuir and Freundlich isotherm models were used to analyze biosorption 1006
data. These models were selected due to their prevalence in biosorption studies (e.g. 1007
enabling comparisons between sorbents) and the relative simplicity of interpreting model 1008
parameters (e.g. sorption capacity and intensity). The Langmuir model, which operates 1009
on the assumptions that adsorption occurs in a monolayer on the solid, all sites are 1010
identical and may sorb only a single molecule, and are independent of adjacent site 1011
sorption, may be described using the following non-linear equation: 1012
𝑞𝑒 =𝑞𝑚𝑏𝐶𝑒𝑞
1+𝑏 𝐶𝑒𝑞 (5) 1013
where qe is metal adsorbed at equilibrium, Ceq is the equilibrium concentration of metal in 1014
solution (μg L-1
), qm is the maximum sorption capacity of surface sites, and b is the 1015
Langmuir equilibrium constant (L μg-1
) which relates to the energy of adsorption and 1016
represents the affinity of the sorbent to the metal. The Freundlich isotherm model is an 1017
empirical formulation which assumes heterogeneous surface adsorption and may be 1018
expressed as: 1019
𝑞𝑒 = 𝐾𝑓𝐶𝑒𝑞1/n
(6) 1020
where qe is metal adsorbed at equilibrium, Kf is a Freundlich constant related to sorption 1021
capacity ((μg g-1
)(μg-1
L)1/n
), and 1/n is a constant related to sorption intensity. 1022
2.4 Statistics: t-tests 1023
34
All the experiments were conducted in duplicate. The obtained data were then 1024
analyzed using paired t-tests to evaluate differences in metal sorption between different 1025
pH levels and different initial metal concentrations. 1026
3 Results and Discussion 1027
3.1Toxicity assays 1028
EC50 assay values in single-metal systems (Figure 1) for Cu was 39.2 mg L-1
and 1029
for Ni was 27.4 mg L-1
. These values were similar to previous studies and increased 1030
from Ni to Cu (Cu 57 mg L-1
and Ni 23 mg L-1
) as reported elsewhere for Euglena 1031
gracilis (Olaveson and Nalewajko 2000). EC50 values reported here suggest that E. 1032
gracilis has the capability to effectively ameliorate toxicity driven by relatively high 1033
concentrations of Cu and Ni. 1034
3.2Sorption Kinetics 1035
Cu kinetics followed the PSO model (r2 > 0.978) whereas Ni exhibited much less 1036
consistency (r2
0.48-0.92) which could be attributed to metabolically driven cell processes 1037
such as active efflux of metal ions or production of metal-binding proteins such as 1038
phytochelatins and/or metallothioneins (Rodriguez-Zavala et al., 2007). Kinetic models 1039
have historically been developed to evaluate the rate-controlling step(s) of the removal 1040
process which may include both the transport of the solute from the aqueous to the solid 1041
phase as well as chemical reactions that may bind or render the solute inert (Febrianto et 1042
al., 2009). Generally, it is assumed that the mass transfer of solutes occurs in two basic 1043
steps: 1) between the liquid phase and the sorbent (fast) and, 2) across the cell boundary 1044
35
layer (slow) (Michalak et al., 2013). The results in this study indicate that the 1045
bioaccumulation of both metals, Cu and Ni, occurred rapidly within 10-30 minutes and 1046
plateaued within 60-90 minutes (Figure 2-3). For this reason, the time duration for the 1047
batch sorption experiments was established at 2h to ensure that equilibrium would be 1048
obtained. It has been suggested that the initial phase may include physical adsorption 1049
(electrostatic attraction) or chemical adsorption (covalent bonding) at the surface of the 1050
cell followed by a slower-paced phase that can include processes such as complexation, 1051
absorption, or the saturation of available binding sites (Gupta et al., 2006). In these 1052
terms, the capacity for metal removal is not only affected by metabolic processes but is 1053
also related to the quantity and nature of active binding sites on the sorbing biomass (Ho 1054
and McKay 1998). The values of qe and k2 (Table 1) were generally found to be in a 1055
similar order of magnitude to those reported in the literature based on living biomass 1056
(Kizilkaya et al. 2012; Markou et al. 2015; Mehta and Gaur 2002). Comparable kinetic 1057
parameters were found using other eukaryotes, green algae, and cyanobacteria (Markou 1058
et al., 2015; Mehta and Gaur 2001; Rafjur et al., 2010). These parameters are an integral 1059
component of designing biologically based water treatment systems in terms of 1060
identifying the rate controlling steps (e.g. mass transport processes and/or chemical 1061
reactions) which not only determine retention times but also contribute to removal 1062
efficacy (Febrianto et al., 2009). The results obtained in this study (Table 1) conform to 1063
published findings regarding the effect of initial metal concentration on algal sorption of 1064
Cu and Ni (Doshi et al. 2008; Kizilkaya et al., 2012; Li et al., 2012; Mehta and Gaur 1065
2001). 1066
36
Although no difference in sorption rate was discerned (p> 0.05), the amount of Cu 1067
and Ni sorbed was found to significantly increase with higher initial concentrations at 1068
both pH levels tested (p< 0.05). Nickel at higher concentrations however (100 mg L-1
), 1069
showed a significant increase in the amount of metal sorbed at a higher pH (p< 0.05). 1070
This may be due to the more severe toxicological effects which Ni has on Euglena as 1071
compared to Cu. The initial Ni2+
concentration (100 mg L-1
) exceeds the EC50 obtained 1072
in this study (27 mg L-1
) thus it may be assumed that the Euglena cells were already 1073
under severe stress, modifying metal sorption. Both the initial concentration of metals in 1074
solution and the pH of the system have been shown to be important factors affecting the 1075
bioaccumulation of metals by living micro-organisms. Initial metal concentration has 1076
also been shown to affect the removal (ie. how sorption rate/capacity changes) by 1077
Euglena gracilis of other heavy metals such as Cd and Cr from aqueous solutions 1078
(Devars et al., 1998; Rocchetta et al., 2003). In general, the sorption of heavy metals 1079
from solution increases as the initial concentration increases until a saturation point is 1080
reached at which all available binding sites are occupied (Mehta and Gaur 2005). 1081
In this study it was found generally that at a higher pH sorption activity was 1082
significantly reduced (p< 0.05) though the rate constant k2 was unaffected (p > 0.05; 1083
Table 1). Previous studies have shown that pH is the most important environmental 1084
parameter affecting the process of biosorption and bioaccumulation in both living and 1085
immobilized algal biosorbents (Al-Rub et al., 2006; Li et al., 2012; Rao et al., 2005). 1086
The pH of a solution controls in large part both the solubility and speciation of metal 1087
ions. Cu2+
is the dominant form of copper ions at pH < 5.5, while Ni2+
is the most 1088
prevalent form of nickel ions at pH < 6.5 whereas at higher pHs oxides and hydroxides 1089
37
are formed and the potential for precipitation increases (Cornelis 2005). Additionally, 1090
the concentration and charge of the main functional groups (e.g. carboxyl groups) and on 1091
the surface of the sorbent varies with pH (Chojnacka et al., 2005; Plaszinski 2013). At 1092
lower pH levels functional groups are occupied by H+
ions that interfere with the binding 1093
of metal cations. As pH increases these functional groups are deprotonated and the 1094
negative charge of the cell surface increases and results in a greater capacity for metal 1095
binding (Mehta et al., 2002). The reduction of metal sorbed at pH 7.5 may be due to the 1096
formation of organic-metal complexes and thus a reduction of free and/or small inorganic 1097
metal species available for uptake by the organism. This result is in contrast with other 1098
reports present in the literature which have generally found that metal sorption in algal 1099
micro-organisms is greater with increasing pH (Mehta and Gaur 2005). A common 1100
response of algae to metal stress is the production of dissolved, organic exudate which 1101
can serve to decrease metal availability by extracellular complexation and/or exporting 1102
complexes from within the cell (Croot and Moffett 2000; Marsalek and Rojickova 1996; 1103
Moffett and Brand 1996; Perales-Vela et al., 2006). E. gracilis also have been shown to 1104
experience reduced growth rates in addition to morphological deformities at pH values 1105
greater than 7 (Olaveson and Nalewajko 2000). 1106
3.3 Sorption equilibria 1107
The sorption of Cu2+
and Ni2+
to the protist Euglena gracilis in single and binary 1108
metal systems was also investigated. The sorption uptake in single-metal solution 1109
increased with increasing equilibrium concentration (Figure 4-5). The degree of fit (r2) to 1110
the Freundlich equation was found to effectively (p<0.001 for both metals) describe the 1111
sorption of Ni (0.958) and Cu (0.876). Freundlich Kf values for Ni and Cu respectively 1112
38
(0.005-0.071: Table 2) were lower than those reported for other species of algae at 1113
various, and generally much higher, (e.g. mg L-1
) concentrations (Doshi et al., 2008; 1114
Markou et al., 2015; Mehta and Gaur 2001; Tien et al., 2002; Wong et al., 2000). Mehta 1115
and Gaur (2001) reported C. vulgaris Kf values for Cu and Ni as 1.22 and 1.19. 1116
Freundlich 1/n values were 0.982 and 1.92 for Cu and Ni respectively. An n value > 1 1117
(ie. 1/n < 1) indicates a favourable sorption of Cu (n=1.02) whereas an n value < 1 (ie. 1118
1/n > 1) which is indicative of a less favourable sorption reaction, was found between Ni 1119
(n=0.521) and the adsorbent. Tien et al., (2002) reported slightly less favourable results 1120
for Cu (n = 0.78) and Wong et al., (2000) found n values for Ni sorption on C. vulgaris 1121
ranging from 1.33-1.51. In these terms, E. gracilis appears to more efficiently remove 1122
Cu from aqueous solution when compared to Ni removal. 1123
The interactive effect of Cu and Ni in a binary metal system was also examined. 1124
Figure 5 presents metal sorption (qe) as a function of the ratio of Cu:Ni initial 1125
concentration. Compared to single-metal solutions (Figure 4) Cu and Ni sorption in 1126
binary solutions was not significantly different (p > 0.05). Total metal uptake of both Cu 1127
and Ni in binary solutions increased when compared to single-metal solutions however 1128
only Cu uptake was significantly higher (p< 0.05). This suggests that different sorption 1129
mechanisms could be involved or that specific binding sites exist for each metal (Chong 1130
et al., 2000; Mallick 2003). Additionally, transition metals have been reported to share 1131
common transporters localized both intracellularly (e.g. P-type ATPase) and at the cell 1132
membrane (e.g. NRAMP, CTR) which could account for higher total metal sorption in 1133
binary solutions (Blaaby-Haas and Merchant 2012). Metal sorption by algal cells has 1134
been characterized as an extremely dynamic process in which a number of different 1135
39
mechanisms may operate and thus may prompt both synergistic and antagonistic 1136
interactions between metals and binding sites (Flouty and Estephane 2012). Sorption of 1137
Cu and Ni at initial concentration ratios both lesser than and greater than one were 1138
comparable. These results suggest that Cu and Ni may not produce inhibitory effects on 1139
the sorption of the other metal. This result is contrary to some Cu/Ni sorption studies of 1140
other eukaryotes (Keshtkar et al., 2015; Mehta et al., 2002). Sorption of metal ions has 1141
been linked with ionic radii and electronegativity which could account for the similar 1142
response between Cu and Ni uptake both of which share comparable physiochemical 1143
characteristics (Flouty and Estephane 2012). Sorption of both metals appears to be 1144
equivalent in binary solutions. This is an important characteristic because interactive 1145
effects of metals can reduce overall metal removal and effluent rarely contains only one 1146
metal contaminant (Hruska and Dube 2004; Mehta et al., 2001). This suggests that 1147
Euglena may be more appropriate for mining effluent metal removal than comparable 1148
organisms due to their ability to simultaneously tolerate, sorb, and accumulate multiple 1149
metals in solution (Devars et al., 2000; Mendoza-Cozatl et al., 2006) in addition to 1150
efficiently reducing nutrient levels and organic contaminants (Kobayashi and Rittmann 1151
1982; Mahaptra et al., 2013). 1152
4 Conclusions 1153
In this study live Euglena gracilis have been shown to exhibit the capacity to 1154
remove Cu and Ni from single and binary solutions in the < 100 mg L-1
range which, to 1155
our knowledge, has not been studied to date. Sorption kinetics followed the PSO model 1156
and single-metal sorption equilibria followed the Freundlich isotherm model which 1157
40
suggests binding sites of living Euglena are heterogeneous. Removal of Cu and Ni 1158
occurred relatively quickly and increased with reduced pH and increasing initial 1159
concentrations of metal; optimizing these conditions could increase Euglena affinity for 1160
these metals. Euglena gracilis showed a greater sorption capacity for Cu as compared to 1161
Ni in both single-metal and binary solutions and could be a potentially economic and 1162
effective biosorbent for Cu removal. Further work should include an assessment of the 1163
capacity for non-living Euglena biomass to remove Cu and Ni from solution and 1164
optimization of operational parameters to maximize removal as well as the evaluation of 1165
E. gracilis removal performance in actual industrial effluent mixtures of metals, 1166
inorganic and organic ligands. 1167
Acknowledgments 1168
The authors would like to thank Antoine Perroud, the Water Quality Centre at Trent 1169
University and Noble Purification Inc. for their assistance in producing this work. This 1170
research was supported by the Ontario Centres of Excellence. 1171
1172
1173
1174
1175
1176
1177
1178
1179
1180
1181
41
Table 1: Pseudo-second-order kinetics parameters for the sorption of Cu2+
and Ni2+
on living Euglena 1182 gracilis cells (±SE).1183
1184
1185
1186
1187
1188
Table 2: Freundlich adsorption isotherm parameters for the biosorption of Cu2+
and Ni2+
on Euglena 1189 gracilis cells at pH 5 (±SE). 1190
1191
1192
1193
1194
42
1195
Figure 1: Experimental data for EC50 toxicity assay on living E. gracilis for (a) Cu2+
and (b) Ni2. 1196
1197
1198
1199
1200
1201
1202
Figure 2: Experimental data for Cu2+
sorption kinetics on living E. gracilis at initial concentrations 1203 of (a) 20 μg L
-1 and (b) 50 μg L
-1. Curves represent pseudo-second-order kinetic model. Error bars 1204
represent SE. 1205
1206
1207
43
1208
Figure 3: Experimental data for Ni2+
sorption kinetics on living E. gracilis at initial concentrations 1209 of (a) 3 mg L
-1 and (b) 100 mg L
-1. Curves represent pseudo-second-order kinetic model. Error 1210
bars represent SE. 1211
1212
1213
1214
1215
1216
1217
Figure 4: Experimental data for a) Cu2+
and b) Ni2+
sorption equilibrium on living E. gracilis. 1218 Curve represents the Freundlich model. 1219
44
1220
Figure 5: Experimental data for Cu2+
and Ni2+
sorption equilibrium from binary-metal solution on 1221 living E. gracilis. Error bars represent SE. 1222
1223
1224
1225
1226
1227
1228
1229
1230
1231
1232
1233
1234
45
References 1235
Adamo, P., Dudka, S., Wilson, M.J., and McHardy, W.J. 2002. Distribution of trace 1236
elements in soils from the Sudbury smelting area (Ontario, Canada). Water, Air, and Soil 1237 Pollution 137(1-4): 95-116. 1238 1239 Al-Rub, F.A.A., El-Naas, M.H., Ashour, I., and Al-Marzouqi, M. 2006. Biosorption of 1240 copper on Chlorella vulgaris from single, binary and ternary metal aqueous solutions. 1241
Process Biochemistry 41(2): 457-464. 1242 1243 Anastopoulos, I., and Kyzas, G.Z. 2015. Progress in batch biosorption of heavy metals 1244 onto algae. Journal of Molecular Liquids 209: 77-86. 1245 1246
Blaby-Haas, C.E., and Merchant, S.S. 2012. The ins and outs of algal metal transport. 1247
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1823(9): 1531-1552. 1248
1249
Borba, C.E., Guirardello, R., Silva, E.A., Veit, M.T., and Tavares, C.R.G. 2006. Removal 1250 of nickel (II) ions from aqueous solution by biosorption in a fixed bed column: 1251 Experimental and theoretical breakthrough curves. Biochemical Engineering Journal 1252
30(2): 184-191. 1253 1254
Bruce, R.D., and Versteeg, D.J. 1992. A statistical procedure for modeling continuous 1255 toxicity data. Environmental toxicology and chemistry 11(10): 1485-1494. 1256 1257
Chojnacka, K., Chojnacki, A., and Gorecka, H. 2005. Biosorption of Cr 3+, Cd 2+ and 1258 Cu 2+ ions by blue-green algae Spirulina sp.: kinetics, equilibrium and the mechanism of 1259
the process. Chemosphere 59(1): 75-84. 1260 1261
Chong, A.M.Y., Wong, Y.S., and Tam, N.F.Y. 2000. Performance of different microalgal 1262 species in removing nickel and zinc from industrial wastewater. Chemosphere 41(1): 1263 251-257. 1264
1265 Cornelis, R. 2005. Handbook of Elemental Speciation, Handbook of Elemental 1266
Speciation II: Species in the Environment, Food, Medicine and Occupational Health. 1267 John Wiley & Sons. 1268 1269
Croot, P.L., Moffett, J.W., and Brand, L.E. 2000. Production of extracellular Cu 1270 complexing ligands by eukaryotic phytoplankton in response to Cu stress. Limnology and 1271 Oceanography 45(3): 619-627. 1272
1273
Devars, S., Hernandez, R., and Moreno-Sanchez, R. 1998. Enhanced heavy metal 1274 tolerance in two strains of photosynthetic Euglena gracilis by preexposure to mercury or 1275 cadmium. Archives of environmental contamination and toxicology 34(2): 128-135. 1276 1277 Febrianto, J., Kosasih, A.N., Sunarso, J., Ju, Y.-H., Indraswati, N., and Ismadji, S. 2009. 1278 Equilibrium and kinetic studies in adsorption of heavy metals using biosorbent: a 1279 summary of recent studies. Journal of Hazardous Materials 162(2): 616-645. 1280
46
Flouty, R., and Estephane, G. 2012. Bioaccumulation and biosorption of copper and lead 1281
by a unicellular algae Chlamydomonas reinhardtii in single and binary metal systems: a 1282 comparative study. Journal of Environmental Management 111: 106-114. 1283 1284
Fu, F., and Wang, Q. 2011. Removal of heavy metal ions from wastewaters: a review. 1285 Journal of Environmental Management 92(3): 407-418. 1286 1287 Gadd, G.M. 2009. Biosorption: critical review of scientific rationale, environmental 1288 importance and significance for pollution treatment. Journal of Chemical Technology and 1289
Biotechnology 84(1): 13-28. 1290 1291 Gupta, V.K., Nayak, A., and Agarwal, S. 2015. Bioadsorbents for remediation of heavy 1292 metals: Current status and their future prospects. Environmental Engineering Research 1293
20(1): 1-18. 1294 1295
Gupta, V.K., Rastogi, A., Saini, V.K., and Jain, N. 2006. Biosorption of copper (II) from 1296 aqueous solutions by Spirogyra species. Journal of Colloid and Interface Science 296(1): 1297
59-63. 1298 1299 Ho, Y.S., Ng, J.C.Y., and McKay, G. 2000. Kinetics of pollutant sorption by biosorbents: 1300
review. Separation & Purification Reviews 29(2): 189-232. 1301 1302
Ho, Y.S., and McKay, G. 1998. A comparison of chemisorption kinetic models applied to 1303 pollutant removal on various sorbents. Process Safety and Environmental Protection 1304 76(4): 332-340. 1305
1306
Ho, Y.S., Wase, D.A.J., and Forster, C.F. 1996. Kinetic studies of competitive heavy 1307 metal adsorption by sphagnum moss peat. Environmental Technology 17(1): 71-77. 1308 1309
Hruska, K.A., and Dubé, M.G. 2004. Using artificial streams to assess the effects of 1310 metal mining effluent on the life cycle of the freshwater midge (Chironomus tentans) in 1311
situ. Environmental toxicology and chemistry 23(11): 2709-2718. 1312 1313
Keller, W., Yan, N.D., Gunn, J.M., and Heneberry, J. 2007. Recovery of acidified lakes: 1314 lessons from Sudbury, Ontario, Canada. In Acid Rain-Deposition to Recovery. Springer. 1315 pp. 317-322. 1316 1317 Kizilkaya, B., Turker, G., Akgul, R., and Dogan, F. 2012. Comparative study of 1318
biosorption of heavy metals using living green algae Scenedesmus quadricauda and 1319 Neochloris pseudoalveolaris: Equilibrium and kinetics. Journal of Dispersion Science 1320
and Technology 33(3): 410-419. 1321 1322 Kobayashi, H., and Rittmann, B.E. 1982. Microbial removal of hazardous organic 1323 compounds. Environmental Science & Technology 16(3): 170A-183A. 1324 1325 Kratochvil, D., and Volesky, B. 1998. Advances in the biosorption of heavy metals. 1326
47
Trends in Biotechnology 16(7): 291-300. 1327
1328 Kreutzweiser, D., Beall, F., Webster, K., Thompson, D., and Creed, I. 2013. Impacts and 1329 prognosis of natural resource development on aquatic biodiversity in Canada’s boreal 1330
zone 1. Environmental Reviews 21(4): 227-259. 1331 1332 Lagergren, S. 1898. About the theory of so-called adsorption of soluble substances. 1333 Kungliga Svenska Vetenskapsakademiens Handlingar 24(4): 1-39. 1334 1335
Li, J., Xie, S., Feng, J., Li, Y., and Chen, L. 2012. Heavy metal uptake capacities by the 1336 common freshwater green alga Cladophora fracta. Journal of Applied Phycology 24(4): 1337 979-983. 1338 1339
Malik, A. 2004. Metal bioremediation through growing cells. Environment International 1340 30(2): 261-278. 1341
1342 Mallick, N. 2003. Biotechnological potential of Chlorella vulgaris for accumulation of 1343
Cu and Ni from single and binary metal solutions. World Journal of Microbiology and 1344 Biotechnology 19(7): 695-701. 1345 1346
Markou, G., Mitrogiannis, D., Celekli, A., Bozkurt, H., Georgakakis, D., and 1347 Chrysikopoulos, C.V. 2015. Biosorption of Cu
2+ and Ni
2+ by Arthrospira platensis with 1348
different biochemical compositions. Chemical Engineering Journal 259: 806-813. 1349 1350 Marsalek, B., and Rojickova, R. 1996. Stress factors enhancing production of algal 1351
exudates: a potential self-protective mechanism? Zeitschrift fur Naturforschung C 51(9-1352
10): 646-650. 1353 1354 Mehta, S.K., and Gaur, J.P. 2001. Removal of Ni and Cu from single and binary metal 1355
solutions by free and immobilized Chlorella vulgaris. European Journal of Protistology 1356 37(3): 261-271. 1357
1358 Mehta, S.K., and Gaur, J.P. 2005. Use of algae for removing heavy metal ions from 1359
wastewater: 1360 progress and prospects. Critical Reviews in Biotechnology 25(3): 113-152. 1361 1362 Mehta, S.K., Tripathi, B.N., and Gaur, J.P. 2002. Enhanced sorption of Cu
2+ and Ni
2+ by 1363
acid-pretreated Chlorella vulgaris from single and binary metal solutions. Journal of 1364
Applied Phycology 14(4): 267-273. 1365 1366
Mendoza-Cozatl, D.G., Rangel-Gonzalez, E., and Moreno-Sanchez, R. 2006. 1367 Simultaneous Cd2+, Zn2+, and Pb2+ uptake and accumulation by photosynthetic 1368 Euglena gracilis. Archives of environmental contamination and toxicology 51(4): 521-1369 528. 1370 1371 Michalak, I., Chojnacka, K., and Witek-Krowiak, A. 2013. State of the art for the 1372
48
biosorption process-a review. Applied biochemistry and biotechnology 170(6): 1389-1373
1416. 1374 1375 Moffett, J.W., and Brand, L.E. 1996. Production of strong, extracellular Cu chelators by 1376
marine cyanobacteria in response to Cu stress. Limnology and Oceanography 41(3): 388-1377 395. 1378 1379 Monteiro, C.M., Castro, P.M.L., and Malcata, F.X. 2012. Metal uptake by microalgae: 1380 underlying mechanisms and practical applications. Biotechnology progress 28(2): 299-1381
311. 1382 1383 Olaveson, M.M., and Nalewajko, C. 2000. Effects of acidity on the growth of two 1384 Euglena species. Hydrobiologia 433(1-3): 39-56. 1385
1386 Paulino, A.T., Minasse, F.A.S., Guilherme, M.R., Reis, A.V., Muniz, E.C., and Nozaki, J. 1387
2006. Novel adsorbent based on silkworm chrysalides for removal of heavy metals from 1388 wastewaters. Journal of Colloid and Interface Science 301(2): 479-487. 1389
1390 Perales-Vela, H.V., Pena-Castro, J.M., and Canizares-Villanueva, R.O. 2006. Heavy 1391 metal detoxification in eukaryotic microalgae. Chemosphere 64(1): 1-10. 1392
1393 Plazinski, W. 2013. Binding of heavy metals by algal biosorbents. Theoretical models of 1394
kinetics, equilibria and thermodynamics. Advances in colloid and interface science 197: 1395 58-67. 1396 1397
Rajfur, M., Klos, A., and Waclawek, M. 2010. Sorption properties of algae Spirogyra sp. 1398
and their use for determination of heavy metal ions concentrations in surface water. 1399 Bioelectrochemistry 80(1): 81-86. 1400 1401
Rao, S.P., Kalyani, S., Suresh Reddy, K.V.N., and Krishnaiah, A. 2005. Comparison of 1402 biosorption of nickel (II) and copper (II) ions from aqueous solution by Sphaeroplea 1403
algae and acid treated Sphaeroplea algae. Separation science and technology 40(15): 1404 3149-3165. 1405
1406 Rocchetta, I., Ruiz, L.B., Magaz, G., and Conforti, V.T.D. 2003. Effects of hexavalent 1407 chromium 1408 in two strains of Euglena gracilis. Bulletin of environmental contamination and 1409 toxicology 70(5): 1045-1051. 1410
1411 1412
Rodriguez-Zavala, J.S., Garcia-Garcia, J.D., Ortiz-Cruz, M.A., and Moreno-Sanchez, R. 1413 2007. Molecular mechanisms of resistance to heavy metals in the protist Euglena 1414 gracilis. Journal of Environmental Science and Health Part A 42(10): 1365-1378. 1415 1416 Tien, C.J. 2002. Biosorption of metal ions by freshwater algae with different surface 1417 characteristics. Process Biochemistry 38(4): 605-613. 1418
49
1419
Volesky, B. 2001. Detoxification of metal-bearing effluents: biosorption for the next 1420 century. Hydrometallurgy 59(2): 203-216. 1421 1422
Wang, J., and Chen, C. 2009. Biosorbents for heavy metals removal and their future. 1423 Biotechnology advances 27(2): 195-226. 1424 1425 Wong, J.P.K., Wong, Y.S., and Tam, N.F.Y. 2000. Nickel biosorption by two chlorella 1426 species, C. Vulgaris (a commercial species) and C. Miniata (a local isolate). Bioresource 1427
Technology 73(2): 133-137. 1428
1429
1430
1431
1432
1433
1434
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1444
1445
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1447
1448
1449
50
Equilibrium and kinetic studies of Cu (II) and Ni (II) biosorption on non-living 1450
Euglena gracilis 1451
Cameron Winters 1452
1453
Environment and Life Sciences Graduate Program, Trent University, 1454
Peterborough, ON, Canada 1455
1456
1457
1458
1459
1460
1461
1462
1463
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1471
51
Abstract 1472
In this study, the use of non- living Euglena gracilis to remove Cu (II) and Ni (II) 1473
ions at environmentally relevant concentrations (0.005 mg L-1
-25 mg L-1
, 0.005 mg L-1-1474
20 mg L-1
, respectively) from aqueous solutions was investigated. Adsorption isotherms 1475
were used in a batch system to describe the kinetic and equilibrium characteristics of 1476
metal removal. Sorption occurred quickly (10-30 min) and both Cu and Ni equilibrium 1477
uptake increased with a concurrent increase of initial metal concentrations. The 1478
biosorption capacity of Cu (II) and) was 3x times greater than that of Ni (II). Structural 1479
characteristics of the biosorbent were assessed using FTIR spectra and showed the 1480
presence of hydrocarbon, carboxylic, and amide functional groups which are involved 1481
with Cu and Ni binding. The pseudo-first-order model was applied to the kinetic data 1482
and the Langmuir and Freundlich models were found to effectively describe single-metal 1483
systems at equilibrium. Euglena showed comparable sorption to other similar 1484
eukaryotic organisms. Biosorption was also studied in Cu + Ni aqueous solutions to 1485
assess competitive uptake in multi-metal systems and showed supressed sorption of one 1486
metal in the presence of relatively high concentrations of the other metal. 1487
1488
1489
1490
1491
1492
52
1 Introduction 1493
Effluent generated from industrial sources has been identified as a major cause of 1494
increasing concentrations of metals in the environment to a point which surpasses the 1495
expected natural abundance of these elements (Nriagu 2006). In Canada, the largest 1496
source of elevated metal release appears to from decommissioned and/or abandoned 1497
mines (e.g. 10,000 inactive mines across Canada) and it has been suggested that peak 1498
concentrations may occur after mining operations have ceased (Ptacek et al., 2004). 1499
Most effluent waters which are discharged from mining operations undergo only primary 1500
treatment (Environment Canada 2002). Their high concentrations (e.g. < 100 mg L-1
) of 1501
dissolved metals can cause serious toxic effects to aquatic organisms (Gopalapillai et al., 1502
2008; Mahdavi et al., 2012; Ponton and Hare 2009). Additionally, expensive effluent 1503
treatments must be applied before discharge to natural water systems. 1504
Several physiochemical methods are currently utilized to ameliorate excessive 1505
concentrations of metals from industrial effluent and reduce the potential for toxic effects 1506
to aquatic and human health (Monteiro et al., 2012). These methods can be highly 1507
effective at removing metals from solution however they are limited by costly 1508
infrastructure and energy expenses (Lesmana et al., 2009). Moreover, chemical methods 1509
of metal remediation have been reported to lack effectiveness at lower (e.g. < 100 mg L-1
) 1510
concentrations and often require more stringent operational parameters than comparable 1511
alternative technologies (Eccles 1995; Vijayaraghavan and Balasubramanian 2015; 1512
Volesky 2001). 1513
As an alternative, the use of biological material for remediation of industrial 1514
wastewater, commonly referred to as biosorption and/or bioaccumulation, offers several 1515
53
advantages over conventional methods which include: the minimization of chemical and 1516
or biological sludge, operation over a broad range of physio-chemical conditions, 1517
relatively low capital investment and operational costs, and an increased efficiency in the 1518
removal of contaminants from dilute effluent (Abbas et al., 2014; Flouty and Estephane 1519
2012). Both living and dead biomass have been successfully utilized for metal removal 1520
and have been studied extensively although the use of dead biomass is becoming the 1521
preferred option in the majority of recent metal removal studies (Fomina and Gadd 2014; 1522
Doshi et al., 2007; Kizilkaya et al., 2012; Kumar et al., 2015; Michalak et al., 2013). 1523
The use of dried biotic material confers several benefits compared to utilizing 1524
metabolically active organisms such as eliminating nutritional requirements, toxicity 1525
thresholds, allowing for desorption and metal recovery processes, and potentially 1526
improving sorption capacity through an increase in the cell surface to area ratio (Donmez 1527
et al., 1999; Kadukova and Vircikova 2005; Mehta and Gaur 2005). Live algal cells are 1528
also limited in terms of metal recovery as ions can be bound intracellularly and/or 1529
metabolic exudates may form complexes with metal ions outside the cell which serve to 1530
retain metals in the aqueous phase (Mehta and Gaur 2005). 1531
Dried biological material has the potential to effectively remove as much as or 1532
greater amounts of metal ions from solution when compared to living algal cells (Burdin 1533
and Bird 1994; Flouty and Estephane 2012; Tien et al., 2005). The characteristics of 1534
dried algae mentioned above contribute in large part to the commercial viability of using 1535
such materials in remediation applications in potentially remote settings where 1536
infrastructure development is constrained by physical and economic limitations. 1537
Additionally, using inert algal material as opposed to living stock for metal removal can 1538
54
result in altered surface chemistry that influences cation sorption (Tien et al., 2005). Cell 1539
surfaces of algal organisms consist of an assortment of polysaccharides, proteins, and 1540
lipids all of which include functional groups (e.g. carboxyl, amino, sulphate and hydroxyl 1541
groups) that are capable of binding metal ions (Crist et al., 1981). Modifications of the 1542
cell surface/structure can occur when utilizing inactivation methods such as heat-drying, 1543
chemical treatments, and vacuum-drying which may result in increased or decreased 1544
sorption capacity and in this sense are dependent on the origin and pre-treatment of the 1545
biological material (Gardea-Torresdey et al., 1990; Mehta and Gaur 2001; Winter et al., 1546
1994). 1547
Euglena gracilis is a free-floating, flagellated unicellular species of protist which 1548
has been found to tolerate and accumulate heavy metals (Rodriguez-Zavala et al., 2007). 1549
Its capacity to tolerate a broad range of pH conditions, in addition to its proven tolerance 1550
to heavy metals such as Cd, Cr, and Hg, identifies Euglena as a candidate for use in 1551
bioremediation of wastewaters (Mendoza-Cozatl et al., 2006; Olaveson and Nalewajko, 1552
2000). The overall goal of this study was to evaluate the performance of inert, freeze-1553
dried Euglena gracilis in sequestering Cu and Ni in single- and binary-metal systems. 1554
Although fundamental sorption processes have been investigated in mono-metallic 1555
solutions, less is known regarding these processes in complex waters (e.g, bi-metallic 1556
system) which are commonly found in wastewaters. Much of the literature regarding 1557
biosorption refers to relatively high concentration ranges (e.g. 10 to > 200 mg L-1
) which 1558
may reflect untreated effluent metal levels but do not correspond to environmentally 1559
relevant ranges (e.g. a threat to aquatic biota) (Gadd 2009). Conventional treatment can 1560
55
remove high metal concentrations (e.g. > 100 mg L-1
) however additional treatments may 1561
be required for effluent to be below water guidelines before release to natural waters. 1562
In this study, kinetic and sorption properties of dried Euglena cells were 1563
assessed in mono (Cu or Ni) and bi-metallic solutions (Cu + Ni) at metal concentrations 1564
typical of wastewaters (Gopalapillai et al., 2008; Mahdavi et al., 2012). Kinetic 1565
modeling of metal sorption is an essential factor in the design, optimization and 1566
commercial application of algal metal removal processes. Kinetic data describes the rate 1567
at which metal ions are taken up by the sorbent and therefore determines the residence 1568
time required for effective removal (Zakhama et al., 2011). 1569
2 Materials and Methods 1570
2.1 Test organism, medium and culture conditions 1571
Euglena gracilis Klebs were obtained from Boreal Laboratory Supplies Ltd (St. 1572
Catharines, ON, Canada). Non-axenic cultures were grown in medium consisting of 0.01 1573
g L CaCl 2 (Bishop Canada Ltd), 1.0 g L
−1 CH3 COONa.3H2O ( Caledon Ltd, Canada), 1574
1.0 g L −1
‘Lab-Lemco’, 2.0 g L −1
tryptone (Oxoid LDD, Basingstoke, Hampshire, 1575
England) and 2.0 g L −1
yeast extract (Oxoid LDD, Basingstoke, Hampshire, England). 1576
All media was prepared using Milli-Q water. The pH of the medium was adjusted using 1577
1M HCl or NaOH after autoclaving and maintained between pH 3-5 at 20°C in a 1578
Conviron (CMP5090) environmental chamber (Controlled Environments Ltd., Winnipeg, 1579
MB, Canada). E. gracilis were grown under a photoperiod of 18:6 (light:dark) at an 1580
intensity of 210 μmol/m2/s. Post-harvest, Euglena biomass was cooled to -80°C (Forma 1581
Scientific, USA) for at least 24 h and subsequently freeze-dried (LabConco, USA) for at 1582
56
least 48 h and milled. Glassware was immersed in 20% HNO3 prior to use for at least 1583
24h and triple-rinsed with Milli-Q water to avoid metal contamination. In addition, any 1584
glassware used for culture growth was autoclaved to mitigate bacterial contamination. 1585
2.2 FTIR analysis 1586
Dried biomass was analysed to identify functional groups with attenuated total 1587
reflectance (ATR) using an ATR-FTIR spectrometer (Nicolet 380, Thermo, USA) in the 1588
absorbance mode (range: 4500-500 cm-1
) with 32 scans at a spatial resolution of 4 cm-1
. 1589
2.3 Experimental procedure 1590
2.3.1 Metal solutions 1591
Cu (II) and Ni (II) stock solutions (0.01mol L-1
) were prepared with CuSO4●5H2O 1592
(Caledon Laboratory Chemicals) and NiSO4●6H2O (BDH Chemicals) respectively. The 1593
pH of working metal solutions was adjusted with 0.1 mol L-1
HCl and 0.1 mol L-1
NaOH 1594
(Accumet, XL15, USA). Metal concentrations were determined utilizing inductively 1595
coupled plasma mass spectrometry (ICPMS) (X Series II, ThermoScientific, USA). 1596
Rhodium was used as an internal standard. The accuracy of the ICP-MS measurements 1597
was assessed using SLEW-3, SLR-4, SLR-5, 1-BIS and 5-BIS certified reference 1598
material (National Research Council, Canada). 1599
1600
1601
1602
1603
57
2.3.2 Cu2+ and Ni2+ Biosorption 1604
The amount of Cu2+
and Ni2+
adsorbed at equilibrium, q (ug g-1
) was calculated 1605
with the following equation: 1606
(1) 1607
where Ci is the initial concentration of the metal ion prior to adsorption (ug L-1
) and Ceq is 1608
the equilibrium concentration of metal ions in the aqueous phase. V is the volume (L) of 1609
the aqueous phase and m is the dry-weight mass of the adsorbent (g). Each experiment 1610
was performed in duplicate and the results are presented as averages. All biosorption 1611
experiments were performed utilizing the batch technique. 1612
2.3.3 Sorption kinetics 1613
Kinetics experiments were performed in duplicate at a constant temperature 1614
(20°C) in 50ml centrifuge tubes (Fisher Scientific) containing E. gracilis (1 g L-1
) 1615
suspended in Milli-Q spiked with metal solutions of either Cu (II) and/or Ni (II). Kinetic 1616
studies were conducted at pH 5.0 and agitated at 70rpm for 240min. Kinetic studies 1617
were carried out for sorption of Cu2+
and Ni2+
as a function of contact time at four initial 1618
concentrations for each metal (Cu2+
= 20 and 50 μg L-1
, 1 and 25 mg L-1
; Ni2+
= 1, 2, 4, 1619
and 20 mg L-1
) at pH 5 on non-living Euglena gracilis. Aliquots (7 mL) were removed 1620
from solution at pre-determined intervals over the time-course of the experiment, 0.7 μm 1621
-filtered (GFF, Merck Millipore, Ireland), acidified (ultrapure HNO3 70%) to pH 2.0 and 1622
metal concentrations were measured by ICP-MS. Adsorption kinetics models were used 1623
to evaluate the overall rate of Cu (II) or Ni (II) removal from Euglena–single metal 1624
𝑞 =(𝐶𝑖 − 𝐶𝑒𝑞)𝑉
𝑚
58
solutions. Samples were taken at time intervals of 0, 10, 20, 30, 60, 90, 120, and 240 1625
minutes. Two different, non-linear models were employed: 1626
The pseudo-first-order kinetic equation (PFO; Lagergren 1898): 1627
𝑞𝑡 = 𝑞𝑒(1 − 𝑒−𝑘𝑡) (2) 1628
where qe is the amount of metal adsorbed (μg g-1
or mg g-1
) at equilibrium, qt is the 1629
amount of metal adsorbed (μg g-1
or mg g-1
) at time t, k1 is the PFO equilibrium rate 1630
constant (min-1
) and t is contact time (min). 1631
The pseudo-second-order kinetic equation (PSO; Ho et al., 1996): 1632
(3) 1633
where qe and qt are metal adsorbed at equilibrium and time t, respectively, k2 is the PSO 1634
equilibrium rate constant (g μg-1
h-1
or g mg-1
h-1
), and t is contact time. 1635
2.3.4 Sorption equilibria 1636
Sorption of Cu (II) and Ni (II) on non-living E. gracilis (1 g L-1
) were examined 1637
in batch adsorption-equilibrium experiments in duplicate (120 min) at a constant 1638
temperature (20°C) and agitated at 70 rpm. Blank trials without Euglena cells and trials 1639
without added metal solution were performed for each tested metal concentration. The 1640
pH levels of both mono- and bimetallic solutions were maintained at 5.0 over the 1641
duration of the experiments with additions of 0.1M HCl and/or 0.1M NaOH. The effect 1642
of metal initial concentration was studied at pH 5.0 in mono-metallic solutions with 1643
values ranging from 5 μg L-1 to 1.5 mg L
-1 (Cu) and 5 μg L
-1 to 1 mg L
-1 (Ni). Bi-1644
𝑞𝑡 =𝑞𝑒𝑘2𝑡
1 + 𝑞𝑒𝑘2𝑡
59
metallic solutions were prepared with initial Cu2+
and Ni2+
concentrations ranging from 1645
10 μg L-1
to 6 mg L-1
and from 2 μg L-1
to 200 μg L-1
respectively. Metal concentrations 1646
were determined using ICP-MS. The Langmuir and Freundlich isotherm models were 1647
used to analyze biosorption data. The Langmuir model, which operates on the 1648
assumptions that adsorption occurs in a monolayer on the solid, all sites are identical and 1649
may sorb only a single molecule, and are independent of adjacent site sorption, may be 1650
described using the following non-linear equation: 1651
𝑞𝑒 =𝑞𝑚𝑏𝐶𝑒𝑞
1+𝑏 𝐶𝑒𝑞 (4) 1652
where qe is metal adsorbed at equilibrium, Ceq is the equilibrium concentration of metal in 1653
solution (μg L-1
), qm is the maximum sorption capacity of surface sites, and b is the 1654
Langmuir equilibrium constant (L μg-1
) which relates to the energy of adsorption and 1655
represents the affinity of the sorbent to the metal. The Freundlich isotherm model is an 1656
empirical formulation which assumes heterogeneous surface adsorption and may be 1657
expressed as: 1658
𝑞𝑒 = 𝐾𝑓𝐶𝑒𝑞1/n
(5) 1659
where qe is metal adsorbed at equilibrium, Kf is a Freundlich constant related to sorption 1660
capacity ((μg g-1
)(μg-1
L)1/n
), and 1/n is a constant related to sorption intensity. 1661
2.4 Statistics: t-tests 1662
Paired t-test analysis was used to evaluate differences in metal sorption between 1663
systems with different initial metal concentrations. 1664
60
3 Results and Discussion 1665
3.1Characterisation of dried Euglena cells 1666
The FTIR spectrum of dried Euglena cells (Figure 1) showed different functional 1667
groups. The intense and strong bands at 3040 and 1640 cm-1
were related to C-H and 1668
C=C in aromatic hydrocarbons. The stretching vibrations of O-H (1700 cm-1
) and C-O 1669
(1380 cm-1
) were attributed to the carboxylic functional group. The C=O in amides was 1670
found at 1640-1660 cm-1
. Overall, dried Euglena cells possessed the main functional 1671
groups (e.g. carboxylic, amino and thiol) for metal binding (Gardea-Torresdey et al., 1672
1990; Mangal et al., 2016). 1673
3.2 Sorption kinetics 1674
Both metals (Cu and Ni) were found to adhere more closely to the PFO model 1675
(Table 1) congruent with previous non-living biomass studies (Liu et al., 2009; Rao et al., 1676
2005; Zakhama et al., 2011). A strong agreement with the PFO model suggested that the 1677
mechanism of adsorption was controlled by the physical attraction of metal ions onto 1678
unoccupied sites on the biomass as opposed to a process of chemisorption (e.g. 1679
agreement to the PSO model) in which metal ions share electrons with functional groups 1680
on the cell surface (Ho and McKay 1998; Plazinski 2013). Sorption of Cu and Ni to 1681
Euglena occurred relatively quickly within 10 to 30 min (Figures 2-3) which indicates an 1682
attraction between the metals and the biomass. A strong affinity between sorbent and 1683
sorbate is an integral component of any application of biosorption to the remediation of 1684
metal-bearing effluent (Volesky 2003). Using biological material to achieve metal 1685
removal requires a mass transfer of metal ions to the biomass from solution driven by a 1686
61
mutually attractive force (e.g. electrostatic, ion exchange). This affinity between 1687
Euglena biomass and metal ions is suggested by the steep initial rise of the curves (Figure 1688
2-3). The PFO kinetic rate (k1) generally increased as the initial concentration of metals 1689
was increased (Table 1 and 2). Previous biosorption studies have reported similar effects 1690
on kinetic constants while others have found higher values at lower concentrations 1691
(Cordero et al., 2004; Jaikumar and Ramamurthi 2009). Amounts of metal sorbed in 1692
kinetic experiments were found to increase with initial metal concentration for both Cu 1693
and Ni (p < 0.05). As the initial concentration of Cu increased from 0.02 mg L-1
to 25 1694
mg L-1
, metal loading also increased from 0.0066 mg g-1
to 8.40 mg g-1
. Similarly, as Ni 1695
initial concentration s increased from 1 to 20 mg L-1
, loading increased from 0.341 mg g-1
1696
to 3.66 mg g-1
(Table 1 and 2). An increase in the initial concentration of Cu (~1250x) 1697
resulted in an approximately proportional increase (~1270x) in the amount of Cu sorbed 1698
to the biomass. In contrast, the amount of Ni sorbed increased ~12x concurrent with a 1699
20x increase in initial concentration. Higher sorption was also reported for Cu as 1700
compared to Ni using algae (Chen et al., 2008; Guler and Sarioglu 2013). A potential 1701
reason for this result could be that ligands which possess a high affinity for Cu are likely 1702
to contain N or S donor atoms (e.g. proteins, amino acids, thiols; Figure 1) which tend to 1703
form stronger bonds with Cu as compared to other groups (Kiefer et al., 1997). 1704
Compared to other algal species, Euglena have been reported to possess higher 1705
proportions of protein and thiol structures which could contribute to the preferential 1706
sorption of Cu over Ni (Mangal et al., 2016). In effect the binding sites on the cell 1707
surface may be more amenable to forming bonds with Cu than with Ni (Chen et al., 1708
2008). Differences is metal concentration may also have an effect on the amount sorbed 1709
62
in terms of which functional groups are available (Kiefer et al., 1997). At higher 1710
concentrations carboxyl groups play a primary role in metal binding whereas at relatively 1711
lower concentrations groups which are not as abundant but more specific and selective in 1712
binding become more significant (Kiefer et al., 1997). In this study, Ni concentrations 1713
used in experiments were generally higher than those for Cu and thus may have been 1714
dependent on a more narrow breadth of appropriate functional groups for binding. 1715
Additionally, these increases may also be due to the increased prevalence of metal ions in 1716
solution generating a greater interaction between metal and biomass particles and in 1717
effect overcoming resistance to the mass transfer of ions from the aqueous to the solid 1718
phase (Chen et al., 2008; Keshtkar et al., 2015). 1719
3.3 Sorption equilibria 1720
In this study, the biosorption of Cu 2+
and Ni2+
was evaluated at industrially 1721
relevant (e.g. wastewater which has undergone primary treatment) concentrations (e.g. < 1722
25 mg L-1
). The biosorption of both Cu2+
and Ni 2+
in single-metal solutions increased 1723
with increasing equilibrium concentration (Figures 2 and 3). The degree of fit (r2) to 1724
both the Langmuir and Freundlich models indicated that the sorption of Cu (0.995 and 1725
0.985 respectively) and Ni (0.996 and 0.985) by non-living Euglena gracilis can be 1726
described appropriately by either model (p < 0.001; both metals and models; Tables 3-4, 1727
Figures 4-5). However, the higher r2 for the Langmuir model compared to Freundlich 1728
indicated that metal ions were being adsorbed in a monolayer with functional groups on 1729
the surface of the biomass (Rao et al., 2005). The comparatively high applicability of 1730
the Freundlich model suggests that sorption also may occur on heterogeneous surfaces on 1731
the cell (Guler and Sarioglu 2013). Comparable Langmuir parameters (k and qmax) were 1732
63
found for both metals (p > 0.05; Table 3). The maximum capacity for biosorption was 1733
89.6 μg/g and 34.1 μg/g for Cu and Ni respectively. Despite differences in metal 1734
concentrations Langmuir parameters (e.g. qmax and b) were analogous to previous studies 1735
(Chen et al., 2008; Fraile et al, 2005; Keshtkar et al., 2015; Lau et al., 1999; Markou et 1736
al., 2015; Rao et al., 2005; Romera et al., 2007; Zakhama et al., 2011). The values of k 1737
ranged from 556 to 625 for Ni and Cu respectively, with no significant differences 1738
between metals (p > 0.05) (Table 3). Euglena gracilis showed a similar affinity for Cu 1739
and Ni as has been previously found with similar organisms (Chen et al., 2008; Fraile et 1740
al, 2005; Keshtkar et al., 2015; Lau et al., 1999; Markou et al., 2015; Rao et al., 2005; 1741
Romera et al., 2007; Zakhama et al., 2011). 1742
The biosorption of Cu and Ni was also examined in binary metal solutions to 1743
better emulate the mixed composition of industrial wastewaters (Figure 6). Sorption was 1744
assessed as a function of the ratio of the initial concentrations of Cu to Ni. When 1745
compared to single-metal solutions, Cu and Ni total metal uptake did not differ 1746
significantly in terms of sorption capacity as a function of initial metal concentrations (p 1747
> 0.05). At Cu:Ni < 1, Ni sorption was greater than Cu (p < 0.05; Figure 6) whereas at 1748
Cu:Ni > 1Cu sorption was higher (p < 0.05). This contrasts with live Euglena cells 1749
wherein Cu and Ni uptake was similar between Cu:Ni ratios (p > 0.05) (Winters et al., 1750
2016). Ni sorption was supressed at high concentrations of Cu (Cu:Ni > 1) whereas the 1751
opposite occurred at relatively higher Ni levels, which suggests a competition between 1752
Cu and Ni ions for commonly shared binding sites referred to as antagonistic biosorption 1753
(Al-Rub et al., 2006; Guler and Sarioglu 2013). The equilibrium uptake (qe) of Ni 1754
64
decreased with the increasing quantity of Cu ions. The antagonistic effect of Cu ions on 1755
the equilibrium Ni uptake was dominant at higher initial Cu concentrations. 1756
4 Conclusion 1757
This work assessed metal sorption at typical concentrations (< 100 mg L-1
) found 1758
in industrial wastewaters after a primary form of treatment. To our knowledge, this is the 1759
first instance in which Euglena has been studied for this purpose. Non-living Euglena 1760
gracilis have demonstrated comparable biosorption capacities (34-89 μg g-1
) to those of 1761
other eukaryotic organisms in the removal of Cu and Ni from aqueous solutions. FTIR 1762
analysis indicated the presence of functional groups (e.g. carboxyl, amide) which have 1763
been previously identified as important metal binding sites for metal biosorption. 1764
Sorption kinetics followed the PFO model suggesting a physically-based form of 1765
biosorption. Both the kinetic rate (k1) and the amount of metal sorbed were found to rise 1766
with increasing initial concentration. Although sorption occurred rapidly (10 – 30 min) 1767
Cu was more efficiently taken out of solution than Ni. In single-metal solutions both Cu 1768
and Ni were found to exhibit a greater degree of fit to the Langmuir model which 1769
suggests that metal ions bind to uniform sites on the cell surface in a monolayer. 1770
Similarly to kinetic results, Cu sorption was found to be greater in single-metal solutions 1771
than Ni although Euglena exhibited a similar affinity for both metals. Langmuir 1772
parameters were found to be comparable to sorption of Cu and Ni by similar organisms. 1773
In binary-metal systems, sorption of Cu was supressed at relatively high concentrations 1774
of Ni and conversely, Ni sorption was inhibited at higher (e.g. > 10) Cu:Ni ratios. 1775
Further investigations of biosorption utilizing Euglena gracilis should include a focus on 1776
65
identifying the overall removal mechanism(s) (e.g. ion exchange, metal-ligand 1777
complexes) in an effort to optimize sorption. Future studies should be conducted using 1778
actual industrial wastewaters to confirm the removal potential of Euglena. 1779
1780
Acknowledgments 1781
The authors would like to thank Antoine Perroud and Vaughn Mangal for their assistance 1782
in producing this work. This research was supported by the Canada Research Chairs 1783
program and the Natural Sciences and Engineering Research Council of Canada. 1784
1785
1786
1787
1788
1789
1790
1791
1792
1793
1794
1795
1796
1797
1798
1799
66
1800
Table 1: Kinetic model parameters for the biosorption of Cu on non-living Euglena 1801 gracilis; pseudo-first-order model1802
1803
1804
1805
1806
Table 2: Kinetic model parameters for the biosorption of Ni on non-living Euglena 1807
gracilis; pseudo-first-order model 1808
1809
1810
1811
1812
1813
1814
67
1815
1816
Table 3: Langmuir adsorption isotherm parameters for the biosorption of Cu and Ni on 1817
non-living Euglena gracilis at pH 51818
1819
1820
1821
1822
1823
1824
Table 4: Freundlich adsorption isotherm parameters for the biosorption of Cu and Ni on 1825
non-living Euglena gracilis at pH 51826
1827
1828
68
1829
Figure 1: FTIR spectra of dried euglena biomass 1830
1831
1832
1833
69
1834
Figure 2: Experimental data for Cu2+
sorption kinetics on non-living E. gracilis at 1835
initial concentrations of (a) 20 ug L-1
and (b) 50 ug L-1
(c) 1 mg L-1
(d) 25 mg L-1
. 1836
Curves represent PFO (a, c) and PSO (b, d) kinetic models. 1837
1838
1839
1840
1841
1842
1843
1844
70
1845
Figure 3: Experimental data for Ni2+
sorption kinetics on non-living E. gracilis at initial 1846
concentrations of (a) 1 mg L-1
and (b) 2 mg L-1
(c) 4 mg L-1
(d) 20 mg L-1
. Curves 1847
represent PFO (b, d) and PSO (a, c) kinetic models. 1848
1849
1850
1851
1852
1853
1854
71
1855
Figure 4: Experimental data for Cu2+
sorption equilibrium on non-living E. gracilis. 1856
Curves represent the Freundlich and Langmuir models. 1857
1858
1859
Figure 5: Experimental data for Ni2+
sorption equilibrium on non-living E. gracilis. 1860
Curves represent the Freundlich and Langmuir models. 1861
1862
1863
72
1864
Figure 6: Experimental data for Cu2+
and Ni2+
sorption (q) from binary-metal 1865
solution on living E. gracilis. Error bars represent SE. 1866
1867
1868
1869
1870
1871
73
References 1872
Abbas, S.H., Ismail, I.M., Mostafa, T.M., and Sulaymon, A.H. 2014. Biosorption of 1873 heavy metals: a review. Journal of Chemical Science and Technology 3(4): 74-102. 1874 1875
Al-Rub, F.A.A., El-Naas, M.H., Ashour, I., and Al-Marzouqi, M. 2006. Biosorption of 1876 copper on Chlorella vulgaris from single, binary and ternary metal aqueous solutions. 1877 Process Biochemistry 41(2): 457-464. 1878 1879 Burdin, K.S., and Bird, K.T. 1994. Heavy metal accumulation by carrageenan and agar 1880
producing algae. Botanica Marina 37(5): 467-470. 1881 1882 Chen, Z., Ma, W., and Han, M. 2008. Biosorption of nickel and copper onto treated alga 1883
(Undaria pinnatifida): application of isotherm and kinetic models. Journal of Hazardous 1884 Materials 155(1): 327-333. 1885 1886
Cordero, B., Lodeiro, P., Herrero, R., de Vicente, S., and Esteban, M. 2004. Biosorption 1887 of cadmium by Fucus spiralis. Environmental Chemistry 1(3): 180-187. 1888 1889
Crist, R.H., Oberholser, K., Shank, N., and Nguyen, M. 1981. Nature of bonding between 1890 metallic ions and algal cell walls. Environmental Science & Technology 15(10): 1212-1891
1217. 1892 1893 Donmez, G., Aksu, Z., Ozturk, A., and Kutsal, T. 1999. A comparative study on heavy 1894
metal biosorption characteristics of some algae. Process Biochemistry 34(9): 885-892. 1895 1896
Doshi, H., Ray, A., and Kothari, I.L. 2007. Bioremediation potential of live and dead 1897 Spirulina: spectroscopic, kinetics and SEM studies. Biotechnology and Bioengineering 1898
96(6): 1051-1063. 1899 1900
Eccles, H. 1995. Removal of heavy metals from effluent streams: why select a biological 1901 process? International Biodeterioration & Biodegradation 35(1): 5-16. 1902 1903 Environment Canada. 2002. Industrial water use 1996. Minister of Public Works and 1904 Government Services, Ottawa, Ontario. 1905
1906 Flouty, R., and Estephane, G. 2012. Bioaccumulation and biosorption of copper and lead 1907 by a unicellular algae Chlamydomonas reinhardtii in single and binary metal systems: a 1908
comparative study. Journal of Environmental Management 111: 106-114. 1909 1910 Fomina, M., and Gadd, G.M. 2014. Biosorption: current perspectives on concept, 1911 definition and application. Bioresource Technology 160: 3-14. 1912
1913 Fraile, A., Penche, S., Gonzalez, F., Blazquez, M.L., Munoz, J.A., and Ballester, A. 2005. 1914 Biosorption of copper, zinc, cadmium and nickel by Chlorella vulgaris. Chemistry and 1915 Ecology 21(1): 61-75. 1916
74
1917
Gadd, G.M. 2009. Biosorption: critical review of scientific rationale, environmental 1918 importance and significance for pollution treatment. Journal of Chemical Technology and 1919 Biotechnology 84(1): 13-28. 1920
1921 Gardea-Torresdey, J.L., Becker-Hapak, M.K., Hosea, J.M., and Darnall, D.W. 1990. 1922 Effect of chemical modification of algal carboxyl groups on metal ion binding. 1923 Environmental Science & Technology 24(9): 1372-1378. 1924 1925
Gopalapillai, Y., Chakrabarti, C.L., and Lean, D.R.S. 2008. Assessing toxicity of mining 1926 effluents: equilibrium-and kinetics-based metal speciation and algal bioassay. 1927 Environmental Chemistry 5(4): 307-315. 1928 1929
Guler, U.A., and Sarioglu, M. 2013. Mono and binary component biosorption of Cu (II), 1930 Ni (II), and Methylene Blue onto raw and pretreated S. cerevisiae: equilibrium and 1931
kinetics. Desalination and Water Treatment 52(25-27): 4871-4888. 1932 1933
Ho, Y.S., and McKay, G. 1998. A comparison of chemisorption kinetic models applied to 1934 pollutant removal on various sorbents. Process Safety and Environmental Protection 1935 76(4): 332-340. 1936
1937 Jaikumar, V., and Ramamurthi, V. 2009. Effect of biosorption parameters kinetics 1938
isotherm and thermodynamics for acid green dye biosorption from aqueous solution by 1939 brewery waste. International journal of chemistry 1(1): p2. 1940 1941
Kadukova, J., and Vircikova, E. 2005. Comparison of differences between copper 1942
bioaccumulation and biosorption. Environment international 31(2): 227-232. 1943 1944 Keshtkar, A.R., Mohammadi, M., and Moosavian, M.A. 2015. Equilibrium biosorption 1945
studies of wastewater U (VI), Cu (II) and Ni (II) by the brown alga Cystoseira indica in 1946 single, binary and ternary metal systems. Journal of Radioanalytical and Nuclear 1947
Chemistry 303(1): 363-376. 1948 1949
Kiefer, E., Sigg, L., and Schosseler, P. 1997. Chemical and spectroscopic characterization 1950 of algae surfaces. Environmental Science & Technology 31(3): 759-764. 1951 1952 Kizilkaya, B., Turker, G., Akgul, R., and Dogan, F. 2012. Comparative study of 1953 biosorption of heavy metals using living green algae Scenedesmus quadricauda and 1954
Neochloris pseudoalveolaris: Equilibrium and kinetics. Journal of Dispersion Science 1955 and Technology 33(3): 410-419. 1956
1957 Kumar, K.S., Dahms, H.-U., Won, E.-J., Lee, J.-S., and Shin, K.-H. 2015. Microalgae-A 1958 promising tool for heavy metal remediation. Ecotoxicology and environmental safety 1959 113: 329-352. 1960 1961 Lau, P.S., Lee, H.Y., Tsang, C.C.K., Tam, N.F.Y., and Wong, Y.S. 1999. Effect of metal 1962
75
interference, pH and temperature on Cu and Ni biosorption by Chlorella vulgaris and 1963
Chlorella miniata. Environmental Technology 20(9): 953-961. 1964 1965 Lesmana, S.O., Febriana, N., Soetaredjo, F.E., Sunarso, J., and Ismadji, S. 2009. Studies 1966
on potential applications of biomass for the separation of heavy metals from water and 1967 wastewater. Biochemical Engineering Journal 44(1): 19-41. 1968 1969 Liu, Y., Cao, Q., Luo, F., and Chen, J. 2009. Biosorption of Cd 2+, Cu 2+, Ni 2+ and Zn 1970 2+ ions from aqueous solutions by pretreated biomass of brown algae. Journal of 1971
Hazardous Materials 163(2): 931-938. 1972 1973 Mahdavi, H., Ulrich, A.C., and Liu, Y. 2012. Metal removal from oil sands tailings pond 1974 water by indigenous micro-alga. Chemosphere 89(3): 350-354. 1975
1976 Mangal, V., Stock, N.L., and Guéguen, C. 2016. Molecular characterization of 1977
phytoplankton dissolved organic matter (DOM) and sulfur components using high 1978 resolution Orbitrap mass spectrometry. Analytical and bioanalytical chemistry: 1-10. 1979
1980 Markou, G., Mitrogiannis, D., Celekli, A., Bozkurt, H., Georgakakis, D., and 1981 Chrysikopoulos, C.V. 2015. Biosorption of Cu
2+ and Ni
2+ by Arthrospira platensis with 1982
different biochemical compositions. Chemical Engineering Journal 259: 806-813. 1983 1984
Mehta, S.K., and Gaur, J.P. 2001. Removal of Ni and Cu from single and binary metal 1985 solutions by free and immobilized Chlorella vulgaris. European Journal of Protistology 1986 37(3): 261-271. 1987
1988
Mehta, S.K., and Gaur, J.P. 2005. Use of algae for removing heavy metal ions from 1989 wastewater: progress and prospects. Critical Reviews in Biotechnology 25(3): 113-152. 1990 1991
Mendoza-Cozatl, D.G., Rangel-Gonzalez, E., and Moreno-Sanchez, R. 2006. 1992 Simultaneous Cd
2+, Zn
2+, and Pb
2+ uptake and accumulation by photosynthetic Euglena 1993
gracilis. Archives of environmental contamination and toxicology 51(4): 521-528. 1994 1995
Michalak, I., Chojnacka, K., and Witek-Krowiak, A. 2013. State of the art for the 1996 biosorption process-a review. Applied biochemistry and biotechnology 170(6): 1389-1997 1416. 1998 1999 Monteiro, C.M., Castro, P.M.L., and Malcata, F.X. 2012. Metal uptake by microalgae: 2000
underlying mechanisms and practical applications. Biotechnology progress 28(2): 299-2001 311. 2002
2003 Nriagu, J.O. 1996. A history of global metal pollution. Science 272(5259): 223. 2004 2005 2006 Olaveson, M.M., and Nalewajko, C. 2000. Effects of acidity on the growth of two 2007 Euglena species. Hydrobiologia 433(1-3): 39-56. 2008
76
2009
Plazinski, W. 2013. Binding of heavy metals by algal biosorbents. Theoretical models of 2010 kinetics, equilibria and thermodynamics. Advances in colloid and interface science 197: 2011 58-67. 2012
2013 Ponton, D.E., and Hare, L. 2009. Assessment of nickel contamination in lakes using the 2014 phantom midge Chaoborus as a biomonitor. Environmental Science & Technology 2015 43(17): 6529-6534. 2016 2017
Ptacek, C., Price, W., Smith, J.L., Logsdon, M., and McCandless, R. 2004. Land-use 2018 practices and changes mining and petroleum production. In Threats To Water 2019 Availability In Canada. p. 67. 2020 2021
Rao, S.P., Kalyani, S., Suresh Reddy, K.V.N., and Krishnaiah, A. 2005. Comparison of 2022 biosorption of nickel (II) and copper (II) ions from aqueous solution by Sphaeroplea 2023
algae and acid treated Sphaeroplea algae. Separation science and technology 40(15): 2024 3149-3165. 2025
2026 Rodriguez-Zavala, J.S., Garcia-Garcia, J.D., Ortiz-Cruz, M.A., and Moreno-Sanchez, R. 2027 2007. Molecular mechanisms of resistance to heavy metals in the protist Euglena 2028
gracilis. Journal of Environmental Science and Health Part A 42(10): 1365-1378. 2029 2030
Romera, E., Gonzalez, F., Ballester, A., Blazquez, M.L., and Munoz, J.A. 2007. 2031 Comparative study of biosorption of heavy metals using different types of algae. 2032 Bioresource Technology 98(17): 3344-3353. 2033
2034
Tien, C.-J., Sigee, D.C., and White, K.N. 2005. Copper adsorption kinetics of cultured 2035 algal cells and freshwater phytoplankton with emphasis on cell surface characteristics. 2036 Journal of Applied Phycology 17(5): 379-389. 2037
2038 Vijayaraghavan, K., and Balasubramanian, R. 2015. Is biosorption suitable for 2039
decontamination of metal-bearing wastewaters? A critical review on the state-of-the-art 2040 of biosorption processes and future directions. Journal of Environmental Management 2041
160: 283-296. 2042 2043 Volesky, B. 2001. Detoxification of metal-bearing effluents: biosorption for the next 2044 century. Hydrometallurgy 59(2): 203-216. 2045 2046
Volesky, B. 2003. Sorption and biosorption. BV Sorbex. Montreal, QC. 2047 2048
Winter, C., Winter, M., and Pohl, P. 1994. Cadmium adsorption by non-living biomass of 2049 the semi-macroscopic brown alga, Ectocarpus siliculosus, grown in axenic mass culture 2050 and localisation of the adsorbed Cd by transmission electron microscopy. Journal of 2051 Applied Phycology 6(5-6): 479-487. 2052 2053 Winters, C., Noble, A., and Guéguen, C., 2016. Equilibrium and kinetic studies of Cu(II) 2054
77
and Ni(II) biosorption on non-living Euglena gracilis (submitted) 2055
Zakhama, S., Dhaouadi, H., and M'Henni, F. 2011. Nonlinear modelisation of heavy 2056 metal removal from aqueous solution using Ulva lactuca algae. Bioresource Technology 2057 102(2): 786-796. 2058 2059
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1 General Conclusion 2083
This study investigated the biosorption and bioaccumulation behaviour of Cu and 2084
Ni on the freshwater protist Euglena gracilis. More specifically, we observed the 2085
sequestration of these metals by Euglena in different environmental conditions (e.g. pH 5 2086
and 7.5), at various initial metal concentrations and in both living and non-living forms. 2087
Kinetic and isotherm analyses were conducted to describe metal ion uptake in single-2088
metal solutions and binary-metal mixtures (Figure 1). Additionally, we assessed Cu and 2089
Ni toxicity to Euglena (e.g. EC50) and characterized functional groups present in Euglena 2090
biomass via FTIR analysis. 2091
1.1 Euglena biomass characterization and toxicity 2092
In the context of sorbent characterization, dried Euglena biomass possessed the 2093
main functional groups (e.g. carboxylic, amino and thiol) which have been identified as 2094
key components for metal binding. The FTIR spectrum of dried Euglena cells (Figure 1; 2095
Chapter 2) showed intense and strong bands at 3040 and 1640 cm-1
which were related to 2096
C-H and C=C in aromatic hydrocarbons. Stretching vibrations of O-H (1700 cm-1
) and 2097
C-O (1380 cm-1
) were ascribed to the carboxylic functional group and C=O in amides 2098
was found at 1640-1660 cm-1
. EC50 toxicity assays were conducted in single-metal 2099
systems and showed a lesser effect for Cu (e.g. 39.2 mg L-1
) as compared to Ni (27.4 mg 2100
L-1
). 2101
2102
79
1.2 Sorption kinetics 2103
In general, both living and non-living Euglena were found to take up metals 2104
relatively quickly within 10-30 minutes and the sorption of both metals increased as the 2105
initial concentration of the system increased. Sorption of both Cu and Ni by active 2106
Euglena was found to be reduced at a higher pH. 2107
1.2.1 Living Euglena 2108
Metabolically active Euglena were found to correlate best with the pseudo-2109
second-order kinetic model which considers the rate of occupation of adsorption sites to 2110
be proportional to the square of the number of unoccupied sites (Ho and McKay 1998). 2111
In this context, chemical sorption involving valence forces (e.g. sharing or exchange of 2112
electrons) is assumed to be the rate-limiting step of the sorption process. 2113
1.2.2 Non-living Euglena 2114
In systems utilizing non-living Euglena, kinetic behaviour was found to be most 2115
appropriately described by the pseudo-first-order model (Table 1). Typically, algal 2116
biosorbents are found to adhere more closely to the PSO model when compared to the 2117
PFO model (Febrianto et al., 2009; Ho 2006). It has been suggested that when the initial 2118
concentration of metals in a system is low (e.g. 0.05 mg/L; Chapter 2) the sorption 2119
process follows the PSO as was reported in this work. Conversely, when the initial 2120
concentration of the solute is higher (e.g. 25 mg/L; Chapter 3) the PFO is more 2121
appropriate (Azizian 2004). Correlation with the PFO model suggests that the rate of 2122
metal binding to metabolically inert Euglena biomass is proportional to the number of 2123
unoccupied sites on the surface of the sorbent and is primarily a physical process based 2124
on electrostatic or Van der Waals’ forces of attraction (Lagergren 1898). An increase in 2125
80
Cu sorption was found to be proportional to a coincidental increase in initial 2126
concentration. However a 20-fold increase in Ni concentration resulted only in a 12-fold 2127
increase in sorption which may indicate that Ni uptake in Euglena may be primarily 2128
mediated by biologically driven processes of accumulation and storage or the presence of 2129
extracellular organic ligands produced by the organism (e.g. EPS). This difference 2130
between living and non-living biomass could be influenced by the higher toxicity of Ni. 2131
1.3 Sorption Isotherms 2132
Adsorption isotherms were generated to describe the relationship between the 2133
amount of Cu or Ni sorbed by a unit weight of Euglena and the amount of metal solute 2134
remaining in the test medium at equilibrium. 2135
1.3.1 Living Euglena 2136
Living Euglena sorption isotherms were found to adhere more closely to the 2137
Freundlich model which indicates that metal binding occurs on heterogeneous sites on the 2138
cell surface and binding strength decreases as site occupation increases (e.g. an 2139
interaction between adsorbed species). Due to the robust characteristics of the 2140
Freundlich equation it is able to fit a wide range of experimental data from many 2141
different types of biosorbents (Febrianto et al., 2009) (Table 2). As the Freundlich 2142
model assumes multilayer sorption and heterogeneous binding sites, it may better 2143
represent the metal binding behaviour of living organisms in terms of both passive 2144
adsorption and metabolically facilitated bioaccumulation (Markou et al., 2015). On the 2145
other hand, the Langmuir model assumes that metal binding occurs in a monolayer 2146
involving a discrete number of binding sites and for this reason may represent sorption by 2147
81
non-living biomass more appropriately than the Freundlich equation. In single-metal 2148
isotherm systems, Cu sorption was more favourable than Ni sorption and both sorption 2149
capacity (e.g. Kf = 0.07 and 0.005 respectively) and sorption intensity (e.g. 1/n= 0.982 2150
and 1.92 respectively) parameters were greater for Cu as compared to Ni. Similarly, in 2151
binary-metal systems this trend continued, Cu sorption was greater than Ni in comparison 2152
to solutions containing one metal only. However, comparing sorption between Cu and Ni 2153
in binary solutions was found to be similar (p > 0.05) suggesting that a synergistic 2154
inhibition of metal uptake may not occur when utilizing living Euglena for metal 2155
removal. This work is the first study assessing Euglena gracilis metal sorption in the 2156
presence of multiple contaminants. 2157
1.3.2 Non-living Euglena 2158
Non-living Euglena biomass exhibited a better correlation with the Langmuir 2159
adsorption model than the Freundlich model indicating that metal binding occurs in a 2160
monolayer to adsorption sites, that Cu and Ni species do not interact with each other, and 2161
do not affect the binding activity of adjacent sites. In the context of non-living 2162
biosorption this suggests the primary mechanism of uptake in this system may be passive 2163
adsorption rather than a process of ion exchange as was found with viable Euglena 2164
biomass. Comparable parameters for both metals were found (p > 0.05): In single-metal 2165
systems maximum uptake (qmax) for Cu was 89.6 μg/g and for Ni was 34.0 μg/g and the 2166
affinity constant (k) was 625 for Cu and 556 for Ni. In comparison to Ni in single-metal 2167
solutions Cu sorption was greater (p <0.05). Comparing single-metal sorption to 2168
sorption in binary-metal solutions, no significant differences in capacity were found for 2169
either Cu or Ni (p > 0.05). In binary solutions, a Cu:Ni of < 1in solution showed greater 2170
82
Ni sorption whereas a Cu:Ni ratio >1 resulted in higher Cu sorption. This suggests that at 2171
relatively high concentration s of one metal, sorption of the other metal is supressed (e.g. 2172
competition for similar binding sites) and that the simultaneous biosorption of Cu and Ni 2173
by non-living Euglena is essentially an antagonistic process. 2174
Overall, it has been shown that living Euglena gracilis remove Cu and Ni from 2175
both single (58% and 44%, respectively) and binary (62% and 58%, respectively) 2176
solutions in greater proportion than non-living biomass in single-metal (49% and 32%, 2177
respectively) and binary-metal (43% and 20%, respectively) solutions. To our 2178
knowledge, the biosorption and bioaccumulation of Cu and Ni by Euglena gracilis for the 2179
purpose of wastewater remediation at environmentally relevant concentrations has not 2180
been investigated previously. Both living and non-living Euglena showed a greater 2181
affinity for Cu in comparison to Ni and removed more of the former from solution. In 2182
comparison with other algae and eukaryotic organisms, Euglena shows an analogous 2183
capacity for the sorption and accumulation of Cu and Ni from solution (Table 2). 2184
Nonetheless, the high efficacy that conventional treatment methods and selected bio-2185
sourced sorbents have established sets a substantial threshold to attain in terms of useful 2186
application to a large-scale bioremediation of industrial wastewater. At this point, it 2187
remains unclear whether Euglena gracilis may be an effective biosorbent suitable for the 2188
removal of Cu and Ni from effluent at a commercial scale, which presents a far more 2189
complex mixture of metals, nutrients, and co-ions (e.g. Ca2+
, Mg2+
). 2190
83
1.4 Significance of the work 2191
It is anticipated that this work should contribute to the identification of baseline 2192
uptake parameters and capacities for Cu and Ni by Euglena as well as to the increasing 2193
amount of research investigating sustainable bioremediation. The evaluation of metal 2194
sorption conducted in this work is a necessary first step towards industrial application. 2195
As government and regulating authorities become more stringent in response to greater 2196
understanding of the consequences of metal deposition in the environment, the need for 2197
more cost-effective solutions to issues of contamination will increase. Current 2198
conventional technologies are either too costly for the high volumes to be treated or are 2199
ineffectual at the concentrations commonly found in metal-bearing wastewaters. Algal 2200
biosorption is a novel treatment which can provide effluent metal removal at a fraction of 2201
the cost of conventional methods and without producing toxic waste material which 2202
would require an additional protocol for disposal. Future research into utilizing Euglena 2203
gracilisfor this purpose should include: 1) a more broad assessment of the effects on 2204
metal removal in terms of environmental conditions such as pH, culture medium, and 2205
potential pre-treatments (e.g. chemical modification), 2) investigation into the possibility 2206
for applying this process at the industrial scale (e.g. column sorption and bioreactors), 2207
and 3) continuing assessment of other toxic metals for which Euglena may be better 2208
suited to sorb, accumulate, and remove. 2209
2210
2211
2212
2213
84
Table 1: Kinetic parameters of biosorbents for Cu and Ni in mono-metallic systems. Co= 2214
initial metal concentration qe= equilibrium concentration k = kinetic rate constant 2215
(d)=dead (l)=live 2216
2217
2218
Table 2: Equilibrium parameters of biosorbents for Cu and Ni in mono-metallic systems. 2219
Co= initial metal concentration qmax= maximum uptake % Rem= percent removal 2220
(d)=dead (l)=live 2221
2222
85
2223
Figure 1: Percentage metal removal of Cu and Ni in mono-metallic and bi-2224
metallic solutions by living and non-living E. gracilis. Error bars represent SE. 2225
Significance represented by: (a, c) = Cu>Ni (p<0.05) (b) = Live>Dead (p<0.05) 2226
2227
2228
2229
2230
2231
2232
2233
2234
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86
References 2238
Azizian, S. 2004. Kinetic models of sorption: a theoretical analysis. Journal of Colloid 2239 and Interface Science 276(1): 47-52. 2240 2241
Febrianto, J., Kosasih, A.N., Sunarso, J., Ju, Y.-H., Indraswati, N., and Ismadji, S. 2009. 2242 Equilibrium and kinetic studies in adsorption of heavy metals using biosorbent: a 2243 summary of recent studies. Journal of Hazardous Materials 162(2): 616-645. 2244 2245 Ho, Y.S., and McKay, G. 1998. A comparison of chemisorption kinetic models applied to 2246
pollutant removal on various sorbents. Process Safety and Environmental Protection 2247 76(4): 332-340. 2248 2249
Lagergren, S. 1898. About the theory of so-called adsorption of soluble substances. 2250 Kungliga Svenska Vetenskapsakademiens Handlingar 24(4): 1-39. 2251 2252
Markou, G., Mitrogiannis, D., Celekli, A., Bozkurt, H., Georgakakis, D., and 2253 Chrysikopoulos, C.V. 2015. Biosorption of Cu
2+ and Ni
2+ by Arthrospira platensis with 2254
different biochemical compositions. Chemical Engineering Journal 259: 806-813. 2255
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