screening of ionic liquids for the extraction of proteins from the … · o 1-etil-3-metilimidazol...

Screening of ionic liquids for the extraction of proteins

from the macroalga Ulva lactuca aiming at an

integrated biorefinery approach

Carlota Fernandes Miranda

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors:

Dr. Corjan van den Berg

Dr. Maria Teresa Ferreira Cesário Smolders

Examination Committee

Chairperson: Dr. Helena Maria Rodrigues Vasconcelos Pinheiro

Supervisor: Dr. Maria Teresa Ferreira Cesário Smolders

Members of the Committee: Dr. Maria Catarina Marques Dias de Almeida

November 2017

iii

Preface The work described in this document was performed under the framework of the curricular

course Master’s Thesis in order to obtain the Master’s degree in Biotechnology at Instituto

Superior Técnico.

All the research work was carried out, from February to July 2017, in the Bioprocess

Engineering Group, part of the Agrotechnology and Food Sciences Group from Wageningen

University and Research (Netherlands), under the supervision of Dr. Corjan van den Berg

(assistant professor) and the PhD student Edgar Suarez Garcia. Dr. Ma Teresa Cesário (post-doc

researcher), from the Bioengineering Department, was the internal supervisor at Instituto Superior

Técnico.

This dissertation consists of a literature review of the research topic under study,

description of the materials and methods used, obtained results and respective discussion.

Finally, conclusions and future prospects are also reported.

v

Acknowledgements

The limited space of this acknowledgment section does not allow me to thank, as I should,

to all of the people that, in the last months, direct or indirectly, helped me fulfilling my expectations

and achieving my goals, finalizing such important phase of my academic life. Hence, I leave here

only a few words, but in a very felt and thankful way.

Firstly, I would like to thank Professor Mª Manuela Fonseca and Dr. Mª Teresa Cesário for

giving me the opportunity to do my master’s dissertation abroad and Dr. Corjan van den Berg for

integrating me in his research group. I believe that the technical and social resources I had

available in Wageningen University and Research were plenty for being able to perform an

excellent work. Furthermore, the six months I spent in the Netherlands highly contributed to my

personal development, and, despite not being an easy journey, I truly believe that my limits were

pushed further, and that now I can do more and better.

To Edgar Suarez, I genuinely thank for the orientation, support, opinions and critics, that

improved my scientific knowledge and, undoubtedly, stimulated my wish of knowing more and

more, and the will to do better. Your vast knowledge in the field and your availability to advise me

and revise my work were crucial to the success of this dissertation project.

I would like to mention again Dr. Mª Teresa Cesário, for constantly keeping up with my

work, always showing an availability to discuss results and suggest new strategies. Thank you

also for having reviewed, in detail, this master’s dissertation report.

I am also eternally grateful to Ana Beatriz Cardoso, my partner on this journey and without

whom this would not have been possible. Thank you for the unconditional patience and support,

for the true friendship, for being my family during the months I have been away from home. I will

never be able to thank you enough for everything you have done for me.

Last, but definitely not the least, I would like to thank to my family, especially my mother,

Luísa, my father, Vasco, my brother, Tomás and my sister, Margarida. Thank you for always

walking by my side - even 2000 km apart from me -, for your unconditional support and

comprehension and for all the sacrifices you made for me. Thank for believing in me more than I

believe in myself, for always pushing me to do better and be better, and for all life teachings. I

could not end without mentioning my grandparents, my aunt Teresa and Dr. Carla, for the

constant encouragement, so to successfully finish this great challenge.

vii

Abstract

Alternative protein sources and production methods are required to fulfil the consumer

needs and to meet the predicted increase of global protein demand. Although seaweeds are

recognized as a valuable source of protein, new techniques for the extraction and separation of

these biomolecules, under mild and stabilizing conditions, are required. Ionic liquids, due to their

unique properties, have been considered promising alternatives for the recovery of value-added

compounds from different sources of biomass.

The green macroalga Ulva lactuca was used in this study. It was composed of 96.50±0.87%

of total solids, of which 22.80±0.35% was ashes, 4.95±0.35% lipids, 17.81±0.79% proteins and

60.92±14.62% carbohydrates. Three methods commonly used for macroalgal protein extraction

were used as reference cases. Extraction by means of aqueous and alkaline solutions

(sequential), mechanical grinding under alkaline conditions and aqueous biphasic system

(PEG/Na2CO3) led to extraction efficiencies of 49.08±6.66%, 6.68±1.09% and 10.52±0.35%,

respectively.

Twenty-five ionic liquids were screened towards the selection of the most suitable for the

disintegration of Ulva lactuca cellular structure. 1-ethyl-3-methylimidazolium dibutyl phosphate

showed the best results, allowing the recovery of 80.62±4.95% of the total protein. Aiming at an

integrated biorefinery approach, an aqueous three-phase partitioning system (1-ethyl-3-

methylimidazolium dibutyl phosphate/K2HPO3) is further proposed as a platform for the single-

step cell disintegration and protein-carbohydrate fractionation. Proteins and carbohydrates are

mainly concentrated in the top and interphase, respectively.

Finally, ultrafiltration was evaluated for the recovery of ionic liquid from macroalgal extracts,

yielding recoveries of ~80% of ionic liquid and ~90% of proteins and carbohydrates.

Keywords

Seaweed | Ionic liquid | Integrated biorefinery | Protein | Carbohydrate | Aqueous biphasic

system

ix

Resumo

Para atender às necessidades dos consumidores e ao previsto aumento global da sua

procura, são necessárias novas fontes de proteína e métodos para a sua produção. Apesar das

macroalgas serem reconhecidas como potenciais fontes de proteína, é preciso desenvolver

novas técnicas para a extração/separação destas biomoléculas, em condições suaves e

estabilizadoras. As propriedades únicas dos líquidos iónicos tornam-nos alternativas promissoras

para recuperar compostos de valor agregado de diferentes fontes de biomassa.

A alga verde Ulva lactuca, usada neste estudo, era composta por 96.50±0.87% de sólidos

totais, dos quais 22.80±0.35% era cinzas, 4.95±0.35% lípidos, 17.81±0.79% proteínas e

60.92±14.62% carboidratos. Três métodos convencionais para a extração de proteínas das

macroalgas foram usados como casos de referência. A extração através de soluções aquosas e

alcalinas (sequencial), de moagem mecânica em condições alcalinas e de um sistema aquoso

bifásico (PEG/Na2CO3) conduziram a eficiências de extração de 49.08±6.66%, 6.68±1.09% e

10.52±0.35%, respetivamente.

Rastrearam-se vinte-cinco líquidos iónicos a fim de selecionar o mais adequado para

desintegrar a estrutura celular da Ulva lactuca. O 1-etil-3-metilimidazol dibutilfosfato mostrou o

melhor resultado, extraindo 80.62±4.95% da proteína total. Visando uma estratégia de

biorrefinaria integrada, é ainda proposto um sistema aquoso de partição trifásico (1-etil-3-metil-

imidazol dibutilfosfato/K2HPO3) como uma plataforma de passo único para a desintegração

celular e separação de proteínas-carboidratos. As proteínas e os carboidratos ficam

concentrados principalmente na fase superior e interfase, respetivamente.

Finalmente, avaliou-se a recuperação do líquido iónico por ultrafiltração a partir dos

extratos algáceos, alcançando-se taxas de recuperação de ~80% de líquido iónico e ~90% de

proteínas/carboidratos.

Palavras-Chave

Alga marinha | Líquido iónico | Biorrefinaria integrada | Proteína | Carboidrato | Sistema

aquoso bifásico

xi

Contents

Acknowledgements ....................................................................................................................... v

Abstract ........................................................................................................................................ vii

Keywords ...................................................................................................................................... vii

Resumo ......................................................................................................................................... ix

Palavras-Chave ............................................................................................................................. ix

Contents ........................................................................................................................................ xi

List of Figures .............................................................................................................................. xiii

List of Tables ................................................................................................................................ xv

Abbreviations .............................................................................................................................. xvii

1. Introduction ........................................................................................................................ 1

1.1 Context ...................................................................................................................... 3

1.2 Objectives .................................................................................................................. 5

2. State of the Art ................................................................................................................... 7

2.1 Biorefinery concept .................................................................................................... 9

2.1.1 Aquatic biomass .............................................................................................. 10

2.2 Macroalgal backgrounds ......................................................................................... 11

2.2.1 Macroalgae as a source of protein .................................................................. 12

2.3 Extraction of macroalgal proteins ............................................................................ 13

2.4 Ionic liquids as new solvents ................................................................................... 16

2.4.1 Ionic liquids for macroalga cell disintegration .................................................. 17

2.4.2 Ionic liquid-based aqueous biphasic systems for biomolecules fractionation . 18

2.4.3 Ionic liquid recovery ......................................................................................... 22

2.5 Macroalgal proteins applications ............................................................................. 24

2.5.1 Macroalgal bioactive proteins .......................................................................... 25

2.5.1.1 Lectins ......................................................................................................... 25

2.5.1.2 Phycobiliproteins ......................................................................................... 26

2.5.2 Macroalgal bioactive peptides ......................................................................... 27

3. Aim of studies .................................................................................................................. 29

4. Materials and Methods .................................................................................................... 33

4.1 Sample collection and preparation .......................................................................... 35

4.2 Biomass characterization ........................................................................................ 35

4.2.1 Total solids and ash......................................................................................... 35

4.2.2 Total lipid content ............................................................................................ 36

4.2.3 Total carbohydrate content .............................................................................. 37

4.2.4 Total protein content ........................................................................................ 37

4.3 Conventional methods for protein extraction ........................................................... 37

4.3.1 Aqueous and alkaline extraction ..................................................................... 37

xii

4.3.2 Extraction by high-shear force under alkaline conditions ................................ 38

4.3.3 Aqueous biphasic system PEG/Na2CO3 ......................................................... 38

4.4 Assays with ionic liquids .......................................................................................... 39

4.4.1 Ionic liquid selection ........................................................................................ 39

4.4.2 Aqueous biphasic system: binodal curve ........................................................ 40

4.4.3 Bead mill experimental setup .......................................................................... 40

4.5 Recovery of ionic liquid............................................................................................ 41

4.6 Analytical methods .................................................................................................. 43

4.6.1 Protein quantification ....................................................................................... 43

4.6.2 Carbohydrate quantification ............................................................................ 43

4.6.3 Ionic liquid quantification ................................................................................. 44

4.6.4 Native-PAGE gel electrophoresis .................................................................... 44

4.7 Statistical Analysis ................................................................................................... 45

5. Results and Discussion ................................................................................................... 47

5.1 Chemical composition of Ulva lactuca ..................................................................... 49

5.2 Reference Cases ..................................................................................................... 52

5.3 Experiments with ionic liquids ................................................................................. 56

5.3.1 Ionic liquid selection ........................................................................................ 56

5.3.2 Extraction and fractioning of protein by IL based-ATPS ................................. 60

5.4 Ionic liquid recovery ................................................................................................. 63

6. Conclusions and future prospects ................................................................................... 65

References .................................................................................................................................. 71

Appendix A Protein, carbohydrate and ionic liquid quantification ......................................... A-1

A.1. Protein Quantification ................................................................................................. A-3

A.2. Carbohydrates Quantification ..................................................................................... A-4

A.3. Ionic liquid Quantification ........................................................................................... A-4

Appendix B Binodal Curve .................................................................................................... B-1

B.1. Binodal curve correlation ............................................................................................ B-3

xiii

List of Figures

Figure 2.1 | Macroalga biomass refinery scheme. Adapted from (IEA, 2016a) .......................... 14

Figure 2.2 | Intra- and intermolecular hydrogen bonds in cellulose. Adapted from (Pinker et al.,

2009). .......................................................................................................................................... 17

Figure 2.3 | Proposed dissolution mechanism of cellulose in ionic liquid 1-Butyl-3-

methylimidazolium chloride ([BMIM][Cl]). Adapted from (Pinker et al., 2009). ........................... 18

Figure 2.4 | Phase diagram for a hypothetical system composed of ionic liquid + salt + water

(weight fraction percentage). TCB: Binodal curve; C: Critical point; TB: Tie-line; T: Composition

of the top phase; B: Composition of the bottom phase; X, Y and Z: total composition of the ATPS.

Adapted from (Raja et al., 2012). ................................................................................................ 19

Figure 2.5 | Ordering of the ions and related properties in the Hofmeister series (Zhang and

Cremer, 2006). ............................................................................................................................ 22

Figure 2.6 | Schematic representation of ultrafiltration. Adapted from (Van Der Bruggen et al.,

2003). .......................................................................................................................................... 23

Figure 3.1 | Seaweed Ulva lactuca (Krisp, 2011). ....................................................................... 31

Figure 3.2 | Distribution of the different Ulva sp. cell wall polysaccharides in a schematic cross

section of a thallus (A) and the proposed associations between the different cell wall

polysaccharides (B). Adapted from: (Lahaye and Robic, 2007). ................................................ 31

Figure 4.1 | DYNO-mill equipment and respective components. ................................................ 41

Figure 4.2 | Schematic diagram of stirred cell ultrafiltration assembly used for recovering the ionic

liquid from the supernatant. Adapted from (Merck Millipore, 2015). ........................................... 42

Figure 5.1 | Percentage of protein recovery by high-shear force, aqueous and alkaline extraction

(sequential) and aqueous biphasic system PEG/Na2CO3 (sum of protein extracted into top and

bottom phases). Protein yield expressed as % of total protein (Protein extracted/Total proteins x

100). Results are the means of triplicate determination for aqueous and alkaline extraction and

duplicate determinations for high-shear force extraction and aqueous biphasic system ±SD; (*) -

means are statistically equal (p>0.05). ........................................................................................ 52

Figure 5.2 | Triplicates of positive controls (algae and milliQ water), negative controls (milliQ

water, PEG1000 and Na2CO3) and ATPS formed by milliQ water + PEG1000 + Na2CO3 + algae.

..................................................................................................................................................... 54

Figure 5.3 | Distribution of extracted proteins and carbohydrates between the top and bottom

phases of the ATPS composed by PEG1000 + water + Na2CO3. .............................................. 54

Figure 5.4 | Electrophoresis gel of protein extracts under non-denaturing conditions. Lane 1:

Protein ladder; Lane 2: Aqueous extract (positive control); Lane 3: Alkaline extract; Lane 4:

Mechanical grinding extract. ........................................................................................................ 55

Figure 5.5 | Suspensions obtained for each ionic liquid after mixing IL + water + algae. A positive

control (+ Ctrl) – algae and water – is placed next to all the samples. ....................................... 57

Figure 5.6 | Percentage of protein recovery by each of the 5 ILs tested (+ control – algae and

water). Protein yield expressed as % of total protein (Protein extracted/Total proteins x 100).

xiv

Results are the means of duplicates for each IL and quadruplicates for positive control ±SD. a, b,

c – means in columns with the same letter are significantly equal (p>0.05). .............................. 59

Figure 5.7 | Binodal curve of [Emim][dbp] and K2HPO4 at 298.15 K. .......................................... 60

Figure 5.8 | ATPS ([Emim][dbp]/K2HPO4) created after biomass processing in bead mill. ......... 61

Figure 5.9 | Distribution of extracted proteins and carbohydrates between the bottom, inter- and

top phases of the aqueous system composed by [Emim][dbp] and K2HPO4. ............................. 61

Figure 5.10 | Recovery of [Emim][dbp] in filtrates and retentates, after ultrafiltration through stirred

ultrafiltration cell and centrifugal filter unit. Results are the means of duplicates ±SD. .............. 63

xv

List of Tables

Table 2.1 | Conventional pre-treatment cell disruption methods and extraction processes for the

isolation of macroalgal proteins. (*) Protein yield expressed as % of total protein (Protein

extracted/Total proteins x 100). ................................................................................................... 15

Table 2.2 | Macroalgal source species and bioactivity of lectins. ............................................... 26

Table 2.3 | Examples of macroalgal peptides exhibiting biological activity. ................................ 27

Table 2.4 | Examples of macroalgal protein hydrolysates exhibiting biological activity. ............. 28

Table 4.1 | Composition of 1 L phosphate buffer saline. ............................................................. 36

Table 4.2 | Reagents and respective purity and supplier used in lysis buffer preparation. ......... 37

Table 4.3 | Ionic liquids tested and respective purity and supplier. ............................................. 39

Table 4.4 | Filtrate and retentate dilutions required for protein, carbohydrate and ionic liquid

quantification. .............................................................................................................................. 42

Table 5.1 | Chemical characteristics of macroalga U. lactuca (% w/w on dry basis). ................. 49

Table 5.2 | Protein content in Ulva lactuca. ................................................................................. 50

Table 5.3 | Ulva lactuca composition determined by thermogravimetric analysis. ..................... 51

Table 5.4 | Ionic liquids selected and respective anion hydrogen bond basicity. ....................... 56

Table 5.5 | Ionic liquids and estimated costs. ............................................................................. 58

Table 5.6 | Output of the third selection. Features of the five ionic liquids selected. .................. 59

Table 5.7 | Rejection coefficients of proteins and carbohydrates for each ultrafiltration setup.

Results are means of duplicates ±SD. ........................................................................................ 63

xvii

Abbreviations

[1,4,4,4-P][MSO4] tributyl(methyl)phosphonium methylsulfate

[6,6,6,14-P][Cl] trihexyltetradecylphosphonium chloride

[6,6,6,14-P][dca] trihexyltetradecylphosphonium dicyanamide

[Bmim][Ac] 1-butyl-3-methylimidazolium acetate

[Bmim][Cl] 1-butyl-3-methylimidazolium chloride

[Bmim][dbp] 1-butyl-3-methylimidazolium dibutyl phosphate

[Bmim][dca] 1-butyl-3-methylimidazolium dicyanamide

[Ch][Ac] Choline acetate

[Ch][Cl] Choline chloride

[Emim][dbp] 1-ethyl-3-methylimidazolium dibutyl phosphate

[P]B Concentration of protein in bottom phase

[P]T Concentration of protein in top phase

AA(s) Amino Acid(s)

ACE Angiotensin Converting Enzyme

AHBB Anion Hydrogen Bond Basicity

AIDS Acquired Immune Deficiency Syndrome

ANOVA Analysis of Variance

ATPS(s) Aqueous Two-Phase System(s)

B Bottom phase

BIL Concentration of ionic liquid in the bottom phase

BPE Bioprocess Engineering

BSA Bovine Serum Albumin

Bsalt Concentration of salt in the bottom phase

CAB Concentration of component A in the bottom phase

CAT Concentration of component A in the top phase

Cf Feed concentration

Cp Permeate concentration

DEA Donor-Electron Acceptor

DW Dry Weight

EAA(s) Essential Amino Acid(s)

xviii

FAO Food and Agriculture Organization

g Centrifugal force

GHG Greenhouse Gases

HIV Human Immunodeficiency Virus

HSD Tukey’s Honest Significant Difference

IEA International Energy Agency

IL(s) Ionic Liquid(s)

K Partition coefficient

M Mixture

p p-value

PEG Polyethylene Glycol

R Rejection coefficient

sB Starting Biomass

SD Standard deviation

SDS Sodium Dodecyl Sulphate

sp Species

STL Slope of the Tie-Line

T Top phase

TGX Tris-Glycine eXtended

TIL Concentration of ionic liquid in the top phase

TL(s) Tie-Line(s)

TLL Tie-Line Length

TPP Three-Phase Partitioning

Tris Tris(hydroxymethyl)aminomethane

Tsalt Concentration of salt in the top phase

U. lactuca Ulva lactuca

ULPC Ultra High-Performance Liquid Chromatography

UNU United Nation University

Vf Final volume

w/w Weight fraction

WHO World Health Organization

wt Weight

1

1 1. Introduction

3

1.1 Context

According to the United Nations, the current world population of 7.3 billion is expected to

reach 9.7 billion in 2050 (FAO, 2017), requiring an estimated 70% increase in food production

(FAO, 2009). In addition, the economic development and the rising in living standards, will result

in changes in food consumption patterns.

It is undeniable that proteins take a crucial role in the human diet. Its global demand is

driven not only by the population increase and socio-economic changes, but also by the growing

recognition of protein role in a healthy diet, particularly for an aging population (Henchion et al.,

2017).

Predictions for protein requirements show that the world demand for animal-derived protein

will have to triple by 2050 (Westhoek et al., 2011), resulting in sustainability and food security

concerns. It is proved that animal-based foods produce higher levels of greenhouse gases (GHG)

than plant-based. These emissions come from, for instance, livestock, manure, feed production

and land conversion (Westhoek et al., 2011). In particular, livestock sector accounts for 14.5% of

all human induced GHG emissions (Gerber et al., 2013), being responsible for substantial

emissions of nitrogen in various forms (ammonia, nitrates), which in turn leads to losses of

terrestrial and aquatic biodiversity (Leip et al., 2015; Westhoek et al., 2011).

Also, the oceans, which are one of the largest sources of protein (fish and seafood), are

reaching the limit of their resources. In 2013, about 89.5% of the global fish stocks were fully or

over exploited (FAO, 2014a), leading to severe consequences for marine ecosystems. Farmed

fish is likely to meet the needs of an ever-growing population; forecasts say it will account for 2/3

of global supply by 2030 (The World Bank, 2013). Nevertheless, it is fast reaching its limit since

farmed fish is heavily dependent on wild caught fish for feed (fishmeal) (FAO, 2014a). Alternative

protein sources and production methods are therefore required to fulfil the demand of consumers

and to meet predicted global protein requirements.

Macroalga, the so called “seaweed”, represents a promising and novel future protein

source. Some species of seaweed, like the red seaweed Porphrya sp., have been shown to

contain levels of protein up to 47% dry weight (wt.) (Marsham et al., 2007). In some cases,

macroalgae can be richer than some conventional protein-rich foods, such as soybean (40%

protein), cereals (15% protein), eggs (9% protein) and fish (25% protein) (Harnedy and

FitzGerald, 2013). In addition, the production of proteins from macroalgae has advantages over

traditional sources in terms of productivity. Macroalgae have higher protein yield per unit area

(2.5-7.5 tons.Ha-1.year-1) compared to terrestrial crops, such as soybean, vegetable seeds, and

wheat (0.6-1.2 tons.Ha-1.year-1, 1-2 tons.Ha-1.year-1, and 1.1 tons.Ha-1.year-1, respectively)

(Krimpen et al., 2013).

Approximately 70% of the total global freshwater is used for agriculture (FAO, 2016) with

animal-based protein production requiring 100 times more water than producing an equivalent

amount of protein from plant sources (Pimentel and Pimentel, 2003). Marine algae do not require

freshwater or arable land to grow, maximising resources that can be used for additional food

production or other cash crops (Bleakley and Hayes, 2017).

4

Although seaweeds can be considered a potential source of proteins, their widespread use

is limited by the availability of scalable production methods for protein isolation. The current

processes are time-consuming, economically unviable and may still compromise the protein

functionality due to the severe conditions applied (Wijffels and Barbosa, 2010). New scalable

biorefining processes for the extraction and separation of macroalgal proteins, which ensure high

recovery yields without compromising the extracts functionality, need to be developed.

Besides being promising to meet the future protein demand, macroalgae are also useful

for bioenergy production based on the carbohydrate fraction. A single-unit operation for cell

disruption and component fractionation – integrated biorefinery – that allows for the isolation of

different macroalgal fractions, is essential for the valorisation of macroalgae as feedstocks (IEA,

2017, 2016a).

Recently, the unique properties of ionic liquids (ILs) have triggered the interest of scientific

community as promising solvents for the extraction and separation of biomacromolecules from

biomass, under mild and stabilizing conditions (MacFarlane and Tan, 2010). Although research

on macroalga cell disruption using these novel solvents is limited to date (Malihan et al., 2014;

Pezoa-Conte et al., 2015), their use for the disintegration of lignocellulosic material and algae has

been already proven for several feedstocks (Brandt et al., 2013; Kim et al., 2012). The use of ILs

is easily scaled up to industrial processes (Socha et al., 2014), therefore, this study can result in

a new integrated biorefinery approach for macroalgal protein valorisation.

5

1.2 Objectives

The main goal of this experimental work was the screening of ionic liquids for the extraction

of proteins from the green macroalga Ulva lactuca. For that, the following objectives should be

met:

1. Biomass characterization in terms of total solids, ash, lipid, carbohydrate and protein

content.

2. Replication of conventional methods, reported in literature, for the extraction of

protein from macroalgae.

3. Screening of ionic liquids for the disintegration of U. lactuca cell wall and the

consequent protein extraction.

4. Development of an aqueous two-phase system based on the most promising ionic

liquid aiming at an integrated fractionation of biomolecules.

5. Study the recovery of ionic liquid from the macroalgal extracts;

6. Perform an economical evaluation of the proposed integrated biorefinery approach.

7

2 2. State of the Art

9

2.1 Biorefinery concept

In the long run, our current way of life is unsustainable. Our consumption and waste levels

cannot be maintained without decreasing the possibilities for next generation. Our usage of

ecosystem services is at such level that the earth is unable to regenerate them at the same pace

(Eickhout, 2012).

We live in a fossil-based economy, in which fossil resources are used for all sorts of

everyday applications, such as electricity, cooling and heating, fuels, materials and chemicals.

They have been used at unprecedented rates (Höök and Tang, 2013) and it is becoming evident

that our economies are unsustainable given that the fossil resources are dwindling and set to get

more expensive. In addition to the non-renewable nature of these resources, their over-

consumption also contributes to serious environmental issues. In 2016, the energy sector

accounted for 68% of the GHG emissions, the main cause of global warming (IEA, 2016b).

The world needs feedstocks that are widely available, relatively inexpensive, renewable

and that can be grown and processed in a sustainable manner. It is undeniable that several sorts

of biomass can fulfil these requirements. The conversion of biomass to energy carriers and a

range of useful products, including food and feed, can be carried out in multi-product biorefineries

(IEA, 2009).

The International Energy Agency (IEA) Bioenergy Task 42 defines biorefinery as the

“sustainable and synergetic processing of biomass into marketable food and feed ingredients,

products (chemicals, materials) and energy (fuels, power, heat)”, which includes systems that

may exist as a concept, a facility, a process, a plant, or even a cluster of facilities (IEA, 2014).

A biorefinery is the integral upstream, midstream and downstream processing of biomass

into a range of products (IEA, 2009). The concept of “integrated biorefinery” describes the main

goal of biorefineries, in which biomass can be used in an optimal way, leading to a longer lifespan

of the resource/product, and thereby increasing its efficient and effective application. Under this

system, several conversion technologies are integrated and designed to maximize the valuable

components, while minimizing the waste streams and processing steps. Biomass is separated

into fractions: the valuable molecules are processed into high-value products such as chemicals

and materials, while the lower quality fractions – side streams – can be used to produce fuels or

to energy recovery (Fernando et al., 2006).

A biorefinery can use all kinds of biomass including forest resources, agricultural crops

such as switchgrass, corn and soybeans, organic residues (both plant and animal-derived, and

industrial and municipal wastes) and aquatic biomass (microalga and seaweed). The major

components of these biomasses include carbohydrates (cellulose and hemicellulose for crop

residues, forage crops and woody crops, starch for grain, and primarily sucrose for sugar crops),

lipids (fats, waxes and oils), proteins, aromatic compounds (primarily lignin), and ash (non-carbon

minerals such as calcium, phosphorus, potassium, etc.) (Dale and Kim, 2008).

The development of biorefineries represents the access key to an integrated production of

food, feed, chemicals, goods, and fuels to meet the future demands.

10

2.1.1 Aquatic biomass

The main driver for the establishment of biorefineries is sustainability, which implies that

biorefinery products should be produced without impacting the economy and ecosystems from

the life cycle perspective (Jung et al., 2013). Therefore, the feedstock supplies must conform to

these parameters.

The use of agricultural substrates – corn, soybean, or sugar cane – to feed biorefineries

(mainly to produce biofuels) is involved in controversial debates due to the “food vs energy”

dilemma. Crop biomass is traditionally destined for food and feed applications and its use as

feedstock competes with both purposes for land, water and other resources, which raises the

question of social sustainability (Fernand et al., 2017).

The use of non-food biomass, such as crop residues (corn stover, manure, straw, waste

wood) and energy crops cultivated on non-arable lands (miscanthus, poplar, willow, switchgrass),

is not in confrontation with food and feed availability. However, their use is not cost effective due

to the abundance of lignin in the composition of these biomasses. Several expensive pre-

treatment steps are required to disintegrate these matrices in order to convert such substrates

into biofuels and bioproducts (Sambusiti et al., 2015).

Although woody biomass is considered a potential feedstock option for biorefineries, its

composition is similar to agricultural residues, meaning that the expensive pre-treatment steps

are still required. In addition, getting this kind of biomass from forests, which are rich in biodiversity

and delicate ecosystems, go against the idea of sustainability (Ghatak, 2011).

Aquatic biomass – microalgae and seaweeds – seems to circumvent the concerns related

with terrestrial biomass. Alga production does not affect the conventional agriculture because

these organisms do not need land or fresh water once they have the ability to grow under harsh

conditions like saline, brackish and coastal seawater (Behera et al., 2015). In addition, aquatic

biomass has a lower risk for the competition for food than other crops, since this application is

important in few East Asia countries (Jung et al., 2013). Other macroalgal uses are for

hydrocolloid extraction, application as fertilizer, and animal feed. It should be noted that

macroalgae do not include lignin in their composition (Saqib et al., 2013) because they do not

need to stand rigidly in the water. This allows the processing of algae in relatively mild conditions

comparing with lignocellulosic biomass. Lower temperatures, less severe acid conditions, and

shorter reaction times are generally needed (Van Hal et al., 2014).

Algae can grow through the year, consequently they have a short harvesting cycle in

comparison to other conventional crops, which have harvesting cycles of once or twice in a year

due to the seasonal variations (Chisti, 2008; Schenk et al., 2008). Moreover, they can convert

solar energy into chemical energy with higher photosynthetic efficiency (6-8%) than terrestrial

biomass (1.8-2.2%) (Ross et al., 2009), therefore they can generate and store higher amounts of

carbon and lipid resources, which can be exploited in biomaterial and bioenergy production.

Amongst aquatic biomass, the use of macroalgae in a bio-based economy is more

advantageous than the use of microalgae because seaweeds show higher volumetric production

rates (biomass per volume per time) and biomass densities (Van Hal et al., 2014).

11

2.2 Macroalgal backgrounds

Seaweeds are the most important organisms in marine ecosystem for the preservation of

marine bio-resources and seawater quality by preventing pollution and eutrophication, as well as

absorbing and fixating CO2 aided by solar energy (Notoya, 2010).

Macroalgae are multicellular organisms and belong to the lower plants, consisting of a leaf-

like thallus instead of roots, stems, and leaves (Cavalier-Smith, 2007; Lobban and Wynne, 1983).

They are photoautotrophic organisms, therefore, produce and store organic carbon by the

conversion of CO2 and HCO3 (Gao and McKinley, 1994). Their photosynthetic rates vary

according to the species but are usually higher than those of terrestrial biomass, like corn and

switchgrass (Chung et al., 2011).

The pigment content, growth rates, and chemical composition of macroalgae are

significantly affected by their habitat conditions such as light, temperature, salinity, nutrient,

pollution and even water motion (Lobban and Wynne, 1983). Light represents the most important

factor affecting pigmentation, allowing to classify macroalgae into Phaeophyta (brown),

Rhodophyta (red) and Chlorophyta (green) classes (Chan et al., 2006). According to the

respective pigments, algae are able to selectively absorb light with specific wavelengths. For

instance, some red algae are able to grow in the deep sea (over 25 m below the surface)

(Santelices, 1991).

In terms of chemical composition, macroalgae have high contents of carbohydrates (20-

60%) (Jung et al., 2013), minerals (10-50% dry wt.) (Ross et al., 2008) and proteins (7-47% dry

wt.) (Jensen, 1993; Marsham et al., 2007); lipids are also present but in very small quantities (1-

5% dry wt) (Jensen, 1993).

The content and specific carbohydrate composition vary significantly among macroalga

classes. As recently reviewed by Jung et al. (Jung et al., 2013), green algae (25-50% dry wt.

carbohydrates) are mainly composed of mannan, ulvan, starch (i.e., α-1,4-glucan) and cellulose.

The proportion of starch is very small (1-4%) and ulvan, which major components are D-

glucoronic acid, D-xylose, L-rhamnose, and sulfate, is a distinctive feature of green algae.

The main polysaccharides of red algae are carrageenan (up to 75% dry wt.), which consists

of repeating D-galactose units and anhydrogalactose, and agar (up to 52% dry wt.), made up of

alternating β-D-galactose and α-L-galactose, with scarce sulfations (Jung et al., 2013). Purified

carrageenan from red algae is generally used to form thick solutions or gels, for instance, it is

used in air freshener gels, toothpaste, firefighting foam, shampoo, cosmetic creams, as food

additives for dairy products, and also in biotechnology as a gel to immobilise cells/enzymes

(Necas and Bartosikova, 2013). In turn, agar is applied in food, pharmaceutical and biological

industries (e.g. it is used as food ingredients, as laxative in pharmaceutical industry, and as solid

media for microbial growth) (McHugh, 2003).

Finally, brown algae are mainly composed of alginic acid (i.e., alginate), laminarin,

fucoidan, cellulose and mannitol. Alginate, composed of mannuronic and guluronic acid blocks,

is the principal constituent of the cell wall (up to 40% dry wt.) and is mostly used in textile (50%)

12

and food (30%) industries (McHugh, 2003). Laminarin, composed of β-1,3-glucan units, accounts

to 35% dry wt. of brown algae (Jung et al., 2013).

Almost 95% of seaweeds used by humans is a result of cultivation activities (FAO, 2014b,

2014c). Seaweed farming is practised in about 50 countries and it expanded 8% per year in the

past decade (FAO, 2014a). Indonesia is the major contributor for aquatic plant production growth,

with an increase in annual farmed seaweeds output by more than 10 times, from less than a

million tonnes in 2005 to 10 million tonnes in 2014 (FAO, 2014a). This impressive expansion is

mainly due to the vast areas of sunlit shallow sea, which are suitable for culture sites of the tropical

seaweed Kappaphycus alvarezii and Eucheuma sp. (FAO, 2014a). In addition to these species,

Laminaria japonica, Gracilaria sp., Undaria pinnatifida and Porphyra sp are also the most

commonly farmed. (FAO, 2014a).

In 2014, 28.5 million tonnes of seaweeds were harvested for direct consumption or further

processing for food (traditionally in Japan, the Republic of Korea and China), fertilizers,

pharmaceuticals, cosmetics and other purposes (FAO, 2014a).

Although the new developments in which seaweeds are considered potential feedstocks

for bioenergy production, it is shown that no commercial process can be developed only based

on the generation of bioenergy (IEA, 2017). However, the maximization of the inherent value of

all macroalgal components in integrated biorefinery approaches could make the process

economically viable.

Growing attention is also focused on the nutritional value of several seaweed species due

to the high content of natural vitamins, minerals, and plant-based proteins. Macroalgae have

shown to provide a rich source of natural bioactive compounds with anti-viral, anti-fungal, anti-

bacterial, anti-oxidant, anti-inflammatory, hypercholesterolemia, hypolipidemic and anti-

neoplastic properties (Plaza et al., 2008).

Recently, different food and drink products containing seaweed flavours have been

commercialized. The majority of seaweed-flavoured food and drink products are currently

launched in the Asia Pacific region, accounting for 88% of global product launched between 2011

and 2015, Europe launched 7% in this time, outpacing both North America (4%) and Latin

America (1%) (Mintel, 2016).

2.2.1 Macroalgae as a source of protein

The protein content and amino acid (AA) composition in seaweeds vary with species and

season (Harnedy and FitzGerald, 2013). The highest protein levels are commonly found during

the period of winter-early spring and the lowest during summer-early autumn (Galland-Irmouli et

al., 1999; Khairy and El-Shafay, 2013; Rouxel et al., 2001). Such fluctuations have been related

to several variables, including nutrient supply, temperature, available light and salinity (Harnedy

and Fitzgerald, 2011). Generally, the protein content is low in brown seaweeds (3-25% dry wt.),

moderate in green algae (9-26% dry wt.) and high in red seaweeds (<47% dry wt.) (Fleurence,

2004).

The bioactive properties of a protein can be determined essentially by the content,

proportion and availability of its amino acids (Millward et al., 2008). The World Health Organization

13

(WHO), Food and Agricultural Organization of the United Nations (FAO) and the United Nation

University (UNU) have been focused on the energy and nutritional needs of the world population

and have produced recommendations on fundamental nutritional requirements, including the

estimation of indispensable amino acid demand (reference pattern of protein in diet)

(WHO/FAO/UNU, 2007).

The comparison between the amino acid composition in seaweeds, reference pattern

(WHO/FAO/UNU, 2007) and other food-proteins, give us a first estimation about the macroalga

protein quality (based only on chemical analysis). However, a biological evaluation including

human and animal feeding studies (to assess in vivo protein digestibility and bioavailability of

essential amino acids) is required to establish the complete nutritional value of macroalgae

(Kumar and Kaladharan, 2007).

The digestibility in vivo of algal proteins is not well studied, and available studies about their

assimilation by humans and animals have not provided conclusive results (Conde et al., 2013).

However, a high rate of algal protein degradation in vitro by proteolytic enzymes such as pepsin,

pancreatin and pronase has been described (e.g. 78%, 87% and 95% for Pyropia tenera, Ulva

pinnatifida and Ulva pertusa, respectively) (Fleurence, 1999).

Despite seasonal and interspecies variability, seaweeds contain all the essential amino

acids (EAAs) and seem to be able to contribute with the adequate levels of EAAs, meeting the

requirements reported in reference pattern (Kumar and Kaladharan, 2007; Matanjun et al., 2009).

Moreover, macroalgae show higher concentrations of EAAs than those commonly present in

other protein sources, such as soy beans and eggs (Bleakley and Hayes, 2017; Lourenço et al.,

2002).

Concerning the EAAs, lysine and tryptophan are often in short supply, in relation to

metabolic requirements, in most alga species (Bleakley and Hayes, 2017). The most frequent

EAA in brown, green and red macroalgae is phenylalanine (Matanjun et al., 2009). Cysteine,

methionine, leucine and isoleucine typically occur at low levels in seaweeds (Bleakley and Hayes,

2017; Cerna, 2011).

In the case of non-essential amino acids, all three classes of seaweeds show similar

profiles (Matanjun et al., 2009), with glutamic acid and aspartic acid representing the highest

fraction. Both AAs reach together up to 23% of total AAs in red algae, 28% in green algae, and

30% in brown algae. They are responsible for the distinctive ‘umami’ taste associated to these

organisms (Biancarosa et al., 2017; Wells et al., 2017).

2.3 Extraction of macroalgal proteins

In general, most of macroalga proteins are located intracellularly, surrounded by a cell wall.

The disintegration of the cell wall and the consequent release of intercellular proteins are very

important to ensure the commercial application of such biomolecules. For instance, seaweeds

have poor protein digestibility in their raw, unprocessed form (Bleakley and Hayes, 2017), which

limits their use in food and feed applications; the extraction of macroalgal proteins will improve

their bioavailability, increasing their commercial value.

14

In addition, as mentioned in section 2.2, although seaweeds are considered potential

feedstocks to produce bioenergy, the economic viability of the process is dependent on the

valorisation of the seaweed remaining components, such as proteins, through integrated

biorefinery approaches – Figure 2.1.

Figure 2.1 | Macroalga biomass refinery scheme. Adapted from (IEA, 2016a)

Algal proteins are conventionally extracted by means of aqueous, acidic, and alkaline

methods, followed by several fractionation techniques such as centrifugation, ultrafiltration,

precipitation and/or chromatography (Kadam et al., 2017). However, one of the most important

factors influencing the successful extraction of proteins is the accessibility of such molecules,

which can be substantially hindered by macromolecular cell wall assemblies, cross-linked via

disulphide bonds to polysaccharides (Harnedy and Fitzgerald, 2015). Therefore, cell-disruption

methods and specific chemical reagents (e.g. reducing agents) are often used to improve the

efficiency of protein extraction.

To date osmotic shock, mechanical grinding, ultrasonic treatment and enzyme aided

digestion are the most common methods used for macroalga cell-disruption - Table 2.1. In

general, after cell disintegration, aqueous protein extracts are first obtained. A second extraction

step under strong alkaline conditions (pH~12) is usually performed to recover the fraction of

insoluble proteins (Harnedy and FitzGerald, 2013).

15

Table 2.1 | Conventional pre-treatment cell disruption methods and extraction processes for the isolation of

macroalgal proteins. (*) Protein yield expressed as % of total protein (Protein extracted/Total proteins x 100).

Cell-disruption method

Extraction Process

Macroalga species

Protein Recovery Yield

Reference

Osmotic shock

Aqueous & alkaline

(sequential)

Palmaria Palmata 6.77±0.22% dry

wt. (Harnedy and

FitzGerald, 2013)

Ulva rigida 26.80±1.3%* (Fleurence et al.,

1995)

Ulva rotundata 36.10±1.4%* (Fleurence et al.,

1995)

Aqueous


1995)


1995)

High shear force Aqueous &

alkaline (Sequential)


wt. (Harnedy and

FitzGerald, 2013)

Porphyra acanthophora var.

acanthophora

8.94±0.53% dry wt.

(Barbarino and Lourenço, 2005)

Sargassum vulgare

6.91±0.15% dry wt.

(Barbarino and Lourenço, 2005)

Ulva fasciata 7.30±0.84% dry

wt. (Barbarino and

Lourenço, 2005)

Enzyme hydrolysis

Aqueous/protein released by action of enzyme activity

& alkaline (sequential)


wt. (Harnedy and

FitzGerald, 2013)

Aqueous/protein released by action of enzyme activity

& acidic (sequential)


1995)


1995)

Sonication Acidic


1995)


1995)

None - direct

Acidic


1995)


1995)

Acidic & alkaline (sequential)


1995)


1995)

Aqueous biphasic system

(PEG/K2CO3)


1995)


1995)

Little or no studies to date have looked at industrial significant protocols for the extraction

of macroalga proteins. Most of the conventional methods described above are laborious and time-

consuming, and there are also scalability concerns associated to some of them. However, the

16

main drawback is the use of alkaline and acidic solutions to mediate the protein extraction in the

majority of these conventional methods. The feasibility of a method for protein extraction depends

mainly on the type of product and the intended downstream application. The use of severe

conditions (extreme pH) can affect the integrity and the consequent functionality of the extracted

proteins.

Novel methods for cell-disruption and extraction of proteins from macroalgae are required.

Since the value of isolated proteins depends on their functional properties, mild operation

conditions for their recovery and separation are desirable. It is important to note that, in addition

to ensure protein functionality, the extractive performance (high protein recovery yields) is also a

crucial parameter to take into account when attempting the development of novel extraction

processes (Harnedy and Fitzgerald, 2015).

2.4 Ionic liquids as new solvents

In the last decades, the world “green” acquired a new meaning in chemistry-related fields,

changing the way by which academia and industry design chemical processes. “Green chemistry”

has demonstrated how fundamental scientific methodologies can protect human health and the

environment in an economically beneficial manner (Anastas and Kirchhoff, 2002). In this context,

besides the development of renewable feedstocks, the design of safer chemicals and

environmentally benign solvents became a priority (Capello et al., 2007). Ionic liquids have

triggered the attention of scientific community as a possible alternative to meet the requirements

of “green chemistry”. At the same time, ILs have been proposed as new solvents for the extraction

and separation of value-added compounds from biomass (Passos et al., 2014), being able to

maintain the stability of extracted molecules, namely, proteins, enzymes and nucleic acids

(Naushad et al., 2012; Vijayaraghavan et al., 2010). These evidences make the use of ILs

promising for the extraction and isolation of proteins from macroalgae.

Ionic liquids are organic salts, with melting points below 100ºC, composed by cations and

anions with low symmetry and low charge density (Seddon, 1997). The choice of these

components determines the distinct physicochemical properties of each IL (melting point, polarity,

viscosity, chemical and thermal stability, solvation properties) (Stark, 2011). They are described

as “designer solvents” since there is a large degree of cation/anion combinations, allowing the

possibility of tuning their characteristics, such as thermo-physical properties, biodegradation

ability or toxicological issues, as well as their hydrophobicity and solution behaviour (Passos et

al., 2014).

ILs’ features and all related advantages make them competitive against conventional

organic solvents (Canales and Brennecke, 2016). They stand out by their negligible vapour

pressure, non-flammability, wide electrochemical window, high chemical and thermal stability and

ability to dissolve a wide range of organic and inorganic compounds, facilitating diverse types of

chemical transformations and separation processes (Bennett and Leo, 2004). The replacement

of volatile organic solvents by non-volatile ILs eliminates solvent losses to the atmosphere,

17

decreasing the environmental footprint. This is the main reason by which ionic liquids are

considered “green” (Zhao et al., 2005).

2.4.1 Ionic liquids for macroalga cell disintegration

The basis of the extraction mechanism proposed in this dissertation lies on the interaction

of ionic liquids with the macroalga cell wall. It is expected that such interaction leads to the

complete or partial cell wall disintegration, allowing access to the desired protein molecules.

The core component of plant cell walls are cellulose microfibrils, accounting to roughly one-

third of the total mass of many plants (Somerville, 2006). Although the proportion of cellulose in

marine algae varies more than in higher plants, cellulose remains a significant fraction in the

majority of seaweeds. In most green algae, the cellulose content can reach up to 70% of the dry

wt. (e.g. Cladophorales and Ulvales) (Baldan et al., 2001). However, in red and brown algae, the

cellulose content is lower and can be lacking in some species, being replaced by xylans and

mannans, which also perform structural function, although with less rigidity than cellulose

microfibrils.

Cellulose consists of a polydisperse linear glucose polymer chain which forms hydrogen-

bonded supramolecular structures (Finkenstadt and Millane, 1998). Glucose units are linked by

1,4-β glycosidic bonds, which stability is reinforced by intrachain hydrogen bonds (Hinterstoisser

and Salmén, 2000).

Although the monomer glucose and short oligomers are water-soluble, cellulose is not.

Reasons for this are its high molecular weight (solubility is usually inversely related to polymer

length) and the comparatively low flexibility of cellulose polymer chains (Arneniades and Baer,

1977), which arises from the strong glycosidic bonds and the huge degree of both intra- and

intermolecular hydrogen bonds (Figure 2.2) (Pinker et al., 2009).

Figure 2.2 | Intra- and intermolecular hydrogen bonds in cellulose. Adapted from (Pinker et al., 2009).

To separate the polymeric structure of cellulose, an excellent solvent must be employed in

order to disrupt the intermolecular H-bond interactions, leading to the polymer dissolution (Mohd

et al., 2017).

In 2002, it was found that several ionic liquids can dissolve large amounts of cellulose

(Swatloski et al., 2002), which opened up the way to the development of new cellulose solvent

systems. The most successful cations for cellulose dissolution are based on methylimidazolium

18

and methylpyridinium cores, with allyl-, ethyl-, or butyl- side chains and the most promising anions

are chloride, acetate, and formate (Pinker et al., 2009).

It is thought that both anions and cations are involved in the cellulose dissolution process.

Figure 2.3 shows the proposed dissolution mechanism of cellulose by ionic liquids, in which

electron donor-electron acceptor (EDA) complexes are formed between the hydroxyl groups of

cellulose and the ionic liquids. In these complexes, the oxygen atoms serve as electron pair

donors and the hydrogen atoms act as electron acceptors. On the other hand, the cations of the

ionic liquid act as electron acceptor centres and the anions as electron donor centres. Both

centres must be located close enough in space to allow the interaction and the formation of EDA

complexes. Upon interaction of the cellulose-OH with the ionic liquid, the oxygen and hydrogen

atoms from hydroxyl groups are separated, resulting in the opening of the hydrogen bonds

between the molecular chains, and their dissolution (Feng and Chen, 2008).

Figure 2.3 | Proposed dissolution mechanism of cellulose in ionic liquid 1-Butyl-3-methylimidazolium chloride

([BMIM][Cl]). Adapted from (Pinker et al., 2009).

It has been demonstrated that the capability of ILs to dissolve cellulose are directly

correlated to their strong hydrogen bond basicity and lower viscosity (Zhang et al., 2017). The

hydrogen bond basicity describes the ability of a molecule to act as a hydrogen bond acceptor

(Oliferenko et al., 2004), a role played by ionic liquid anions according to the proposed

mechanism. In this sense, the better the hydrogen bond acceptor ability of the IL anion, the higher

is its ability to solubilize cellulose (Stark et al., 2012). On the other hand, the chemical structure

of cations also has an important effect on the cellulose dissolution. In general, when the chain of

alkyl groups or the symmetry of the cations increases, the dissolution rate of cellulose in ILs

decreases, due to the rise of the viscosity and/or decrease of cation hydrogen bond acidity (Zhang

et al., 2017).

2.4.2 Ionic liquid-based aqueous biphasic systems for biomolecules

fractionation

Aqueous two-phase systems (ATPSs) have been used for the recovery of a broad array of

biomolecules (e.g. proteins, polysaccharides, nucleic acids) through their partition between the

two aqueous liquid phases (Liu et al., 2012), which makes its use a promising strategy for refining

other seaweed components beyond the desired proteins. This allows the maximization of the

inherent value of several components from seaweed, which may be crucial for the profitable use

of seaweeds as raw-materials (Van Hal et al., 2014).

19

ATPS consists of two immiscible aqueous-rich phases based on polymer-polymer,

polymer-salt or salt-salt combinations. Although the most investigated ATPSs are the polymer-

based systems, in the last decade, ionic liquids were proposed as alternative phase-forming

components (Gutowski et al., 2003).

In an ATPS, both solutes that make up the system are water soluble, they separate into

two coexisting phases above their critical concentration, in which each phase will contain

predominantly only one type of solute (Freire et al., 2012). The coexisting phases will offer

different physicochemical environments for the biomolecules, driving them more extremely to one

phase, according to the biomolecule properties (e.g. hydrophobicity, charge, size) (Andrews and

Asenjo, 1989).

The phase diagram can be considered as a fingerprint of an ATPS under specific conditions

(e.g. temperature and pH). It is unique and shows the potential working area of the ATPS,

providing information about the required concentration of phase-forming components for phase

splitting, the concentration of phase-components in the top and bottom phases, and the ratio of

phase volumes (Raja et al., 2012). Figure 2.4 represents a phase diagram for a hypothetical

system, in which the binodal curve (TCB) indicates the boundaries of mono- and biphasic regions.

For mixture compositions above the binodal curve, there is the formation of a two-phase system,

while mixture compositions below this curve fit into the monophasic region and will not phase

separate. The three systems X, Y and Z differ in their initial compositions and in volume ratios.

However, in equilibrium, all the systems have the same top phase composition (T IL,Tsalt) and the

same bottom phase composition (BIL,Bsalt). This is because they are lying on the same tie-line

(TL) – TB in Figure 2.4 –, whose end points determine the equilibrium phase composition. Point

C on the binodal curve is called critical point, just above this point the volume of both phases is

theoretically equal, and the tie-line length (TLL) is 0.

Figure 2.4 | Phase diagram for a hypothetical system composed of ionic liquid + salt + water (weight fraction

percentage). TCB: Binodal curve; C: Critical point; TB: Tie-line; T: Composition of the top phase; B:

Composition of the bottom phase; X, Y and Z: total composition of the ATPS. Adapted from (Raja et al.,

2012).

20

The units of TLL are % w/w, same as the component concentrations, and it can be

estimated by using the ratio of the mass compositions (Equation 2.1) (Freire, 2016a):

𝑉𝑇𝜌𝑇

𝑉𝐵𝜌𝐵

=𝑋𝐵

𝑋𝑇

Equation 2.1

in which V and ρ stands for volumes and densities of top (T) and bottom (B) phases while XB and

XT are the segment lengths of the tie-line as shown in Figure 2.4.

Equation 2.2 describes an alternative and more accurate way for the TLL determination

based on the equilibrium phase compositions (Freire, 2016a):

𝑇𝐿𝐿 = √[𝐵𝑠𝑎𝑙𝑡 − 𝑇𝑠𝑎𝑙𝑡]2 + [𝑇𝐼𝐿 − 𝐵𝐼𝐿]2

Equation 2.2

Bsalt and Tsalt are the concentrations of salt in the bottom and top phases, respectively,

whereas TIL is the concentration of IL in the top phase, and BIL is the concentration of ionic liquid

in the bottom phase.

The tie-lines are commonly parallel, and their slope (STL) can be determined by

Equation 2.3 (Freire, 2016a), facilitating the construction of further TLs.

𝑆𝑇𝐿 =[𝑇𝐼𝐿 − 𝐵𝐼𝐿]

[𝐵𝑠𝑎𝑙𝑡 − 𝑇𝑠𝑎𝑙𝑡]=

∆𝐼𝐿

∆𝑠𝑎𝑙𝑡

Equation 2.3

The knowledge of the binodal curve is essential when working with ATPS because the

composition of the two phases must be known in order to understand the partition of biomolecules.

The binodal curve can be determined by three methods: turbidimetric titration, cloud-point and

node determination methods (Iqbal et al., 2016).

In most of IL-based ATPS studies, the binodal experimental data is usually fitted according

to Equation 2.4 (Merchuk et al., 1998), in which Y and X are the mass fraction percentages of the

ionic liquid and the salting-out agent, respectively. a, b and c are fitting parameters obtained by

least squares regression.

𝑌 = 𝑎𝑒𝑥𝑝[(𝑏 × 𝑋0.5) − (𝑐 × 𝑋3)]

Equation 2.4

The fitting of the binodal data allows the determination of TLs by a weight balance

relationship. The compositions of the top and bottom phases and the overall system composition

are calculated by the lever-arm rule. Commonly, the following system of four unknown constants

is applied to fit the tie-line data (Equation 2.5 to Equation 2.8) (Merchuk et al., 1998): T, B and M,

designate the top phase, the bottom phase and the mixture, respectively; Y represents the IL

weight fraction percentage, whereas X represents the salting-out agent; α is the ratio between the

mass of the top phase and the total mass of the mixture; and A, B and C are the fitted constants

obtained by the application of Equation 2.4.

21

𝑌𝑇 = 𝑎𝑒𝑥𝑝[(𝑏 × 𝑋𝑇0.5)] − (𝑐 × 𝑋𝑇

3)]

Equation 2.5

𝑌𝐵 = 𝐴𝑒𝑥𝑝[(𝐵 × 𝑋𝐵0.5)] − (𝐶 × 𝑋𝐵

3)]

Equation 2.6

𝑌𝑇 =𝑌𝑀

𝛼−

1 − 𝛼

𝛼× 𝑌𝐵

Equation 2.7

𝑌𝑇 =𝑋𝑀

𝛼−

1 − 𝛼

𝛼× 𝑋𝐵

Equation 2.8

The ratio of equilibrium compositions in the ATPS top and the bottom phases determines

the partition coefficient (K) of biomolecules, which is given by Equation 2.9.

𝐾 =𝐶𝐴𝑇

𝐶𝐴𝐵

Equation 2.9

where CAT is the equilibrium concentration of component A in the top phase and CAB is the

equilibrium concentration of A in the bottom phase. The partition coefficient is often used to

evaluate the extension of biomolecules separation in the ATPS. The more different the K of a

target biomolecule from the K of the remaining biomolecules present in the system, the better the

extraction. In other words, K values greater than one indicate the effectiveness of partitioning in

the ATPS (Mazzola et al., 2008).

IL-based ATPSs have been firstly proposed in 2003 (Gutowski et al., 2003), and have been

widely used in biological separation and purification fields (Chen et al., 2014). The system is

usually formed by water + ionic liquid + inorganic salt, owing to the high ability of salt ions to

induce the salting-out of IL. This way, the phase formation is due to the preferential hydration of

the high charge-density salt ions, over the low-symmetry and charge delocalized IL ions, which

are only able of weak directional intermolecular interactions (and hence weakly hydrated as

compared to the common salting-out inducing salts) (Freire, 2016b).

Obviously, the nature of the inorganic salt used as salting-out agent plays a crucial role in the

definition of any ATPS. The ATPS formation trend follows closely the well-known Hofmeister

series, which ranks the relative influence of ions on the physical behaviour of a wide variety of

aqueous processes (Zhang and Cremer, 2006). Generally, this influence is more pronounced for

anions than for cations (Schneider et al., 2011), and the typical ordering of the ions and some of

its related properties are shown in Figure 2.5. The species in the green region are described as

kosmotropes, while those in red region are chaotropes. These terms refer to the ion ability to alter

the hydrogen bonding network of water (Collins and Washabaugh, 1985). The kosmotropes,

which are believed to be “water structure makers”, are strongly hydrated and have stabilizing and

salting-out effects on proteins. On the other hand, chaotropes (“water structure breakers”) are

known to destabilize folded proteins and give rise to salting-in behaviour.

22

Figure 2.5 | Ordering of the ions and related properties in the Hofmeister series (Zhang and Cremer, 2006).

Several studies have reported the successful application of IL-based ATPS for protein

partition. The proteins were mainly concentrated in the IL-rich phase (top phase) and, although

the partition mechanism involved in the ATPS is still poorly understood, hydrophobic and

electrostatic interactions, as well as salting-out effects are suggested to be the main driving forces

responsible for such behaviour (Ventura et al., 2017). The formation of IL aggregate-protein

complexes has also been proposed as the driving force for the selective separation of proteins

(Pei et al., 2010).

From an engineering point of view, the extraction and separation methods should be

connected, preferably merged and conducted in a single-step. Therefore, high expectations are

placed on the use of ionic liquids as novel solvents once they can provide a promising platform,

by means of an ATPS, for the simultaneous extraction and selective separation of compounds

from macroalgae.

2.4.3 Ionic liquid recovery

Ionic liquids remain to date the most expensive research-grade solvents, under

investigation, for the dissolution of biomass, which is one of the major drawbacks when envisaging

their large-scale application (George et al., 2015). In addition, when considering their application

at an industrial scale, the possible environmental impact must be considered. Although the lack

of volatility of ionic liquids does not contribute to the release of harmful vapour into the

atmosphere, there still exist concerns about their toxicity and biodegradability, which can cause

severe water contamination upon the release to aquatic environments (Frade and Afonso, 2010).

In this sense, it is of the most importance the recovery of ionic liquids from the algal extracts,

so that they can be reused in further extractions/separations steps, saving the overall costs of the

process and avoiding the associated environmental impact. Depending on the nature of the ILs

and the target molecules to extract, a wide range of recovery methods have been proposed in

literature (Mai et al., 2014). However, barrier processes stand out from competitive techniques

because, in addition to their high-performance (Kuzmina, 2016), membrane separations are a

well-known procedure, commercially available to industrial deployment and considered a low-

23

energy demanding process, in which no chemicals are added, decreasing the costs (Haerens et

al., 2010; Wu et al., 2009).

A membrane process performs a certain separation by means of a membrane capable of

retaining specific compounds, while allowing the passage of others. In pressure-driven membrane

processes (reverse osmosis, nanofiltration, ultrafiltration and microfiltration), when a force is

exerted across the membrane, the feed stream goes through it and is split into permeate and

retentate. Both fractions contain the same solvent, permeate is rich in lower size molecules

whereas the retentate is concentrated with larger size molecules (Beier, 2015).

Particles and dissolved components are (partially) retained based on the properties of the

membrane such as size, shape and charge. The separation efficiency is expressed by the

rejection coefficient (R) of a given compound (Equation 2.10):

𝑅 = 1 −𝐶𝑝

𝐶𝑓

Equation 2.10

where Cp stands for permeate concentration and Cf stands for feed concentration of the specific

compound. The rejection coefficient ranges from 0 for complete permeation to 1 for complete

rejection (Van Der Bruggen et al., 2003).

Pressure-driven membrane processes can be classified by several criteria: the

characteristics of the membrane (pore size), size and charge of the retained particles or

molecules, and pressure exerted on the membrane (Beier, 2015). This classification distinguishes

microfiltration, ultrafiltration, nanofiltration, and reverse osmosis. In the specific case of this

master’s dissertation, ultrafiltration was performed in order to recover the ionic liquid from the

macroalga extracts (Figure 2.6). Ultrafiltration membranes have pores with 2-100 nm that allow

the retention of macromolecules (e.g. proteins) while it is permeable to salts (e.g. ionic liquid)

(Van Der Bruggen et al., 2003).

Figure 2.6 | Schematic representation of ultrafiltration. Adapted from (Van Der Bruggen et al., 2003).

24

2.5 Macroalgal proteins applications

Seaweeds are rich sources of proteins and contain all the essential amino acids at various

concentrations (Kumar and Kaladharan, 2007; Matanjun et al., 2009), which suggest the high-

quality of macroalgal proteins. Although further studies in in vivo digestibility of seaweed proteins

are necessary to evaluate their functional properties, macroalga proteins seem to be suitable for

the formulation of rich-protein supplements for human and animal dietary or to enhance the

protein content of foods and feed, which will be useful to fulfil the future needs. In addition, it is

also important to note that proteins are an essential nutritional component in the diet of athletes,

required to repair and build muscle during exercise (Kerksick et al., 2006). The American College

of Sports Medicine recommends a protein intake of 1.2-1.7 g.kg-1.day-1 for endurance- and

resistance-trained athletes (Gerovasili et al., 2009). Once again, algae could represent a valuable

resource for athletes requiring high levels of protein, especially for vegan athletes for whom eggs

and dairy whey protein may not be suitable (Bleakley and Hayes, 2017).

Macroalgal protein extracts can also be a potential source for valuable amino acids for food

and feed supplements. The presence of essential amino acids in right quantities is crucial to make

quality proteins and they cannot be replaced by other “less-valuable” building-blocks (Holdt and

Kraan, 2011).

Besides the nutritional purposes in food, proteins can also provide and/or stabilize the

characteristic structure of individual foods. The functional properties of proteins are mainly

associated with their ability to form and/or stabilize networks (gels and films), foams, emulsions

and sols, which is often revealed by their tertiary structure (Foegeding and Davis, 2011). For

instance, in milk proteins, the intrinsically unfolded structure of caseins, in contrast to the compact

globular structure of whey proteins, alters their film forming ability and thereby foaming properties

(Marinova et al., 2009).

During the last decade, consumer requirements in the field of food production have

changed considerably (Siró et al., 2008). Consumers increasingly believe that food contribute

directly to their health and, today, foods are no more intended to only satisfy hunger and to provide

the necessary nutrients, but also and specially to prevent nutrition-related diseases and to

improve physical and mental well-being (Bigliardi and Galati, 2013). In this regard, there is a

market trend in the food industry towards the development and manufacturing of functional

products1 (Plaza et al., 2010). The general contribution of proteins/peptides with specific

bioactivities is of increasing importance prompting the development of food products based on

protein-related health issues (Kitts and Weiler, 2003).

1 Functional products are considered to be fortified, enriched, altered or enhanced foods that provide an additional health or well-being benefit besides the energetic and nutritional aspects that every food must confer (Hasler, 2002).

25

Some macroalgal proteins (lectins and phycobiliproteins) are considered bioactive

compounds2 and therefore, they have great potential to be used as ingredients for functional

foods, in addition to be useful for pharmacologic proposes (Samarakoon and Jeon, 2012).

Seaweed proteins are also sources of bioactive peptides that, although inactive within the parent

proteins, can be released through microbial fermentation or enzymatic hydrolysis (Daliri et al.,

2017; Jiménez-Escrig et al., 2011). These protein fragments have useful properties for human

health, such as anti-microbial, anti-fungal, anti-viral, anti-oxidative, anti-thrombotic, anti-

hypertensive, anti-tumor, immunomodulatory and appetite suppression activities (Korhonen and

Pihlanto, 2006; Perez Espitia et al., 2012).

Algal proteins and derivatives are also suitable for several cosmetic applications. For

instance, these biomolecules are important for moisture retention on hair and skin. On the other

hand, they also show a strong affinity for both, improving their nourishments. In the sense that

cosmeceuticals are supposed to be involved in healing and repairing damage skin, not only by

moisturizing, but also maintaining the nourishment, proteins and peptides derived from algae can

be applied in cosmeceutical products (Hagino and Salto, 2004).

The anti-oxidative marine algal peptides might be an interesting source of primary

ingredients for the formulation of future cosmeceuticals due to its protective effect from the

reactive oxygen species damaging activities (Samarakoon and Jeon, 2012).

2.5.1 Macroalgal bioactive proteins

2.5.1.1 Lectins

Lectins are glycoproteins that selectively recognize and reversibly bind to carbohydrates

without initiating their further modifications through the associated enzymatic activity (Weis and

Drickamer, 1996). They are involved in many biological processes, such as host-pathogen

interactions, cell-to-cell recognition and signalling, induction of apoptosis, cancer metastasis, cell

growth and differentiation (Ziółkowska and Wlodawer, 2006). Furthermore, due to their high-

specific carbohydrate binding capacity, lectins have been found to increase the agglutination of

blood cells (erythrocytes). They are also useful as cancer biomarkers or as targets for drug

delivery and in the detection of disease-related alterations of glycan synthesis, including infectious

agents such as viruses, bacteria, fungi and parasites (Holdt and Kraan, 2011; Naeem et al., 2007).

Nevertheless, other bioactive properties exhibited by macroalgal lectins include antibiotic,

mitogenic, cytotoxic, anti-nociceptive, anti-inflammatory, anti-adhesion, anti-human

immunodeficiency virus (HIV), and human platelet aggregation inhibition activities (Harnedy and

Fitzgerald, 2011) – Table 2.2.

2 Bioactive compounds are essential and non-essential compounds that occur in nature, are part of the food chain, and have an effect on human health (Biesalski et al., 2009).

26

Table 2.2 | Macroalgal source species and bioactivity of lectins.

Macroalgal source species Bioactivity Reference

Caulerpa cupressoides Anti-nociceptive and anti-

inflammatory activity (Vanderlei et al., 2010)

Codium fragile Host-pathogen interactions (Hori et al., 2000; Smit, 2004)

Eucheuma amakusaensis Induction of apoptosis,

metastasis and differentiation (Hori et al., 2000)

Eucheuma cottonii Recognition and binding of

carbohydrates, including virus, bacteria, fungi and parasites

(Bird et al., 1993; Cardozo et al., 2007; Hori et al., 2000)

Eucheuma serra

Induction of apoptosis in several cancer cell-lines (including

Colo201 and HeLa) (Sugahara et al., 2001)

Suppression of colonic carcinogenesis in mice

Growth inhibition of 35 human cancer cell-lines

(Hori et al., 2007)

Antibacterial against fish pathogen Vibrio vulnificus

(Liao et al., 2003)

Galaxaura marginata Anti-HIV (Mori et al., 2005; Smit, 2004)

Gracilaria sp.

Antibacterial against fish pathogen Vibrio vulnificus

(Liao et al., 2003)

Griffithsia sp. Anti-adhesion (Smit, 2004)

Hypnea japonica

Inhibition of human platelet aggregation

(Matsubara et al., 1996)

Cytotoxic (Smit, 2004)

Solieria robusta Mitogenic activity on mouse

spleen lymphocytes (Hori et al., 1988)

Ulva sp. Antibiotic (Smit, 2004)

2.5.1.2 Phycobiliproteins

Phycobiliproteins are water-soluble, coloured and fluorescent proteins with an important

role in photosynthesis (Glazer, 1994). These molecules can constitute a major proportion of red

alga cell proteins, with reported values of 12% (w/w) dry wt. in Palmaria palmata (Guangce et al.,

2002). There are four main groups of phycobiliproteins – phycoerythrin, phycocyanin,

allophycocyanin and phycoerythrocyanin – which are classified based on their colour and

absorption characteristics (Sekar and Chandramohan, 2008).

Phycobiliprotein pigment molecules spontaneously fluoresce in vivo and in vitro (Aneiros

and Garateix, 2004; Fleurence, 2004) and, therefore, they are used in fluorescent immunoassays,

fluorescent immunohistochemistry assays, biomolecule (proteins, antibody, nucleic acid)

labelling, and fluorescent microscopy (Harnedy and Fitzgerald, 2011). They are also currently

used as natural pigments for food (chewing gums and dairy products) and in cosmetic applications

(Sekar and Chandramohan, 2008; Viskari and Colyer, 2003). Niohan Siber Hegner Ltd. and

Dainippon Ink & Chemical Inc., two companies in Tokyo, market the phycobiliprotein phycocyanin

as a natural food dye (Houghton, 1996). Nevertheless, seventeen patents for the therapeutic

application of phycobiliproteins have been submitted worldwide due to the anti-oxidant, anti-

27

inflammatory, neuroprotective, hypocholesterolaemic, hepatoprotective, anti-viral, anti-tumour,

liver-protecting, atherosclerosis treatment, serum lipid-reducing and lipase inhibition activities that

these biomolecules have shown (Sekar and Chandramohan, 2008).

2.5.2 Macroalgal bioactive peptides

Several protein hydrolysates and associated peptides exhibiting bioactive properties have

been found in marine macroalgae - Table 2.3 and Table 2.4. Therefore, they have potential to be

applied as functional food ingredients (Harnedy and Fitzgerald, 2011). It is important to note that

several studies have reported that alga-derived peptides are able to resist gastrointestinal

digestion from enzymes such as trypsin, pepsin, and chymotrypsin. This is an essential trait for

bioactive peptides in order to achieve their physiological effect at their site of action (Bleakley and

Hayes, 2017).

Table 2.3 | Examples of macroalgal peptides exhibiting biological activity.

Macroalga Species Sequence/Name Biological Activity Reference

Bryopsis sp. Kahalalide A and F

Anti-cancer, anti-tumour and anti-AIDS

(acquired immune deficiency syndrome)

(Smit, 2004)

Galaxaura filamentous Galaxamide Anti-Cancer (Xu et al., 2008)

Hizikia fusiformis Gly-Lys-Tyr; Ser-Val-Tyr;Ser-Lys-Thr-Tyr

Anti-hypertensive (Suetsuna, 1998a)

Palmaria palmata (dulse)

Val-Tyr-Arg-Thr; Leu-Asp-Tyr; Leu-Arg-Tyr; Phe-Glu-Gln-Trp-Ala-

Ser

Anti-hypertensive (Furuta et al., 2016)

Asn-Ile-Gly-Gln Anti-inflammatory (Fitzgerald et al., 2013)

Porphyra yezoensis

Ile-Tyr; Met-Lys-Tyr; Ala-Lys-Tyr-Ser-Tyr;

Ley-Arg-Tyr Anti-hypertensive

(Cha et al., 2006; Suetsuna, 1998b)

Ala-Lys-Tyr-Ser-Tyr Anti-hypertensive (Saito and Hagino,

2005)

Ulva sp. Glu-Asp-Arg-Ley-Lys-

Pro Mitogenic in skin

fibroblasts (Ennamany et al.,

1998)

Undaria pinnatifida Val-Tyr; Ile-Tyr; Phe-

Tyr; Ile-Trp

Reduction of blood pressure in

spontaneously hypertensive rats

(Sato et al., 2002)

Undaria pinnatifida Val-Tyr; Ile-Tyr; Ala-

Trp; Phe-Tyr; Val-Trp; Ile-Trp; Leu-Trp

Anti-hypertensive (Sato et al., 2002)

28

Table 2.4 | Examples of macroalgal protein hydrolysates exhibiting biological activity.

Macroalga species Biological activity Reference

Caulerpa racemosa Anti-oxidant, anti-cancer (Lakmal et al., 2014)

Costaria costata Anti-oxidant, anti-tumour, ACE

(angiotensin converting enzyme)-inhibitory

(Lee et al., 2005)

Ecklonia cava Anti-hypertensive (Cha et al., 2006)

Antioxidant (Heo et al., 2003)

Enteromorpha prolifera Anti-hypertensive, anti-oxidant,

anti-tumour (Lee et al., 2005)

Grateloupia filicina Anti-hypertensive, anti-oxidant,

anti-tumour (Lee et al., 2005)

Hizikia fusiformis Anti-hypertensive (Suetsuna, 1998a)

Ishige okamurae Anti-oxidant (Heo et al., 2003)

Sargassum coreanum Anti-oxidant (Heo et al., 2003)

Sargassum fullvelum Anti-oxidant (Heo et al., 2003)

29

3 3. Aim of studies

31

Although seaweeds show great potential as sources of proteins for both human and animal

nutrition, to date there is no feasible procedure for the recovery of these proteins (Bleakley and

Hayes, 2017). In order to meet this gap, this master’s dissertation aims to study a new integrated

biorefinery approach for the single-step extraction and separation of proteins from macroalga Ulva

lactuca, using ionic liquids as new solvents.

Among the seaweed species available in European Atlantic waters, Ulva sp. (Figure 3.1)

is extensively characterised (Fleurence et al., 1995), having a high protein content (up to 44% dry

wt.) (Holdt and Kraan, 2011). In addition, this species is successfully cultivated in integrated multi-

trophic aquaculture systems, enabling not only scalable and controllable cultivation conditions

(Marinho et al., 2013; Robertson-Andersson et al., 2008) but also the removal of N- and P-rich

compounds in waste water from land-based aquaculture (Lawton et al., 2013). On the other hand,

since Ulva sp. is the world’s most abundant seaweed, this can have a negative impact on the

environment and tourism in the coastline, making its harvesting essential (Briand and Morand,

1997).

Figure 3.1 | Seaweed Ulva lactuca (Krisp, 2011).

One of the limitations in protein extraction from seaweed is the presence of

polysaccharides as structural components of macroalga cell walls (Harnedy and Fitzgerald,

2015). The main polysaccharides in U. lactuca cell wall are ulvan and cellulose, which have a

structural behaviour as described in Figure 3.2.

Figure 3.2 | Distribution of the different Ulva sp. cell wall polysaccharides in a schematic cross section of a

thallus (A) and the proposed associations between the different cell wall polysaccharides (B). Adapted from:

(Lahaye and Robic, 2007).

32

Ulvan is a water-soluble polysaccharide, which main constituents are sulphate, rhamnose,

xylose, and glucoronic acid (Lahaye and Robic, 2007). On the other hand, cellulose is insoluble

in water and in most common organic liquids, mainly due to the many intermolecular hydrogen

bonds present in its structure (Swatloski et al., 2002). The insoluble character of cellulose is a big

challenge in cell disruption methods, especially when the desired molecules are proteins, which

functionality can be easily compromised by aggressive pre-treatment and extraction processes.

Fortunately, ionic liquids have demonstrated a good performance in the complete dissolution of a

wide range of biomass matrices mainly due to their ability to dissolve cellulose (Brandt et al.,

2013). Moreover, they have also been proposed as promising alternative solvents for the

extraction and separation of added-value compounds from biomass. Ionic liquids can provide a

non-denaturing environment for biomolecules, maintaining the protein structure and, if applicable,

ensuring their enzymatic activity (Passos et al., 2014).

In this perspective, this thesis focuses on the development of a process in which U. lactuca

biomass is processed for protein extraction in the presence of a suitable ionic liquid. An aqueous

biphasic system aiming to separate the proteins from the carbohydrates is also studied. Hereby,

the proposed biorefinery approach would allow not only the valorisation of seaweed proteins but

also of carbohydrates for further use, for instance, for bioenergy generation. Finally, the recovery

of the ionic liquid from the macroalga extracts is evaluated by means of ultrafiltration.

33

4 4. Materials and Methods

35

4.1 Sample collection and preparation

Ulva lactuca was kindly provided by Dr. Willem de Visser from Wageningen Plant

Research. In short, cultures were maintained in 1 m3 tanks containing filtered sea water in a

greenhouse property of Wageningen University and Research (Nergena, Wageningen – The

Netherlands). Samples were collected every month in the period June-August 2016. After

biomass collection, the excess water was removed and the samples were freeze dried in a

sublimator 2x3x3 (Zirbus Technology GmbH, Germany) for 72 hours. Dried samples were stored

in sealed bags and maintained in the dark, at room temperature, until further use. Before starting

the experiments, the dried alga was milled in a coffee grinder to obtain a fine and homogeneous

powder, which was stored under the same conditions and regarded as starting biomass (sB).

4.2 Biomass characterization

The algal batch under study was characterized in terms of ash and moisture content. The

quantification of total lipids, carbohydrates and proteins was also performed.

4.2.1 Total solids and ash

The dry weight concentration and ash content were determined according to NREL

(National Renewable Energy Laboratory) analytical procedure, specific for algal biomass (Van

Wychen and Laurens, 2013). For total solids determination, three aluminum trays were weighed

in an analytical balance (ME 23 SP, Santorius, Germany), and the weight of each aluminum tray

was recorded. An amount of 100 mg of sB was weighted in the pre-weighed trays (experiment

performed in triplicate); the weight of each tray and sample was then recorded. Samples were

placed in a convection drying oven (Nabertherm, Germany), and dried for 18 hours at 105ºC and

atmospheric pressure. Afterwards, trays were removed and cooled to room temperature in a

desiccator. Trays and dried samples were weighed and the respective weights recorded. In order

to determine the percentage of total solids and moisture content, Equation 4.1 and Equation 4.2

were, respectively, used. All the results reported in this dissertation are expressed on dry wt. basis

(DW), defined as the weight of biomass mathematically corrected for the amount of moisture in

the sample (Equation 4.3).

% 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠 = 𝑊𝑒𝑖𝑔ℎ𝑡𝑎𝑙𝑢𝑚𝑖𝑛𝑢𝑚 𝑡𝑟𝑎𝑦+𝑑𝑟𝑦 𝑠𝑎𝑚𝑝𝑙𝑒 − 𝑊𝑒𝑖𝑔ℎ𝑡𝑎𝑙𝑢𝑚𝑖𝑛𝑢𝑚 𝑡𝑟𝑎𝑦

𝑊𝑒𝑖𝑔ℎ𝑡𝑠𝐵

× 100

Equation 4.1

% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 = 100 − % 𝑇𝑜𝑡𝑎𝑙 𝑠𝑜𝑙𝑖𝑑𝑠

Equation 4.2

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒 =𝑊𝑒𝑖𝑔ℎ𝑡𝑠𝐵 × % 𝑇𝑜𝑡𝑎𝑙 𝑆𝑜𝑙𝑖𝑑𝑠

100

Equation 4.3

To access the ash content, two glass tubes (VWR International, USA) were weighed in an

analytical balance, and each glass tube weight was recorded. An amount of 100 mg of sB was

36

weighted in the pre-weighted glass tube (experiment performed in duplicate). Samples were

ignited and incinerated using an ash-oven (muffle furnace L 24/11, Nabertherm, Germany)

equipped with a ramping program: temperature was ramped to 105ºC and maintained at this

temperature for 30 minutes; temperature was ramped again until 575ºC in 4 hours and maintained

at this temperature for more 4 hours; temperature was finally dropped until 105ºC in 4 hours.

Samples were removed and cooled down to room temperature in a desiccator. The weight of

glass tubes and respective ashes was recorded. The ashes were expressed as a percentage of

dry matter according to Equation 4.4.

%𝐴𝑠ℎ =𝑊𝑒𝑖𝑔ℎ𝑡𝑔𝑙𝑎𝑠𝑠 𝑡𝑢𝑏𝑒+𝑎𝑠ℎ − 𝑊𝑒𝑖𝑔ℎ𝑡𝑔𝑙𝑎𝑠𝑠 𝑡𝑢𝑏𝑒

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒

× 100

Equation 4.4

4.2.2 Total lipid content

The total lipid content was determined according to Folch et al. (Folch et al., 1987). An

amount of 100 mg of sB was weighed in a glass tube, using an analytical balance (experiment

performed in duplicate). The total lipid extraction began with the addition of 1 mL of chloroform

(HPLC grade, Biosolve Chimie SARL, France), followed by the addition of 1 mL of methanol

(HPLC supra gradient, Biosolve Chimie SARL, France). The mixture was vortexed for 3 minutes,

after which 1 mL of chloroform was added; the mixture was again vortexed for 3 minutes. A

volume of 0.8 mL of phosphate buffer saline (prepared according to Table 4.1) was added and

the resulting suspension was mixed for 45 minutes in an orbital shaker (LD79, Labinco,

Netherlands) at 150 rpm.

Table 4.1 | Composition of 1 L phosphate buffer saline.

Components Purity Supplier

0.21 g KH2PO4 ≥ 99% Merck Millipore, Germany

0.48 g Na2HPO4.2H2O ≥ 98% Merck Millipore, Germany

9.00 g NaCl ≥ 99% Merck Millipore, Germany

1 L distilled water

Samples were then centrifuged at 1000xg (Allegra X-30R Centrifuge, Beckman Coulter,

USA) for 1 minute to accelerate phase separation, and the bottom layer was collected to a pre-

weighed glass tube. The steps described before were performed four times, using the middle and

upper layers as starting material. The bottom layers of the following four extractions were also

collected and the solvent was evaporated using a rotational vacuum concentrator for 45 minutes

(RVC 2-25 CDplus, Martin Christ Freeze Dryers, Germany). Samples were left overnight, at room

temperature, under N2 flow to remove any remaining traces of solvent. The weight of glass tubes

and respective lipids was recorded, and the total lipid content was determined by Equation 4.5.

% 𝑇𝑜𝑡𝑎𝑙 𝑙𝑖𝑝𝑖𝑑𝑠 =𝑊𝑒𝑖𝑔ℎ𝑡𝑔𝑙𝑎𝑠𝑠 𝑡𝑢𝑏𝑒+𝑙𝑖𝑝𝑖𝑑𝑠 − 𝑊𝑒𝑖𝑔ℎ𝑡𝑔𝑙𝑎𝑠𝑠 𝑡𝑢𝑏𝑒

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒

× 100

Equation 4.5

37

4.2.3 Total carbohydrate content

Concerning the determination of total carbohydrate content in macroalgae, 1 mg of sB was

weighed in lysing matrix D tubes (MP Biomedicals, USA), followed by the addition of 1 mL milliQ

water (experiment performed in triplicate). In order to get a homogeneous suspension, the tubes

were placed in a tissue homogenizer (Precellys 24 Homogenizer, Bertin Instruments, France) and

bead beaten for 3 cycles of 60 seconds at 6500 rpm, with 120 seconds break between cycles.

Samples were diluted 10x with milliQ water and analyzed for carbohydrates as described in

section 4.6.2.

4.2.4 Total protein content

To determine the total protein content, two different colorimetric procedures were used,

namely the Lowry’s method and the Bradford assay, each one requiring a particular sample

preparation (experiment performed in triplicate and quadruplicate, respectively). For the first

method, 1 mg of sB was resuspended in 1 mL of lysis buffer (60 mM Tris pH 9, 2% SDS – Table

4.2) in lysing matrix D tubes, whereas for the second assay, the biomass was resuspended in 0.1

M NaOH (Sigma Aldrich, Germany). Tubes were placed in the tissue homogenizer and bead

beaten for 3 cycles of 60 seconds at 6500 rpm, with 120 seconds break between cycles, followed

by 30 minutes of incubation at 100ºC in a block heater (SBH200D/3, Stuart, UK). Then, samples

were centrifuged for 10 minutes at 3500 rpm (Centrifuge 5424R, Eppendorf, Germany), and

analyzed for protein quantification as described in section 4.6.1.

Table 4.2 | Reagents and respective purity and supplier used in lysis buffer preparation.

Reagent Purity Supplier

Tris(hydroxymethyl)aminomethane (Tris) ≥ 99.9% Sigma Aldrich, Germany

Sodium dodecyl sulphate (SDS) ≥ 98.5% Sigma Aldrich, Germany

4.3 Conventional methods for protein extraction

After literature review for the most common methods applied in protein extraction from

macroalgae, three conventional procedures were replicated to be used as reference cases.

4.3.1 Aqueous and alkaline extraction

Aqueous and alkaline extraction (sequential) was performed according to Fleurence et al.

(Fleurence et al., 1995). 1 g of sB was resuspended in 20 mL of milliQ water, and gently stirred

overnight at 4ºC (experiment performed in triplicate). The suspension was centrifuged for 20

minutes at 10000xg. The supernatant was collected for further analysis (aqueous extract)

whereas the pellet was resuspended in 10 mL of NaOH 0.1 M. The resulting suspension was

gently stirred, at room temperature, for 1 hour in a tube shaker (Multi Reax, Heidolph, Germany)

and subsequently centrifuged at 10000xg for 20 minutes. The supernatant, corresponding to

alkaline extract, was collected for further analysis.

38

Both aqueous and alkaline extracts were analyzed for protein by Bradford assay, as

described in section 4.6.1.

The pH was measured in alkaline extract using a pH electrode (SI-600, Sentron,

Netherlands).

4.3.2 Extraction by high-shear force under alkaline conditions

High-shear disruption of macroalgal cells was performed based on Harnedy and FitzGerald

(Harnedy and FitzGerald, 2013) (experiment performed in duplicate). An amount of 2.5 g of sB

was resuspended in 50 mL of a pH 8 solution, and the biomass was processed using an Ultra-

turrax (Ultra-Turrax® T25, IKA®, Germany), at 6400 rpm, for 10 minutes, at room temperature.

The beaker was placed in an ice bath to avoid sudden temperature fluctuations, which were

controlled with a thermometer along the experiment.

After mechanical grinding, the suspension was centrifuged at 10000xg for 20 minutes and

the supernatant was collected for protein analysis. The concentration of protein in the resulting

extract as determined by Bradford assay as described in section 4.6.1.

4.3.3 Aqueous biphasic system PEG/Na2CO3

Aqueous two-phase system was created based on the binodal curves reported by Snyder

et al. (Snyder et al., 1992). The system was prepared at room temperature by mixing the required

amount of 9.90% (w/w) Polyethylene Glycol 1000 (PEG) (Sigma Aldrich, Germany), 10.90% (w/w)

Na2CO3 (≥ 99%, Sigma Aldrich, Germany), and milliQ water in microtubes (Eppendorf, Germany)

to obtain the final weight of 1.5 mg (experiment performed in duplicate). Afterwards, 45 mg of sB

was added to the tube, which was thoroughly stirred for 20 minutes in the tube shaker. Then it

was centrifuged at 4500xg for 20 minutes to speed up the phase formation. The volumes of each

phase were determined, and the content of both phases was analysed for protein and

carbohydrate concentration. A system composed by 9.90% (w/w) PEG 1000, 10.90% (w/w)

Na2CO3, and milliQ water was prepared as negative control, as well as a mixture composed by

45 mg of algal biomass and 1.5 mL of milliQ water as a positive control. The concentration of

protein in each phase was determined by Lowry’s method (section 4.6.1) requiring a 2x dilution

with lysis buffer (120 mM TRIS pH 9, 4% SDS). For carbohydrate quantification (section 4.6.2),

samples were diluted 20x with milliQ water. The negative control was used as blank in both

analytical methods.

The partition coefficient of protein in the formed system was determined by Equation 4.6

𝐾 =[𝑃]𝑇

[𝑃]𝐵

Equation 4.6

where [P]T and [P]B are the protein concentration in the top phase and bottom phase,

respectively.

39

4.4 Assays with ionic liquids

4.4.1 Ionic liquid selection

The screening of ionic liquids for protein extraction and fractionation comprised three

stages. Firstly, using the physicochemical properties of the ionic liquids to predict their ability to

interact with the macroalga cell wall, ten ionic liquids were selected among the twenty-five

available in Bioprocess Engineering (BPE) group from Wageningen University and Research.

Several reports have identified features of ionic liquids associated with cellulose dissolution. For

instance, the anion hydrogen bond basicity (AHBB) is one of the properties that most influence

the ability of certain ionic liquids to dissolve cellulose (i.e. the better the hydrogen bond acceptor

ability of the anion, the higher the solubility of cellulose) (Stark et al., 2012). In this perspective,

based on a BPE group’s database3 comprising different physicochemical properties of several

ionic liquids and respective cations and anions, the ten ILs selected were those with the highest

values of anion hydrogen bond basicity.

A second selection allowed to reduce the number of candidates from ten to five. This

selection was based on a qualitative analysis of ionic liquid-macroalga interaction, IL costs

(information from suppliers) and toxicity (information from literature). In order to evaluate

qualitatively the interaction of ionic liquids and macroalgae, solutions of 40% (w/w) IL were

prepared for each of the 10 ILs (Table 4.3) in 2 mL-microtubes and 10 mg of sB was added to all

of them. Positive controls, composed by sB and milliQ, and negative controls, composed by 40%

(w/w) IL and milliQ water, were also prepared and the samples were then vortexed for 2 minutes

– the experiment was performed in triplicate for each ionic liquid.

Under this experimental setting, each suspension had a particular behaviour, and by its

observation (e.g. phase formation; supernatant colour development), the interaction of the tested

ionic liquids and alga was evaluated.

Table 4.3 | Ionic liquids tested and respective purity and supplier.

Ionic Liquid Purity Supplier

Choline acetate ([Ch][Ac]] 98% Iolitec, Germany

1-butyl-3-methylimidazolium acetate ([Bmim][Ac]) 95% Iolitec, Germany

1-ethyl-3-methyl-imidazolium dibutyl phosphate ([Emim][dbp]) 97% Iolitec, Germany

1-butyl-3-methylimidazolium dibutyl phosphate ([Bmim][dbp]) 98% Iolitec, Germany

1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) 98% Iolitec, Germany

Choline chloride ([Ch][Cl]) 98% Sigma Aldrich, Germany

trihexyltetradecylphosphonium chloride ([6,6,6,14-P][Cl]) 97% Iolitec, Germany

tributyl(methyl)phosphonium methylsulfate ([1,4,4,4-P][MSO4]) 98% Sigma Aldrich, Germany

1-butyl-3-methylimidazolium dicyanamide ([Bmim][dca]) 95% Iolitec, Germany

trihexyltetradecylphosphonium dicyanamide ([6,6,6,14-P][dca]) 95% Iolitec, Germany

3 The databased was made by Edgar Suarez, member of BPE group of Wageningen University and Research, based on Cho et al. (Cho et al., 2012, 2011).

40

Finally, the third stage of the screening was carried out by quantitative analysis, and

allowed the selection the most suitable ionic liquid, among the five previously chosen, for the

following experiments. A mixture of 40% (w/w) IL and 10 mg of sB was prepared in lysing matrix

D tubes for each of the five ionic liquids (positive controls, composed by sB and milliQ water, and

negative controls, composed by 40% (w/w) IL and milliQ water, were also prepared) – the

experiment was performed in duplicate for each ILs and quadruplicate for positive controls.

Suspensions were placed in the tissue homogenizer and processed under a program composed

by three cycles of 60 seconds at 6500 rpm, with 120 seconds break between cycles. Samples

were then centrifuged for 10 minutes at 3500 rpm and analyzed for proteins by Bradford assay

(section 4.6.1), requiring a dilution of 5x with milliQ water. The ionic liquid that led to the highest

fraction of released proteins was selected to the following experiments.

4.4.2 Aqueous biphasic system: binodal curve

After the selection of the most suitable IL for the extraction of proteins, an aqueous biphasic

system composed by ionic liquid and salt was created for the extraction and separation of

proteins, requiring the construction of a binodal curve. Aqueous solutions of 50% (w/w) K2HPO4

(≥ 99%, Merck Millipore, Germany) and 60% (w/w) IL were prepared for the determination of the

corresponding binodal curve, which was established at room temperature and at atmospheric

pressure through the cloud point titration method (Kaul, 2000). Repetitive dropwise addition of the

aqueous salt solution to the ionic liquid aqueous solution (with known weight), was followed by

vortexing and time to settle. Upon settling, if a cloudy solution was formed (i.e. biphasic region),

the cloud point is considered reached and the concentration of phase-forming components was

dertermined based on the mass of aqueous salt solution added. The mixture was then diluted

with milliQ water until the formation of a clear solution (i.e. monophasic region), the weight of

milliQ water added was recorded and the above process was repeated until enough points were

measured for an accurate curve. All the measurements were taken as weight percentage.

The experimental binodal curve was fitted by least-squares regression according to

Equation 4.7 (Merchuk et al., 1998) (correlation shown in Appendix B).

[𝐼𝐿] = 𝑎𝑒𝑥𝑝[(𝑏 × [𝐾2𝐻𝑃𝑂4]0.5) − (𝑐 × [𝐾2𝐻𝑃𝑂4]3)]

Equation 4.7

4.4.3 Bead mill experimental setup

The DYNO-Mill Research Lab (Willy A. Bachofen AG Maschinenfabrik, Switzerland) was

used to conduct simultaneous disintegration, extraction and fractionation of compounds from

macroalgae. The DYNO-mill (Figure 4.1) consists of a horizontal milling chamber (Vchamber = 80

mL), with the engine connected over a central shaft. 65% (v/v) of the milling chamber volume was

filled with 1 mm ZrO2 beads (YTZ® beads, Tosoh, Japan) and a suspension composed of 1%

(w/w) sB, 11% (w/w) IL and 24% (w/w) K2HPO4 (Vf = 200 mL) was fed to the system. The beads

were accelerated in the opposite direction of the shaft (the average of agitator shaft speed was

2039 rpm) by a single DNYO-accelerator (Ø 56.20 mm). Different layers of beads were formed

41

moving at different speeds causing a grinding effect responsible for cell disintegration. The major

part of the energy introduced (energy input ~0.04 kWh) was converted to heat due to friction and,

therefore, a cooling jacket integrated in the milling chamber and a cooling coil in the feed funnel

were used to remove the heat, avoiding temperatures that could lead to protein denaturation (the

average temperature was ~24ºC). The algal suspension and grinding beads were separated by

a sieve plate at the outlet, and the algal suspension was fed back to the feed funnel. The bead

mill was operated under batch recirculation mode (i.e. the algal suspension was recirculated,

passaging multiple times through milling chamber) for 1 hour. In the end of the experiment, the

processed mixture was removed from the bead mill to 50 mL-centrifuge tubes (Sarstedt,

Germany). After time to settle and phase formation, the weight of each phase was noted down

and the top and bottom fractions were analysed for proteins and carbohydrates, requiring a 2x

dilution with milliQ water for the former and a 100x dilution for the latter. A negative control

composed by 11% (w/w) IL, 24% (w/w) K2HPO4 and water was used as blank in both analytical

methods.

Figure 4.1 | DYNO-mill equipment and respective components.

4.5 Recovery of ionic liquid

A bead mill experiment was run under the same conditions described in section 4.4.3,

however the feed suspension was composed by 15% (w/w) IL and 10% (w/w) sB. The resulting

mixture was centrifuged, at 4700 rpm, for 20 minutes (Alegra X-30R, Beckman Coulter, USA) and

aliquots of supernatant were analysed for protein (sample previously diluted 3x with milliQ water).

An ultrafiltration process was carried out in order to recover the ionic liquid from the algal

extract (previously diluted 2x with milliQ water). Two different setups were tested: a stirred

ultrafiltration cell (model 8050, EMD Millipore AmiconTM Bioseparations, USA) – Figure 4.2 – and

a centrifugal filter unit (Amicon Ultra-4 centrifugal filter with Ultracel-3 membrane, Millipore, USA)

– both setups were performed in duplicate.

In the first setup, a Biomax polyethersulfone membrane (EMD Millipore AmiconTM

Bioseparations, USA) with a pore size of 10 kDa was used. The stirred cell was filled with 10 mL

42

of supernatant, and the stir bar/pressure cap assembly was fitted onto the cell. The stirred cell

was placed on a magnetic stirrer plate with the stirring speed set to 600 rpm, preventing the build-

up of a biomass layer at the membrane surface. The pressure inside the chamber was maintained

at 32.50 psi by nitrogen gas, which provided the driving force for the filtration. The separation

process was carried out at a constant flow rate (0.10 mL.min-1) until the retentate volume is

reduced to approximately 2 mL.

Figure 4.2 | Schematic diagram of stirred cell ultrafiltration assembly used for recovering the ionic liquid from

the supernatant. Adapted from (Merck Millipore, 2015).

In the second setup, a centrifugal filter unit equipped with an ultracel-3 membrane (pore

size of 3 kDa) was filled with 4 mL of supernatant and spun, at 4000xg, for 10 minutes (Allegra

X-30R Centrifuge, Beckman Coulter, USA). The centrifugation step was repeated 4 times until

the filtrate volume reached 3 mL.

The concentration of ionic liquid, protein and carbohydrates in retentates and filtrates were

determined. Proteins were quantified by Bradford assay (section 4.6.1), whereas carbohydrates

and ionic liquid were quantified according to section 4.6.2 and section 4.6.3, respectively. The

required dilutions for each assay are described in Table 4.4.

Table 4.4 | Filtrate and retentate dilutions required for protein, carbohydrate and ionic liquid quantification.

Protein quantification Carbohydrate quantification IL quantification

Retentates 3x 500x 50x

Filtrates Without dilution 50x 50x

The rejection coefficients of proteins and carbohydrates in both membranes were determined

according to Equation 4.8, in which Cp and Cf stand for the concentration of protein/carbohydrate

in the permeate and in the feed, respectively.

𝑅 = 1 −𝐶𝑝

𝐶𝑓

Equation 4.8

43

4.6 Analytical methods

4.6.1 Protein quantification

Lowry’s method or Bradford assay were used for protein quantification depending on the

assay. For both methods, a calibration curve was prepared using bovine serum albumin (BSA)

(Sigma Aldrich, Germany) as standard, for a working range of 0–1.40 mg.mL-1 for Lowry’s method

and 0–2 mg.mL-1 for Bradford assay.

Lowry’s method was performed, with a commercial kit (DCTM Protein Assay, Bio-Rad,

USA), following the microplate assay protocol. 10 µL of each standard and unknown samples

were transferred into a 96-well plate (Greiner Bio-One International, Austria), and after the

addition of Lowry’s reagents, as described in the protocol, the microplate was covered with

aluminium foil and incubated, at room temperature, for 30 minutes. The absorbance was then

measured at 750 nm using a microplate reader (Infinite M200, Tecan, Switzerland).

The Bradford assay was also performed following the microplate assay protocol of a

commercial kit (PierceTM Coomassie protein assay, Thermo Fisher Scientific, USA). 5 µL of

standards and unknown samples were added to a 96-well microplate and after the addition of 250

µL of Coomassie reagent, the microplate was shaken for 30 seconds and incubated 10 minutes

at room temperature. The absorbance was measured at 595 nm in the microplate reader.

According to the experiment, the concentration of protein was determined by direct

interpolation from the calibration curves described in Appendix A.1. The total protein content in

macroalgae and the fraction of released proteins in each extraction procedure were determined

by Equation 4.9 and Equation 4.10, respectively.

% 𝑇𝑜𝑡𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =[𝑃𝑟𝑜𝑡𝑒𝑖𝑛](𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑜𝑙𝑢𝑚𝑒𝑠𝑎𝑚𝑝𝑙𝑒(𝑚𝐿)

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒(𝑚𝑔)× 100

Equation 4.9

% 𝑅𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑒𝑥𝑡𝑟𝑎𝑐𝑡(𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑜𝑙𝑢𝑚𝑒𝑒𝑥𝑡𝑟𝑎𝑐𝑡(𝑚𝐿)

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒(𝑚𝑔) ×%𝑇𝑜𝑡𝑎𝑙 𝑃𝑟𝑜𝑡𝑒𝑖𝑛

100

× 100

Equation 4.10

4.6.2 Carbohydrate quantification

The quantification of carbohydrates was carried out according to Dubois et al. (Dubois et

al., 1956). Calibration curves were prepared using glucose (Sigma Aldrich, Germany) as

standard, for a working range of 0-0.10 mg.mL-1. 40 µL of standard and unknown samples were

transferred to a 96-well microplate, followed by the addition of 40 µL of 5% (w/w) phenol solution

(molecular biology grade, Sigma Aldrich, Germany), and 200 µL of concentrated H2SO4 (95-98%,

Sigma Aldrich, Germany). The microplate was covered by aluminum foil and incubated, at room

temperature, for 10 minutes, followed by an incubation step for 35 minutes, at 35ºC. The

absorbance was measured at 483 nm in the plate reader.

The concentration of carbohydrates was determined, according to the experiment, by direct

interpolation from the calibration curves described in Appendix A.2. The percentage of total

44

carbohydrate content in macroalgae and the fraction of released carbohydrates in each extraction

procedure were determined by Equation 4.11 and Equation 4.12, respectively.

% 𝑇𝑜𝑡𝑎𝑙 𝑐𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠 =[𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠](𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑜𝑙𝑢𝑚𝑒𝑠𝑎𝑚𝑝𝑙𝑒

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒(𝑚𝑔)× 100

Equation 4.11

% 𝑅𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝑐𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠 =[𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠]𝑒𝑥𝑡𝑟𝑎𝑐𝑡(𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑜𝑙𝑢𝑚𝑒𝑒𝑥𝑡𝑟𝑎𝑐𝑡(𝑚𝐿)

𝐷𝑊𝑠𝑎𝑚𝑝𝑙𝑒(𝑚𝑔) ×%𝑇𝑜𝑡𝑎𝑙 𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠

100

× 100

Equation 4.12

4.6.3 Ionic liquid quantification

The concentration of ionic liquid in the fractions resulting from the ultrafiltration experiment

was estimated using an Ultra High-Performance Liquid Chromatography (UPLC) apparatus

(Nexera X2, Shimadzu, USA) equipped with a RezexTM ROA-Organic Acid H+ 8% (300mm x

7.8mm) column (Phenomenex, USA), coupled with a security guard (Phenomenex, USA), a pump

(LC-30AD, Shimadzu, USA), an autosampler (SIL-30AC, Shimadzu, USA) and a refractive index

detector (RID-20A, Shimadzu, USA). The injection volume was 20 µL and the isocratic elution

was performed with 0.005 N H2SO4 solution, as mobile phase. The column was kept at 60ºC,

under a pressure of 55 bar, and the pump operated at a flow rate of 0.60 mL.min-1.

The concentration of ionic liquid was determined by direct interpolation of the calibration

curve described in Appendix A.3.

The ionic liquid recovery was calculated according to Equation 4.13.

𝐼𝐿𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦(%) =[𝐼𝐿]𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑒(𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑒(𝑚𝐿)

[𝐼𝐿]𝑓𝑒𝑒𝑑(𝑚𝑔. 𝑚𝐿−1) × 𝑉𝑓𝑒𝑒𝑑(𝑚𝐿)× 100

Equation 4.13

4.6.4 Native-PAGE gel electrophoresis

Native-PAGE electrophoresis was performed to study how the conditions used in aqueous

and alkaline extraction and mechanical grinding extraction affect the protein conformation.

Aliquots from extracts were diluted with milliQ water in order to obtain solutions with the same

concentration of protein. Afterwards, those samples were diluted with native sample buffer

(BioRad, USA) in a proportion of 1:2. After mixing, 25 µL of each sample were transferred into a

4-20% Criterion Tris-Glycine eXtended (TGX) precast gel (BioRad, USA), as well as 5 µL of

NativeMark™ Unstained Protein Ladder (Invitrogen, EUA). Samples were run in 10x Tris/Glycine

buffer (BioRad, USA), at 125 V, for 75 minutes.

Native gel was stained using the Pierce® silver stain kit (Thermo Fisher Scientific, USA),

following the respective user guide. The image of native gel were acquired with LabScan 6.0

software, using the ImageScanner III (GE Healthcare, UK).

45

4.7 Statistical Analysis

R-studio was used to carry out the statistical analysis of results. Replicates were performed

for almost all experiments and data are presented as mean values ±SD (standard deviation).

Experimental data were analysed by one-way analysis of variance (ANOVA) with the significance

level of p=0.05. When significant differences were found (i.e. p<0.05), the Tukey’s Honest

Significant Difference (HSD) test was used to detect significant differences among treatments.

47

5 5. Results and Discussion

49

5.1 Chemical composition of Ulva lactuca

The freeze-dried Ulva lactuca was analysed and its composition determined. The average

composition is shown in Table 5.1.

Table 5.1 | Chemical characteristics of macroalga U. lactuca (% w/w on dry basis).

Components Values

Total Solids (%) 96.50±0.87

Ash 22.80±0.35% dry wt.

Protein 17.81±0.79% dry wt. (Lowry’s method)

3.70±0.30% dry wt. (Bradford assay)

Lipid 4.95±0.35% dry wt.

Carbohydrate 60.92±14.62% dry wt.

The moisture content (3.50±0.87% dry wt.) is very low because the starting algal biomass

has been lyophilized previously. Lyophilization (freeze-drying) is a gentle dehydration process

that allows the removal of cellular water, leading to a residual moisture contents in the biomass

(Dejoye et al., 2011; Wessman et al., 2013).

The ash content (22.80±0.35% dry wt.) is comparable with values published in previous

studies. For instance, Wong and Cheung measured 21.30±2.78% dry wt. of ash in U. lactuca,

whereas Yaich et al. determined 19.59±0.51% dry wt. of ash in the same species (Wong and

Cheung, 2000; Yaich et al., 2015).

The lipid content (4.95±0.35% dry wt.) is found to be significantly higher than that usually

found in U. lactuca, which varies between 0.30% dry wt. and 1.79% dry wt.(Ortiz et al., 2006;

Tabarsa et al., 2012; Wong and Cheung, 2000). This difference could be attributed to factors such

as the conditions for seaweed growth such as climate and geography, as well as the timing for

harvest or the method used for lipid extraction (Yaich et al., 2015).

The carbohydrate content determined in this study (60.92±14.62% dry wt.) agrees with

values reported in literature for U. lactuca (De Pádua et al., 2004; Ortiz et al., 2006). However,

although the value is within expectations, it is important to note the large standard deviation

obtained, which indicates some variability in the value of carbohydrate content determined

between the different biological replicates. In order to validate this analysis, the experiment should

be performed again.

The total protein content in U. lactuca varies considerably between published studies

(Table 5.2). In addition to season’s related variability, the method applied for protein quantification

strongly affects the reliability of the determined value (Cerna, 2011).

50

Table 5.2 | Protein content in Ulva lactuca.

Protein content in U. lactuca

% (w/w) dry wt. Reference

8.46±0.01 (Yaich et al., 2015)

10.69±0.67 (Tabarsa et al., 2012)

7.06±0.06 (Wong and Cheung, 2000)

27.02±1.10 (Ortiz et al., 2006)

18.35 (De Pádua et al., 2004)

18.10 (Shuuluka et al., 2013)

17.88±0.10 (Khairy and El-Shafay, 2013)

The determination of total protein content in this study was carried out by two different

colorimetric methods: the Lowry’s method and the Bradford assay. The total protein content

determined by Lowry’s method is substantially higher than that determined by Bradford assay –

17.81±0.79% dry wt. and 3.70±0.30% dry wt., respectively. Several authors have reported that

Bradford assay generates lower protein values for a large number of organisms compared to

Lowry’s method (Berges et al., 1993; Clayton Jr. et al., 1988; Eze and Dumbroff, 1982). The

comparison between both methods was already performed in other macroalgal species and the

results confirmed the general trends found in this study, with a Lowry:Bradford ratio varying from

1.50 to 3.20 (Barbarino and Lourenço, 2005). Actually, in this study, the Lowry:Bradford ratio is

found to be slightly higher (4.80), which might be due to the procedure followed for sample

preparation. In the Lowry’s method, the biomass was resuspended in lysis buffer containing SDS,

an anionic surfactant/detergent that assists in the dissolution of membranes by disrupting the

hydrophobic-hydrophilic interactions among their molecules (Brown and Audet, 2008).

Nevertheless, the interference of ionic detergents with Coomassie reagent, prevents the use of

SDS in samples prepared for Bradford assay (Marshall and Williams, 2004). In order to ensure

that all the proteins present in macroalgae were quantified, as it is assumed to happen with the

Lowry’s quantification, the biomass was suspended in 0.1 M NaOH, assuming that such alkaline

environment was sufficiently harsh for algal cells as SDS. However, it might not be and cell

membranes were not completely disintegrated, avoiding the release of all the proteins present in

seaweeds. In consequence, the value of total protein measured by Bradford may be even more

underestimated than expected, explaining the higher Lowry:Bradford ratio.

The difference between the alga protein content measured by Bradford assay and Lowry’s

method could be also explained by the interaction of respective reagents with proteins. The

development of colour in Bradford protein assay is associated with the binding of Coomassie dye

to basic and aromatic amino acids (Compton and Jones, 1985). Most of seaweeds show relatively

low concentrations of basic amino acids, such as lysine and histidine, as well as aromatic amino

acids, tyrosine and tryptophan (Bleakley and Hayes, 2017). Therefore, the binding with proteins

occurs mainly with arginine and phenylalanine residues, a fact that may contribute to an

underestimation of protein content. On the other hand, the Folin-Ciocalteu reagent used in the

Lowry’s method interacts with all peptide bonds and the colour development can still be enhanced

51

by the presence of certain amino acids (e.g. tyrosine, tryptophan, cysteine and asparagine)

(Legler et al., 1985). Therefore, the quantification of protein tends to be greater.

According to Barbarino and Lourenço, the Lowry’s method is the most reliable way to

determine the protein content in algae since they found out that results generated with Lowry’s

method were more similar to the data obtained from the sum of the amino acid residues

(Barbarino and Lourenço, 2005). In this sense, it is assumed that the total protein content in Ulva

lactuca is 17.81±0.79% dry wt., the value determined by Lowry’s method. The total protein content

is comparable with certain values reported in literature (Table 5.2). In particular, with the value

determined by Khairy and El-Shafay (Khairy and El-Shafay, 2013), which results from the analysis

of U. lactuca collected during the summer, as the samples used in this study.

The Bradford analysis is nevertheless needed for the determination of the protein recovery

yield when using ionic liquids. This is because of the interference between Lowry’s reagents and

ionic liquids, which prevents the use of this method in IL-based experiments. Since the fraction of

protein released by each extraction procedure is calculated using the total protein content in

macroalga (Equation 4.10), for accurate and reliable results, the quantification of protein in the

extracts should be performed with the same method used for total protein quantification.

The composition of the algal batch under study was also evaluated by thermogravimetric

analysis4 and the results are presented in Table 5.3. The protein content is similar to the one

obtained by the Lowry’s method. The amount of carbohydrates determined by thermogravimetric

analysis is slightly lower than the one determined by the Dubois method. However, besides the

inherent differences between the principles of each method and the absence of thermogravimetric

analysis replicates to validate the results, one must consider that Dubois’ quantification has a

large SD associated, which leads to concerns about the reliability of results. The lipid and ash

contents also present some variation. Replicates of thermogravimetric analysis are recommended

to confirm the results obtained.

Table 5.3 | Ulva lactuca composition determined by thermogravimetric analysis.

Component % (w/w) dry wt.

Ash 19.36

Protein 16.00

Lipid 7.60

Carbohydrate 53.80

To conclude, it is important to note that, with the percentages obtained for ashes, proteins,

carbohydrates and lipids, it is possible to close the mass balance of U. lactuca composition,

suggesting the reliability and accuracy of the experiments performed for biomass

characterization.

4 Experiment carried out at Instituto Superior Técnico, Department of Chemical Engineering, by Dr. Ana Paula Vieira Soares Pereira Dias (Ferreira et al., 2016).

52

5.2 Reference Cases

As mentioned in Materials and Methods (section 4.3), three conventional methods used for

macroalga protein extraction were replicated in this study. Extractions by means of aqueous and

alkaline solutions (sequential), high-shear force and aqueous biphasic system (PEG/Na2CO3)

were used as reference cases because, among the protein extraction techniques reported in

literature, these methods have led to higher yields of protein recovery in Ulva species (Table 2.1).

Aqueous and alkaline extraction (sequential) showed better performance compared to

high-shear force and ATPS extractions (Figure 5.1).

Figure 5.1 | Percentage of protein recovery by high-shear force, aqueous and alkaline extraction (sequential)

and aqueous biphasic system PEG/Na2CO3 (sum of protein extracted into top and bottom phases). Protein

yield expressed as % of total protein (Protein extracted/Total proteins x 100). Results are the means of

triplicate determination for aqueous and alkaline extraction and duplicate determinations for high-shear force

extraction and aqueous biphasic system ±SD; (*) - means are statistically equal (p>0.05).

The overall fraction of protein extracted by aqueous and alkaline extraction (sequential)

(49.08±6.66%) seems to be significantly higher than that obtained by the same method for Ulva

rigida (26.80±1.3%) and Ulva rotundata (36.10±1.4%) (Fleurence et al., 1995). Although these

authors used the same procedure to extract the protein fraction from both Ulva species, the

recovery yields varied considerably between species. One can thus conclude that the protein

recovery yield attained using this procedure is a function of the green alga species.

Separately, osmotic shock (aqueous extraction) led to a protein recovery yield of

5.04±0.95%, whereas the alkaline extraction led to 45.42±8.81%, suggesting that the majority of

macroalgal proteins are insoluble in water. At high pH (~12), most of the proteins are negatively

charged due to the deprotonation of amine groups, which enhances the protein-solvent

interaction, thereby increasing the protein solubility (Valenzuela et al., 2013). In addition, since

many water-insoluble polysaccharides are solubilized under basic pH (Hostettmann, 2014; Knill

and Kennedy, 2003), the alkaline conditions may also have aided in cell wall disruption, leading

to a higher protein recovery yield (Zhang et al., 2015).

0%

10%

20%

30%

40%

50%

60%

High-shear forceextraction

Aqueous biphasicsystem

Aqueous & alkalineextraction

Pro

tein

Recovery

*

*

*

*

53

Concerning the extraction by high-shear force, Barbarino and Lourenço extracted protein

from Ulva fasciata with a recovery yield of 7.30±0.84 g protein/100 g dry wt. (Barbarino and

Lourenço, 2005). On the other hand, the same method was used to extract proteins from the red

seaweed Palmaria Palmata, obtaining a recovery yield of 6.92±0.12 g protein/100 g dry wt.

(Harnedy and FitzGerald, 2013). In this study, only 0.25±0.04 g of protein was extracted per 100

g alga dry wt. (6.68±1.09%), which is an extremely low yield compared to the literature. The

procedure used in this study was adapted from both published works (Barbarino and Lourenço,

2005; Harnedy and FitzGerald, 2013), in which the mechanical grinding was followed by

extraction under strong alkaline conditions (pH~12), known to lead to higher recovery yields. In

order to avoid the expected protein denaturation due to the extremely basic pH (Marambe and

Wanasundara, 2017), the procedure followed in this study did not include the strong alkaline

conditions. Alternatively, the algae were previously resuspended in a pH~8 solution, providing

mild alkaline conditions, which could improve the protein solubility without compromising their

functionality, and subsequently processed by mechanical grinding, saving time and costs due to

a single-step process. However, the modified procedure fell short of expectations, displaying

much lower yields, which suggests that the pH was not high enough to solubilize the proteins.

It should also be noted that the high-shear homogenization is supposed to damage the

alga cells inducing the protein release (e.g. by reducing the particle size, enhancing mass transfer

between solvent and algae and/or fractionate large agglomerates), however when comparing the

recovery yield obtained by mechanical grinding (6.68±1.09%) and aqueous extraction

(5.04±0.95%), there is not a significant difference between both procedures (p=0.17). This fact

suggests that only the effect of the solvent was playing a role while Ultra-Turrax homogenizer

worked exclusively as a mixing device. A reasonable explanation for this unexpected result is the

coffee grinder use in alga sample preparation, which may have reduced the algae to the minimum

size, making the Ultra-Turrax action useless.

The overall fraction of protein extracted by ATPS was 10.52±0.35% (sum of protein

quantified in both formed phases (9.11±0.32% in the top phase and 1.42±0.03% in the bottom

phase). The same procedure was used by Fleurence et al. to extract proteins from Ulva rigida

and Ulva rotundata, achieving recovery yields of 19.10±1.1% and 31.56±2.1%, respectively

(Fleurence et al., 1995). In Figure 5.2, it is possible to observe that, in addition to the top and

bottom phases formed, there was also the formation of an interphase in sample tubes. The

interphase was solid and, although it was not analysed for proteins, these molecules may be part

of its composition. In this sense, besides the influence of the species on the protein recovery yield,

the overall fraction of protein extracted may be underestimated because the contribution of

interphase proteins was not considered.

54

Figure 5.2 | Triplicates of positive controls (algae and milliQ water), negative controls (milliQ water, PEG1000

and Na2CO3) and ATPS formed by milliQ water + PEG1000 + Na2CO3 + algae.

In addition to extract proteins from algae, the ATPS can still separate them from other

components, for instance, carbohydrates. The phase separation results from the salting-out of

the hydrophilic polymer PEG and leads to two phases of different hydrophilic character. Protein

partitioning is known to be highly selective in PEG/salt phase systems (i.e. the partition

coefficients are high (K>>1)) (Iqbal et al., 2016). Generally, the proteins concentrate in the PEG-

rich phase, with a more hydrophobic character, while most of the polysaccharides remains in the

salt-rich phase (Jordan and Vilter, 1991). This trend was confirmed in this study, as displayed in

Figure 5.3, which describes the distribution of the extracted proteins and carbohydrates between

the top and bottom phases. Most of the proteins were concentrated in the top phase: i.e. the PEG-

rich phase (K=11.25), whereas the carbohydrates had higher affinity to the bottom phase, the

salt-rich phase.

Figure 5.3 | Distribution of extracted proteins and carbohydrates between the top and bottom phases of the

ATPS composed by PEG1000 + water + Na2CO3.

The proteins were quantified in the supernatant of positive controls. The protein recovery

yield of 9.98±0.83% means that the presence of PEG or salt in the solution did not significantly

increase the percentage of protein released, since the overall recovery yield in the ATPS was

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Proteins Carbohydrates

Bottom phase Top phase

55

10.52±0.35% (p=0.46). In this perspective, the importance of the PEG/Na2CO3 ATPS lies mainly

in its ability to separate the proteins from carbohydrates rather than in its use for protein extraction.

To evaluate how the conditions used in high-shear force extraction and aqueous and

alkaline extraction affect the protein conformation, a Native-PAGE electrophoresis was

performed, and the results are presented in Figure 5.4.

Figure 5.4 | Electrophoresis gel of protein extracts under non-denaturing conditions. Lane 1: Protein ladder;

Lane 2: Aqueous extract (positive control); Lane 3: Alkaline extract; Lane 4: Mechanical grinding extract.

In the native-PAGE electrophoresis, the proteins are separated according to the net

charge, size, and shape of their native structure. The electrophoretic migration occurs because

most proteins carry a net negative charge in alkaline running buffers (the higher the negative

charge density, the faster a protein will migrate). At the same time, the gel matrix acts as a

molecular sieve, regulating the movement of proteins according to their size and three-

dimensional shape (small proteins face only a small frictional force, migrating more, while higher

proteins face a large frictional force, migrating less) (Nelson and Cox, 2008).

Considering the protein pattern of aqueous extract (lane 2) as a positive control (performed

under mild conditions in order to ensure protein conformation), it is possible to draw conclusions

about how the extraction under strong alkaline conditions (pH~12) and the mechanical grinding

under mild alkaline conditions (pH~8) can affect the algal proteins conformation. The protein

pattern of alkaline extract (lane 3) is clearly different from the positive control, suggesting that

proteins lose their native structure during the extraction. Although the alkaline extraction leads to

the highest recovery yields (~45%), there are strong evidences that the functionality of proteins

may be compromised, making this method unfeasible for applications in which the native structure

of proteins is needed.

On the other hand, the mild alkaline conditions used in mechanical grinding did not

compromise the native structure of the proteins since the protein pattern (lane 4) is similar to

positive control. However, this method leads to low yields of protein recovery (~7%) as previously

discussed.

56

5.3 Experiments with ionic liquids

5.3.1 Ionic liquid selection

The selection of the most suitable ionic liquid, among those available to test, comprised

three steps. Table 5.4 shows the output of the first selection, in which the 10 ILs selected and the

respective anion hydrogen bond basicity are enumerated.

Table 5.4 | Ionic liquids selected and respective anion hydrogen bond basicity.

Ionic Liquid Anion hydrogen bond basicity

[Ch][Ac] 4.84

[Bmim][Ac] 4.84

[Emim][dbp] 4.36

[Bmim][dbp] 4.36

[Bmim][Cl] 4.25

[Ch][Cl] 4.25

[6,6,6,14-P][Cl] 4.25

[1,4,4,4-P][MSO4] 2.81

[Bmim][dca] 1.84

[6,6,6,14-P][dca] 1.84

The second selection included the qualitative analysis of the IL-alga interaction, the cost

and toxicity of each ionic liquid. As described in Materials and Methods (section 4.4.1), solutions

of 40% of ionic liquid were prepared for the experiments because the use of pure ILs is only viable

when they are liquid at room temperature and have low viscosities. Otherwise, the extraction

efficiencies will be low due to the limited mass transfer coefficients and the poor penetration of

ILs into the biomass structure (Passos et al., 2014). Although high temperatures could be applied

when working with pure ILs, either to overcome their melting points or to reduce their viscosities,

they might lead to the thermal degradation of target molecules as well as represent further

economic concerns. Therefore, to overcome these issues, the ionic liquids were diluted in water,

reducing their viscosity and allowing the extraction process to occur at room temperature. Besides

the lower energy consumption, there is also a reduction on the overall solvent cost.

The suspensions obtained after mixing IL + water + algae are shown in Figure 5.5. After

visual inspection it was possible to infer about the IL-alga interaction. Certain pigments located

inside the cells of Ulva lactuca are responsible for its green colour. Therefore, the development

of such colour in the supernatants suggests the release of those pigments, implying the

destabilization of the cell wall. In this sense, it is possible to conclude that the greener the

supernatant, the larger the pigment release, and the greater the interaction of the IL with the cell

structures. Reminding the first selection in which the ability of the IL anion to act as hydrogen

bond acceptor was used to predict the IL-macroalga interaction, it is expected that ionic liquids

with higher AHBB (Table 5.4) lead to a greener supernatant.

57

Figure 5.5 | Suspensions obtained for each ionic liquid after mixing IL + water + algae. A positive control (+

Ctrl) – algae and water – is placed next to all the samples.

[Choline][Ac] and [Bmim][Ac] are the ionic liquids with the highest AHBB (Table 5.4),

however their supernatants were almost uncoloured. On the other hand, [1,4,4,4-P][MSO4] had

one of the greenest supernatants and has a low AHBB. These evidences contradict what was

expected and, although ILs with higher AHBB are the most effective in cellulose dissolution, it is

not possible to predict their interaction with macroalgae based only on this parameter. However,

there are some relations that can be made based on the data reported in literature and the results

obtained in this study. For instance, it is recognized that the choice of the IL cation also affects

the cellulose solubility and, although data are not always consistent, it is generally accepted that

the length of the cation’s alkyl chains progressively reduces the dissolution of cellulose (Pinker et

al., 2009). The [Emim][dbp] supernatant is greener than the [Bmim][dbp] supernatant, suggesting

a greater interaction of macroalgae and [Emim][dbp]. This behaviour can be easily explained by

the shorter alkyl chain of [Emim] cation in comparison to [Bmim], increasing the ability of the IL to

solubilize cellulose.

Comparing two ILs with the same cation and different anions, for instance [Bmim][dbp] and

[Bmim][Cl], it is evident how the AHBB affects the interaction of both ILs with macroalgae.

Although the slight difference between the AHBB of [Bmim][dbp] and [Bmim][Cl] (4.36 and 4.25,

respectively), it is sufficient for the [Bmim][dbp] supernatant to be much greener than [Bmim][Cl]

supernatant.

58

There was phase formation in the mixtures composed by [6,6,6,14-P][Cl] and [6,6,6,14-

P][dca], indicating the highly hydrophobic character of both ionic liquids. Their immiscibility in

water coupled with the high viscosity arising from the long alkyl chain (Zhang et al., 2017), makes

difficult the use of both ionic liquids in macroalga cell disintegration, which may only be achieved

under intensive mixing, for instance, using Ultra-Turrax. Considering an IL-based ATPS, only ILs

miscible in water can be used for its formation because, when dealing with highly hydrophobic

ILs, two phases already exist before the addition of any salting-out agent, and one of the phases

is far from being aqueous due to these ILs’ low solubility in water and vice-versa (Freire et al.,

2007). Moreover, experimental studies have clearly documented the correlation between

increased hydrophobicity and increased cytotoxicity (Ranke et al., 2007). Hydrophobic ionic

liquids have a greater ability to interact with hydrophobic patches on proteins and cell membranes

in living cells, which facilitates the uptake of ILs into organisms (Matzke et al., 2010), and a

consequent inhibition of the enzyme activity (Jing et al., 2016). All the mentioned issues

contributed to the exclusion of both ILs from the list of suitable candidates.

[Bmim][dca] and [Choline][Ac] were excluded due to their high cost (Table 5.5) and lack of

colour development in the supernatants. On the other hand, and although its supernatant was

one of the greenest, [1,4,4,4-P][MSO4] was also excluded because it is recognized that

phosphonium-based ionic liquids are generally more toxic than imidazolium-based ILs (Ventura

et al., 2012).

Table 5.5 | Ionic liquids and estimated costs.

Ionic Liquid Cost (€.g-1) Reference

[Ch][Cl] 0.06 (Sigma Aldrich, 2017a)

[Bmim][Ac] 0.78 (Sigma Aldrich, 2017b)

[Bmim][Cl] 1.31 (Sigma Aldrich, 2017c)

[6,6,6,14-P][Cl] 1.63 (Sigma Aldrich, 2017d)

[1,4,4,4-P][MSO4] 2.15 (Gute chemie abcr, 2017)

[6,6,6,14-P][dca] 3.22 (Sigma Aldrich, 2017e)

[Ch][Ac] 7.04 (Sigma Aldrich, 2017f)

[Emim][dbp] 8.72 (Sigma Aldrich, 2017g)

[Bmim][dbp] 9.20 (Santa Cruz Biotechnology, 2017)

[Bmim][dca] 16.22 (Sigma Aldrich, 2017h)

Therefore, the 5 ILs selected for the following analysis were [Ch][Cl], [Emim][dbp],

[Bmim][dbp], [Bmim][Ac] and [Bmim][Cl]. There was no colour formation in the [Ch][Cl]

supernatant, however it is the cheapest IL and choline-based ILs are considered biodegradable

and non-toxic (Gadilohar and Shankarling, 2017), reasons why it was considered for the following

analysis. The supernatants of [Emim][dcp] and [Bmim][dcp] were the greenest ones, hence they

were considered promising candidates. In turn, the supernatants of [Bmim][Ac] and [Bmim][Cl]

were colourless, but both ILs have already been applied in food and bioproduct industries (Toledo

Hijo et al., 2016). This is an indication of the applicability of these ILs in the food sector, the same

area of application of the proteins that will be extracted from macroalgae to be used as functional

ingredients in food.

59

Table 5.6 | Output of the third selection. Features of the five ionic liquids selected.

[Emim][dbp] [Bmim][Ac] [Bmim][Cl] [Bmim][dbp] [Ch][Cl]

Hydrophilic

Lowest Cost

Non-toxic

Biodegradable

Applied in food industry

Greenest supernatants

The ability of the selected ILs to extract macroalgal proteins was then evaluated. According

to Figure 5.6, [Emim][dbp] appears to be the most suitable candidate, extracting 80.62±4.95% of

the total proteins present in Ulva lactuca.

Figure 5.6 | Percentage of protein recovery by each of the 5 ILs tested (+ control – algae and water). Protein

yield expressed as % of total protein (Protein extracted/Total proteins x 100). Results are the means of

duplicates for each IL and quadruplicates for positive control ±SD. a, b, c – means in columns with the same

letter are significantly equal (p>0.05).

No significant difference is observed in terms of protein recovery yield between [Bmim][Cl],

[Bmim][dbp] and [Emim][dbp]. However, the cost of [Bmim][dbp] is higher than [Emim][dbp] (Table

5.5) and the larger alkyl chains of both [Bmim][Cl] and [Bmim][dbp] increase their toxicity, facts

that strengthen the potential of [Emim][dbp] as the most suitable ionic liquid.

In turn, [Ch][Cl] and [Bmim][Ac] extract almost the same amount of protein as the positive

control – no significant differences were observed between the protein recovery yields of [Ch][Cl]

vs positive control (p=0.99) and [Bmim][Ac] vs positive control (p=0.87) – indicating the

ineffectiveness of these ILs for U. lactuca protein extraction.

Concerning the reference cases, the extraction efficiency obtained by [Emim][dbp] is

significantly higher than that obtained by any of the conventional methods tested. Moreover,

although the effect of the ionic liquid in protein conformation has not been evaluated in this

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

(+) Control [Ch][Cl] [Bmim][Ac] [Bmim][Cl] [Bmim][dbp] [Emim][dbp]

Pro

tein

Recovery

c

c

a a b

a b

b c

60

experimental work, published studies have shown that certain ILs improve the stability and half-

lives of various proteins and enzymes and allow some enzymes to display superactivity (Ventura

et al., 2017), which support the applicability of ionic liquids for the purpose under study.

A recent economic analysis assessment demonstrates that the application of ILs for the

extraction of compounds from biomass is only viable when the concentration of extracted

compounds is considerably higher (<5% dry wt.) or when they are truly value-added compounds

(Passos et al., 2014). In the case under study, the use of ILs seems to outweigh the requirements,

since, in addition to the concentration of extracted components ranges from 3 to 14% dry wt.,

such components are proteins with a high potential for downstream applications.

5.3.2 Extraction and fractioning of protein by IL based-ATPS

The economic and environmental feasibility of the use of ionic liquids to extract proteins as

a single product from seaweeds needs to be assessed. Cascade biorefinery approaches aiming

at the maximization of the inherent value of all the components present in the biomass would

certainly improve the overall economics of the biorefinery. An integrated biorefinery process

where the extracted proteins are the high-value components but also the carbohydrate fraction is

used, for instance, for the production of biofuels, chemicals or materials, might meet the

expectations for using seaweed as a viable feedstock for biorefineries. In this perspective, an

aqueous biphasic system composed by [Emim][dbp], K2HPO4 and water was studied for the

protein-carbohydrate separation in macroalga extracts. K2HPO4 was the inorganic salt selected

as salting-out agent because it is the most used in IL based-ABSs for protein extraction (Desai et

al., 2016).

The binodal curve obtained for the ATPS composed by [Emim][dbp] and K2HPO4 is shown

in Figure 5.7. Although different ATPS compositions could be selected, a low concentration of

ionic liquid (11% (w/w) [Emim][dbp]) and a high concentration of salt (24% (w/w) K2HPO4) were

used for the ATPS formation in the bead-mil experiment because the stock of [Emim][dbp]

available was lower than that of K2HPO4.

Figure 5.7 | Binodal curve of [Emim][dbp] and K2HPO4 at 298.15 K.

0

10

20

30

40

50

60

0 5 10 15 20 25 30

[Em

im] [d

bp]

% (

w/w

)

K2HPO4 % (w/w)

61

As described in Materials and Methods (section 4.4.3), the [Emim][dbp] based-ATPS

creation (Figure 5.8) was a single-step process, in which all the reagents were added at the same

time (alga, ionic liquid, water and salt) and the biomass was processed inside a milling equipment.

Figure 5.8 | ATPS ([Emim][dbp]/K2HPO4) created after biomass processing in bead mill.

In Figure 5.8 it is possible to distinguish two aqueous phases (top phase and bottom phase)

and a solid pellet and interphase. While both top and bottom phases were collected for protein

and carbohydrate analysis, the consistency of the interphase (solid gum) did not allow the

quantification of both biomolecules by the available methods. Therefore, taking the starting

amount of protein and carbohydrates as 100%, the extraction of both molecules in the interphase

was calculated subtracting the mass of protein quantified in the top and bottom phases. On the

other hand, the pellet was also not analysed because it was most probably precipitated salt.

Figure 5.9 | Distribution of extracted proteins and carbohydrates between the bottom, inter- and top phases

of the aqueous system composed by [Emim][dbp] and K2HPO4.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Carbohydrates Proteins

Bottom phase Interphase Top phase

62

In the several published studies in which IL based-ATPSs were applied as fractionating

strategy to separate proteins from polysaccharides, the proteins were preferentially concentrated

in the IL-rich phase, whereas the polysaccharides were accumulated into the bottom phase

(Ventura et al., 2017).

As displayed in Figure 5.9, although the distribution of proteins agrees with literature

(preferentially concentrated in the top phase), most of the carbohydrates, instead of being

concentrated in the bottom phase, were enriched into the interphase.

Like inorganic salts, polysaccharides are also kosmotropic (Wu et al., 2008) and, although

their presence is not sufficient to form an ATPS in macroalga extracts, they assist the K2HPO4 in

the phase separation. The interaction between water and saccharides are strong enough to enrich

the sugars in the bottom phase (Pei et al., 2010), however it has been shown that the extraction

efficiency of carbohydrates into the bottom phase decreases with increasing concentrations of

salt (Tan et al., 2012). This may be attributed to the salting-out effect, in which the increase in salt

concentration, decreases the solubility of carbohydrates due to the competition of salt molecules

for intermolecular hydrogen bonds (McKee and McKee, 1999), meaning less free water available

to dissolve saccharides. This phenomenon may explain the separation of carbohydrates into the

additional interphase, which should hold insoluble material with high water content, but not dense

enough to settle. Instead, the water activity of this material is such that it is trapped between the

water activities of the top and bottom phases (i.e. the water activity in the bottom phase was lower

because the water molecules were bound to the salt molecules – density of bound water is greater

than that of free water (Vaclavik and Christian, 2008)). The formation of the interphase turns the

ATPS into a three-partitioning phase (TPP) system and several TPP systems have already been

reported in literature for the extraction of carbohydrates into the interfacial layer (Coimbra et al.,

2010; Sharma and Gupta, 2002; Tan et al., 2015).

The ATPS negative control was used as blank in protein and carbohydrate quantification

to avoid interferences from the top and bottom phases, however, concerns about the

quantification of such molecules in the interphase arise from its unknown composition in terms of

ionic liquid and salt. Therefore, sophisticated analytical methods for the quantification of protein

and carbohydrates in the three-phases formed should be attempted to a more accurate and

reliable results. Moreover, replicates of the system should be performed to validate the

experiment.

It is also important to keep in mind that the stability of proteins in this specific ionic liquid is

a prime requirement to ensure the viability of the proposed process. Techniques such as infrared

spectroscopy, NMR spectroscopy, dynamic light scattering, and circular dichroism yield important

information on protein stability, which makes them suitable to evaluate the effect of the IL based-

ATPS in the extracted proteins.

Once the aforementioned issues are assessed, the optimization of separation conditions is

crucial to increase the extraction efficiency of protein in the top phase. Parameters such as the

concentration of phase-forming components, pH and temperature affect the partition coefficient

of protein in IL based-systems (Pei et al., 2010; Yan et al., 2014). Thus, a screening of the suitable

63

conditions for this particular separation process should be carried out. The biomass loading has

important repercussions for the throughput and economics of the process as there would be cost

saving if less of the solvent can be used and more biomass processed per batch. In this sense,

the maximum amount of biomass that can be processed without compromising the protein

extraction efficiency should be evaluated.

5.4 Ionic liquid recovery

As planned, the recovery of IL was further studied by ultrafiltration. The aim was to recover

the IL in the filtrate, while molecules, such as proteins and carbohydrates, would be rejected by

the membrane. Retentate and filtrate mass balances allowed the evaluation of each membrane

performance in terms of [Emim][dbp] recovery. The results showed that both membranes are

permeable to IL, allowing the recovery of 80-85% [Emim][dbp] (Figure 5.10).

Figure 5.10 | Recovery of [Emim][dbp] in filtrates and retentates, after ultrafiltration through stirred

ultrafiltration cell and centrifugal filter unit. Results are the means of duplicates ±SD.

On the other hand, the separation efficiency of proteins/carbohydrates from the IL solution

was analysed by the rejection coefficient. As it is shown in Table 5.7, the rejection coefficients for

both filtration setups were close to 1, meaning that the proteins and the carbohydrates were

effectively retained by the membranes.

Table 5.7 | Rejection coefficients of proteins and carbohydrates for each ultrafiltration setup. Results are

means of duplicates ±SD.

Ultrafiltration setup Protein rejection coefficient Carbohydrate rejection coefficient

Stirred ultrafiltration cell 0.91±0.04 0.89±0.01

Centrifugal filter unit 0.99±0.01 0.99±0.02

Based on these results, one should conclude that ultrafiltration can effectively separate

ionic liquids from other compounds in mixture, as far as the selection of the membrane considers

the size of the target molecule. Improved recoveries can be achieved by optimizing the operation

parameters (e.g. flow rate, concentration ratio, temperature) (Liu and Wu, 1998). Since the

0%

20%

40%

60%

80%

100%

Stirred ultrafiltration cell Centrifugal filtration unit

IL r

ecovery

Retentate Filtrate

64

efficiency of IL recovery by both ultrafiltration setups was not significantly different (p=0.36),

considering the possible scale-up of the process, stirred ultrafiltration cell setup might be more

suitable in terms of equipment requirements and time-consumption.

65

6 6. Conclusions and future

prospects

67

The commercialization of macroalgal proteins depends on the development of new

techniques for the extraction and separation of these biomolecules, under mild and stabilizing

conditions. The work developed in this thesis aimed at using ionic liquids to mediate the extraction

of macroalgal proteins. After screening twenty-five different ILs, [Emim][dbp] was selected as the

most suitable for the disintegration of U. lactuca cells and subsequent protein extraction. A protein

extraction efficiency of 80.62±4.95% was obtained which is significantly higher than that obtained

by reference cases: extraction by high-shear force under alkaline conditions, ATPS

(PEG/Na2CO3) and sequential aqueous and alkaline extraction, with protein recovery yields of

6.68±1.09%, 10.52±0.35% and 49.08±6.66%, respectively.

Aiming at an integrated biorefinery approach and the valorisation of the different algal

components, a strategy was proposed that involved the single-step processing of algae under

mechanical grinding and in the presence of an aqueous solution of [Emim][dbp] and a certain

amount of K2HPO4 capable to induce phase formation. A TPP system was created, allowing the

selective separation of proteins and polysaccharides, which were mainly concentrated in the top

phase (IL-rich phase) and interfacial layer, respectively. Despite this, only ~50% of the extracted

proteins were enriched into the top phase, meaning that, although [Emim][dbp] extracts ~80% of

the total protein content of U. lactuca, the addition of an aqueous solution of K2HPO4 permitted

the recovery of only ~40% of the total proteins. Therefore, in order to improve the performance of

the system under study, it is of utmost importance to increase the extraction efficiency of the

proteins into the top phase by optimizing the conditions used for the TPP system formation.

For an economically sustainable process, it is crucial that the IL can be easily separated

from the product and recycled for further use. Ultrafiltration showed great potential for the recovery

of ionic liquids from macroalgal extracts, with IL recovery yields of almost 80% in the filtrate while

allowing the concentration of proteins and carbohydrates in the retentate. In this sense, one

should conclude that, if ultrafiltration would be applied only to the top phase of the proposed TPP

system (rich in IL and proteins), it should lead to the isolation of the desired proteins while making

the IL available for reuse.

The results attained with the TPP system for cell disintegration and protein/carbohydrate

fractionation, along with ultrafiltration for IL recovery, are encouraging for a large-scale

application. However, several items must be addressed in addition to the experimental issues

discussed in section 5.

The purity levels required for biocompounds or refined extracts in cosmetic, food and

mainly pharmaceutical and medical sectors are high. This means that the requirements of industry

and markets are crucial when attempting the design and development of separation and

purification processes based on ILs. The scarce information on the stability and bioactivity of ILs

limits their application in such target industries, so further studies in this area are needed.

The development of reliable models that predict the partitioning behaviour of proteins or

polysaccharides in TPP systems would be a major breakthrough for the rational design of these

separation processes and would help the establishment of TPP systems as a standard and large-

68

scale unit operation. In addition, special attention should also be paid to the environmental

concerns that could arise from phase-forming component waste streams.

Although ILs have been claimed as “green” solvents due to their negligible volatility, there

are several studies reporting their negative impact in water ecosystems (Frade and Afonso, 2010).

Therefore, a detailed life-cycle assessment on [Emim][dbp] is necessary in order to claim its

sustainability from a “green” point of view. However, the integration of an additional ultrafiltration

unit operation is a promising option to recovering and recycling [Emim][dbp], minimizing the

environmental concerns of its disposal. Moreover, the high salt concentrations used in TPP

systems can also create waste disposal concerns, for instance due to enhancing eutrophication

phenomenon in water (Ratanapongleka, 2010). This limitation may be overcome by recycling the

salts, using biodegradable salts (e.g. sodium citrate) or gentler salting-out species, such as amino

acids, carbohydrates and polymers (Freire et al., 2012; Raja et al., 2012). Nevertheless, the ability

of these alternative components to induce the phase-formation in [Emim][dbp]-based ATPSs

needs to be evaluated because they suffer the drawback on only being able to form ATPS with a

limited number of ILs due to their low salting-out ability (carbohydrates, amino acids, and polymers

versus salts) (Desai et al., 2016).

Finally, it is of the utmost importance to ascertain the economic viability of the proposed

biorefinery approach, which was one of the objectives of this research project. However, the

obtained results did not allow the accurate establishment of the process mass and energy

balances. Due to the impossibility of protein quantification in the interphase, the non-optimized

partition conditions in the ATPS, the lack of replicates and the unknown amount of biomass that

can be processed at the same time, a proper economic appraisal of the project was deemed not

feasible.

Upon the optimization of the proposed biorefinery strategy and with reliable data available

for the establishment of mass and energy balances, it will be possible to estimate the costs of raw

materials (biomass, [Emim][dbp] and K2HPO4), as well as utilities (water supply, energy

consumption and nitrogen gas/compressed air). Concerning the required equipment (bead mill,

settler, filtration unit), it should be properly scaled up in order to serve the needs of the bioprocess

maximizing its performance while also managing to do so in the most cost-effective manner.

Once the process equipment and utility requirements are determined, the costs associated

towards the implementation of the projected plant design should be defined. In this sense, one

must look at the benefits and impacts of the process through provisional demonstrations of

expected costs and monetary yield. The economic outlook should be based on parameters such

as capital investment, estimate production costs, provisional balances and ultimately, profitability

analysis. This study should determine the success or downfall of the downstream process under

development.

As final remarks, although not all the objectives of the study proposed in the beginning of

this thesis were fully met, the outcome of the present work presents important insights toward the

valorisation of seaweeds as feedstocks for integrated biorefineries. [Emim][dbp] seems to play a

69

remarkable role in the extraction of macroalgal proteins, which widespread use is envisaged in

the near future to boost the quality of modern society.

71

References

Anastas, P.T., Kirchhoff, M.M., 2002. Origins, current status, and future challenges of green

chemistry. Acc. Chem. Res. 35, 686–694.

Andrews, B.A., Asenjo, J.A., 1989. Aqueous Two-Phase Partitioning, in: Harris, E.L. V,

Angal, S. (Eds.), Protein Purification Methods: A Pratical Approach. IRL Press, Oxford, pp. 161–

174.

Aneiros, A., Garateix, A., 2004. Bioactive peptides from marine sources: Pharmacological

properties and isolation procedures. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 803, 41–

53.

Arneniades, C.D., Baer, E., 1977. Transitions and relaxations in polymers, in: Kaufman,

H.S., Falcetta, J.J. (Eds.), Introduction to Polymer Science and Technology: SPE Textbook.

Wiley-Interscience Publications, New York, pp. 239–301.

Baldan, B., Andolfo, P., Navazio, L., Tolomio, C., Mariani, P., 2001. Cellulose in algal cell

wall : An “in situ” localization. Eur. J. Histochem. 45, 51–56.

Barbarino, E., Lourenço, S.O., 2005. An evaluation of methods for extraction and

quantification of protein from marine macro- and microalgae. J. Appl. Phycol. 17, 447–460.

Behera, S., Singh, R., Arora, R., Sharma, N.K., Shukla, M., Kumar, S., 2015. Scope of

Algae as Third Generation Biofuels. Front. Bioeng. Biotechnol. 2, 1–13.

Beier, S.P., 2015. Pressure Driven Membrane Processes: Downstream processing, 3rd ed.

BookBoon.

Bennett, M.D., Leo, D.J., 2004. Ionic liquids as stable solvents for ionic polymer

transducers. Sensors Actuators, A Phys. 115, 79–90.

Berges, J.A., Fisher, A.E., Harrison, P.J., 1993. A comparison of Lowry, Bradford and Smith

protein using different protein standards and protein isolated from the marine diatom

Thalassiosira pseudonana assays. Mar. Biol. 115, 187–193.

Biancarosa, I., Espe, M., Bruckner, C.G., Heesch, S., Liland, N., Waagbø, R., Torstensen,

B., Lock, E.J., 2017. Amino acid composition, protein content, and nitrogen-to-protein conversion

factors of 21 seaweed species from Norwegian waters. J. Appl. Phycol. 29, 1001–1009.

Biesalski, H.K., Dragsted, L.O., Elmadfa, I., Grossklaus, R., Müller, M., Schrenk, D., Walter,

P., Weber, P., 2009. Bioactive compounds: Definition and assessment of activity. Nutrition 25,

1202–1205.

Bigliardi, B., Galati, F., 2013. Innovation trends in the food industry: The case of functional

foods. Trends Food Sci. Technol. 31, 118–129.

Bird, K., Chiles, T., Longley, A., Kendrick, A., Kinkema, M., 1993. Agglutinins from marine

macroalgae of the southeastern United States. J. Appl. Phycol. 5, 213.

Bleakley, S., Hayes, M., 2017. Algal Proteins: Extraction, Application, and Challenges

Concerning Production. Foods 6, 1–34.

Brandt, A., Gräsvik, J., Hallett, J.P., Welton, T., 2013. Deconstruction of lignocellulosic

biomass with ionic liquids. Green Chem. 15, 550.

72

Briand, X., Morand, P., 1997. Anaerobic Digestion of Ulva sp. 1. Relationship between Ulva

composition and methanisation. J. Appl. Phycol. 9, 259–279.

Brown, R.B., Audet, J., 2008. Current techniques for single-cell lysis. J. R. Soc. Interface

5, S131–S138.

Canales, R.I., Brennecke, J.F., 2016. Comparison of Ionic Liquids to Conventional Organic

Solvents for Extraction of Aromatics from Aliphatics. J. Chem. Engeneering data 61, 1685–1699.

Capello, C., Fischer, U., Hungerbühler, K., 2007. What is a green solvent? A

comprehensive framework for the environmental assessment of solvents. Green Chem. 9, 927.

Cardozo, K.H.M., Guaratini, T., Barros, M.P., Falcão, V.R., Tonon, A.P., Lopes, N.P.,

Campos, S., Torres, M.A., Souza, A.O., Colepicolo, P., Pinto, E., 2007. Metabolites from algae

with economical impact. Comp. Biochem. Physiol. - C Toxicol. Pharmacol. 146, 60–78.

Cavalier-Smith, T., 2007. Evolution and relationships, in: Brodie, J., Lewis, J. (Eds.),

Unravelling the Algae_ the Past, Present, and Future of Algal Systematics. CRC Press, Boca

Raton, FL, USA, p. 21.

Cerna, M., 2011. Seaweed proteins and amino acids as nutraceuticals, 1st ed, Advances

in Food and Nutrition Research. Elsevier Inc.

Cha, S.H., Ahn, G.N., Heo, S.J., Kim, K.N., Lee, K.W., Song, C.B., Cho, S.K., Jeon, Y.J.,

2006. Screening of Extracts from Marine Green and Brown Algae in jeju for Potential Marine

Angiotensin-I Converting Enzyme (ACE) Inhibitory Activity. J. Korean Soc. Food Sci. Nutr. 35,

307–314.

Chan, C.X., Ho, C.L., Phang, S.M., 2006. Trends in seaweed research. Trends Plant Sci.

11, 165–166.

Chen, J., Wang, Y., Zeng, Q., Ding, X., Huang, Y., 2014. Partition of proteins with extraction

in aqueous two-phase system by hydroxyl ammonium-based ionic liquid. Anal. Methods 6, 4067.

Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–

131.

Cho, C.W., Jungnickel, C., Stolte, S., Preiss, U., Arning, J., Ranke, J., Krossing, I.,

Thoming, J., 2012. Determination of LFER descriptors of 30 cations of ionic liquids-progress in

understanding their molecular interaction potentials. ChemPhysChem 13, 780–787.

Cho, C.W., Jungnickel, C., Stolte, S., Preiss, U., Arning, J., Ranke, J., Krossing, I.,

Thoming, J., Klamt, A., Krossing, I., Thoming, J., 2011. Ionic Liquids: Predictions of

Physicochemical Properties with Experimental and/or DFT-Calculated LFER Parameters To

Understand Molecular Interactions in Solution. J. Phys. Chem. B 115, 6040–6050.

Chung, I.K., Beardall, J., Mehta, S., Sahoo, D., Stojkovic, S., 2011. Using marine

macroalgae for carbon sequestration: A critical appraisal. J. Appl. Phycol. 23, 877–886.

Clayton Jr., J.R., Dortch, Q., Thoresen, S.S., Ahmed, I., 1988. Evaluation of methods for

the separation and analysis of proteins and free amino acids in phytoplankton samples. J.

Plankton Res. 10, 341–358.

Coimbra, C.G. d O., Lopes, C.E., Calazans, G.M.T., 2010. Three-phase partitioning of

hydrolyzed Levan. Bioresour. Technol. 101, 4725–4728.

73

Collins, K.D., Washabaugh, M.W., 1985. The Hofmeister effect and the behaviour of water

at interfaces. Q. Rev. Biophys. 18, 323.

Compton, S.J., Jones, C.G., 1985. Mechanism of dye response and interference in the

Bradford protein assay. Anal. Biochem. 151, 369–374.

Conde, E., Balboa, E.M., Parada, M., Falqué, E., 2013. Algal proteins, peptides and amino

acids, in: Dominguez, H. (Ed.), Functional Ingredients from Algae for Foods and Nutraceuticals.

Woodhead Publising, pp. 135–180.

Dale, B.E., Kim, S., 2008. Chapter 2. Biomass Refining Global Impact-The Biobased

Economy of the 21st Century, in: Biorefineries-Industrial Processes and Products. pp. 41–66.

Daliri, E., Oh, D., Lee, B., 2017. Bioactive Peptides. Foods 6, 32.

De Pádua, M., Growoski Fontoura, P.S., Mathias, A.L., 2004. Chemical composition of

Ulvaria oxysperma (Kützing) bliding, Ulva lactuca (Linnaeus) and Ulva fascita (Delile). Brazilian

Arch. Biol. Technol. 47, 49–55.

Dejoye, C., Vian, M.A., Lumia, G., Bouscarle, C., Charton, F., Chemat, F., 2011. Combined

extraction processes of lipid from Chlorella vulgaris microalgae: Microwave prior to supercritical

carbon dioxide extraction. Int. J. Mol. Sci.

Desai, R.K., Streefland, M., Wijffels, R., Eppink, M., 2016. Extraction of Proteins with ABS,

in: Freire, M. (Ed.), Ionic-Liquid-Based Aqueous Biphasic Systems. Green Chemistry and

Sustainable Technology. Springer, Berlin, Heidelberg, pp. 123–134.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric Method

for Determination of Sugars and Related Substances. Anal. Chem. 28, 350–356.

Eickhout, B.B., 2012. A strategy for a bio-based economy, Green new deal series.

Ennamany, R., Saboureau, D., Mekideche, N., Creppy, E.E., 1998. SECMA 1, a mitogenic

hexapeptide from Ulva algeae modulates the production of proteoglycans and

glycosaminoglycans in human foreskin fibroblast. Hum. Exp. Toxicol. 17, 18–22.

Eze, J.M.O., Dumbroff, E.B., 1982. A comparision of the Bradford and Lowry methods for

the analysis of protein in chlorophyllous tissue. Can. J. Bot. 60, 1046–1049.

FAO, 2017. World Population Prospects: The 2017 Revision, Key Findings and Advance

Tables.

FAO, 2016. AQUASTAT website [WWW Document]. URL

http://www.fao.org/nr/water/aquastat/water_use/index.stm (accessed 9.7.17).

FAO, 2014a. The state of world fisheries and aquaculture, Food and Agriculture

Oraganization of the United Nations.

FAO, 2014b. World aquaculture production of aquatic plants by producers in 2014, in: FAO

Yearbook - Fishery and Aquaculture Statistics.

FAO, 2014c. Seaweeds and other aquatic plants - Capture production by countries or

areas, in: FAO Yearbook - Fishery and Aquaculture Statistics.

FAO, 2009. Global agriculture towards 2050, High Level Expert Forum-How to feed the

world 2050.

74

Feng, L., Chen, Z., 2008. Research progress on dissolution and functional modi fi cation of

cellulose in ionic liquids. J. Mol. Lipids 142, 1–5.

Fernand, F., Israel, A., Skjermo, J., Wichard, T., Timmermans, K.R., Golberg, A., 2017.

Offshore macroalgae biomass for bioenergy production: Environmental aspects, technological

achievements and challenges. Renew. Sustain. Energy Rev. 75, 35–45.

Fernando, S., Adhikari, S., Chandrapal, C., Murali, N., 2006. Biorefineries: Current Status,

Challenges, and Future Direction. Energy Fuels 20, 1727–1737.

Ferreira, A.F., Paula, A., Dias, S., Silva, C.M., Costa, M., 2016. Effect of low frequency

ultrasound on microalgae solvent extraction : Analysis of products , energy consumption and

emissions. Algal Res. 14, 9–16.

Finkenstadt, V.L., Millane, R.P., 1998. Crystal Structure of Valonia Cellulose I.

Macromolecules 31, 7776–7783.

Fitzgerald, C., Gallagher, E., O’Connor, P., Prieto, J., Mora-Soler, L., Grealy, M., Hayes,

M., 2013. Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH)

inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the

Zebrafish larvae assay. Peptides 50, 119–124.

Fleurence, J., 2004. Seaweed proteins, in: Yada, R.Y. (Ed.), Proteins in Food Processing.

Woodhead Publishing Limited, pp. 197–211.

Fleurence, J., 1999. Seaweed proteins : biochemical, nutritional aspects and potential

uses. Trends Food Sci. Technol. 10, 25–28.

Fleurence, J., Le Coeur, C., Mabeau, S., Maurice, M., Landrein, A., 1995. Comparison of

different extractive procedures for proteins from the edible seaweeds Ulva rigida and Ulva

rotundata. J. Appl. Phycol. 7, 577–582.

Foegeding, E.A., Davis, J.P., 2011. Food protein functionality: A comprehensive approach.

Food Hydrocoll. 25, 1853–1864.

Folch, J., Lees, M., Sloane Stanley, G.H., 1987. A simple method for the isolation and

purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509.

Frade, R.F., Afonso, C.A., 2010. Impact of ionic liquids in environment and humans: An

overview. Hum. Exp. Toxicol. 29, 1038–1054.

Freire, M.G., 2016a. Introduction to Ionic-Liquid-Based Aqueous Systems (ABS), in: Freire,

M.G. (Ed.), Ionic-Liquid- Based Aqueous Biphasic Systems: Fundamentals and Applications.

Springer, Berlin, Heidelberg, pp. 1–26.

Freire, M.G., 2016b. Introduction to Ionic-Liquid-Based Aqueous Biphasic Systems (ABS),

in: Freire, M.G. (Ed.), Ionic-Liquid-Based Aqueous Biphasic Systems. Green Chemistry and

Sustainable Technology. Springer, Berlin, Heidelberg, pp. 1–25.

Freire, M.G., Cláudio, A.F.M., Araújo, J.M.M., Coutinho, J.A.P., Marrucho, I.M., Lopes,

J.N.C., Rebelo, L.P.N., 2012. Aqueous biphasic systems: a boost brought about by using ionic

liquids. Chem. Soc. Rev. 41, 4966.

75

Freire, M.G., Neves, C.M.S.S., Carvalho, P.J., Gardas, R.L., Fernandes, A.M., Marrucho,

I., Santos, L., Coutinho, J.A.P., 2007. Mutual Solubilities of Water and Hydrophobic Ionic Liquids.

J. Phys. Chem. B 111, 13082–13089.

Furuta, T., Miyable, Y., Kinoshita, Y., H., K., 2016. Angiotensin I Converting Enzyme

Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata. Mar. Drugs 14, 1–

10.

Gadilohar, B.L., Shankarling, G.S., 2017. Choline based ionic liquids and their applications

in organic transformation. J. Mol. Liq. 227, 234–261.

Galland-Irmouli, A.V., Fleurence, J., Lamghari, R., Lu??on, M., Rouxel, C., Barbaroux, O.,

Bronowicki, J.P., Villaume, C., Gu??ant, J.L., 1999. Nutritional value of proteins from edible

seaweed Palmaria palmata (Dulse). J. Nutr. Biochem. 10, 353–359.

Gao, K., McKinley, K.R., 1994. Use of macroalgae for marine biomass production and CO2

remediation: a review. J. Appl. Phycol. 6, 45–60.

George, A., Brandt, A., Tran, K., Zahari, S.M.S.N.S., Klein-Marcuschamer, D., Sun, N.,

Sathitsuksanoh, N., Shi, J., Stavila, V., Parthasarathi, R., Singh, S., Holmes, B.M., Welton, T.,

Simmons, B.A., Hallett, J.P., 2015. Design of low-cost ionic liquids for lignocellulosic biomass

pretreatment. Green Chem. 17, 1728–1734.

Gerber, P.J., Steinfeld, H., Henderson, B., Mottet, A., Opio, C., Dijkman, J., Falcucci, A.,

Tempio, G., 2013. Tackling climate change through livestock - A global assessment of emissions

and mitigation opportunities, Food and Agriculture Organization of the United Nations.

Gerovasili, V., Stefanidis, K., Vitzilaios, K., Karatzanos, E., Politis, P., Koroneos, A.,

Chatzimichail, A., Routsi, C., Roussos, C., Nanas, S., 2009. Electrical muscle stimulation

preserves the muscle mass of critically ill patients: a randomized study. Crit. Care 13, 1–8.

Ghatak, H.R., 2011. Biorefineries from the perspective of sustainability: Feedstocks,

products, and processes. Renew. Sustain. Energy Rev. 15, 4042–4052.

Glazer, A.N., 1994. Phycobiliproteins - a family of valuable, widely used fluorophores. J.

Appl. Phycol. 6, 105–112.

Guangce, W., Haibao, S., Xiao, F., Chengkui, T., 2002. Large-scale isolation and

purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed

adsorption method. Acta Bot. Sin. 44, 541–546.

Gute chemie abcr, 2017. Tributylmethylphosphonium methyl sulfate; 95% [WWW

Document]. URL https://www.abcr.de/shop/en/catalog/product/view/sku/AB289826 (accessed

10.1.17).

Gutowski, K.E., Broker, G.A., Willauer, H.D., Huddleston, J.G., Swatloski, R.P., Holvrey,

J.D., Rogers, R.D., 2003. Controlling the aqueous miscibility of ionic liquids: aqueous biphasic

systems of water-miscible ionic liquids and water-structuring salts for tecycle, metathesis, and

separation. J. Am. Chem. Soc. 125, 6632–6633.

Haerens, K., Van Deuren, S., Matthijs, E., Van der Bruggen, B., 2010. Challenges for

recycling ionic liquids by using pressure driven membrane processes. Green Chem. 12, 2182.

Hagino, H., Salto, M., 2004. Use of alagal proteins in cosmetics. EP 1 433 463 A1.

76

Harnedy, P.A., Fitzgerald, R.J., 2015. Extraction and enrichment of protein from red and

green macroalgae, in: Stengel, D.B., Connan, S. (Eds.), Methods in Molecular Biology. Humana

Press, pp. 103–108.

Harnedy, P.A., Fitzgerald, R.J., 2011. Bioactive proteins, peptides, and amino acids from

macroalgae. J. Phycol. 47, 218–232.

Harnedy, P.A., FitzGerald, R.J., 2013. Extraction of protein from the macroalga Palmaria

palmata. LWT - Food Sci. Technol. 51, 375–382.

Hasler, C.M., 2002. Functional foods: benefits, concerns and challenges-a position paper

from the american council on science and health. J. Nutr. 132, 3772–81.

Henchion, M., Hayes, M., Mullen, A., Fenelon, M., Tiwari, B., 2017. Future Protein Supply

and Demand: Strategies and Factors Influencing a Sustainable Equilibrium. Foods 6.

Heo, S.-J.S.J., Lee, K.-W., Song, C.B.C.B., Yeon, Y.-J., Lee, G.W., Song, C.B.C.B., Jeon,

Y.J., 2003. Antioxidant Activity of Enzymatic Extracts from Brown Seaweeds. Algae 18, 71–81.

Hinterstoisser, B., Salmén, L., 2000. Application of dynamic 2D FTIR to cellulose. Vib.

Spectrosc. 22, 111–118.

Holdt, S.L., Kraan, S., 2011. Bioactive compounds in seaweed: Functional food

applications and legislation. J. Appl. Phycol. 23, 543–597.

Höök, M., Tang, X., 2013. Depletion of fossil fuels and anthropogenic climate change-A

review. Energy Policy 52, 797–809.

Hori, K., Ikegami, S., Miyazawa, K., Ito, K., 1988. Mitogenic and antineoplastic

isoagglutinins from the red alga Solieria robusta. Phytochemistry 27, 2063–2067.

Hori, K., Matsubara, K., Miyazawa, K., 2000. Primary structures of two hemagglutinins from

the marine red alga. Biochim. Biophys. Acta - Gen. Subj. 1474, 226–236.

Hori, K., Sato, Y., Ito, K., Fujiwara, Y., Iwamoto, Y., Makino, H., Kawakubo, A., 2007. Strict

specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra

lectin. Glycobiology 17, 479–491.

Hostettmann, K., 2014. Handbook of Chemical and Biological Plant Analytical Methods:

Volume 1, 1st ed. John Wiley & Sons Ltd., Chichester.

Houghton, J.D., 1996. Haems and bilins, in: Houghton, J.D., Hendry, G.A.F. (Eds.), Natural

Food Colorants. Blackie (Chapman and Hall), Glasgow, pp. 157–196.

IEA, 2017. State of Technology Review - Algae Bioenergy, IEA Bioenergy: Task 39.

IEA, 2016a. Proteins for Food, Feed and Biobased Applications.

IEA, 2016b. CO2 emissions from fuel combustion 2016.

IEA, 2014. IEA Bioenergy: Task42 Biorefining Brochure.

IEA, 2009. Biorefineries : Adding Value to the Sustanable Utilisation of Biomass.

Iqbal, M., Tao, Y., Xie, S., Zhu, Y., Chen, D., Wang, X., Huang, L., Peng, D., Sattar, A.,

Shabbir, M.A.B., Hussain, H.I., Ahmed, S., Yuan, Z., 2016. Aqueous two-phase system (ATPS):

an overview and advances in its applications. Biol. Proced. Online 18, 18.

Jensen, A., 1993. Present and future needs for algae and algal products. Hydrobiologia

260, 15–23.

77

Jiménez-Escrig, A., Gómez-Ordóñez, E., Rupérez, P., 2011. Seaweed as a source of novel

nutraceuticals: Sulfated polysaccharides and peptides. Adv. Food Nutr. Res. 64, 325–337.

Jing, B., Lan, N., Qiu, J., Zhu, Y., 2016. Interaction of Ionic Liquids with a Lipid Bilayer: A

Biophysical Study of Ionic Liquid Cytotoxicity. J. Phys. Chem. B 120, 2781–2789.

Jordan, P., Vilter, H., 1991. Extraction of proteins from material rich in anionic mucilages:

Partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae

Laminaria digitata and L. saccharina in aqueous polymer two-phase systems. BBA - Gen. Subj.

1073, 98–106.

Jung, K.A., Lim, S.R., Kim, Y., Park, J.M., 2013. Potentials of macroalgae as feedstocks

for biorefinery. Bioresour. Technol. 135, 182–190.

Kadam, S., Alvarez, C., Tiwari, B., O’Donnell, C., 2017. Extraction and characterization of

protein from Irish brown seaweed Ascophyllum nodosum. Food Res. Int. 99, 1021–1027.

Kaul, A., 2000. The Phase Diagram, in: Hatti-Kaul, R. (Ed.), Aqueous Two-Phase Systems:

Methods and Protocols. Methods in Biotechnology. Humana Press, pp. 11–21.

Kerksick, C., Rasmussen, C., Lancaster, S., Magu, B., 2006. The effects of protein and

amino acids supplementation of performance and training adaptations during ten weeks of

resistance training. J. Strength Cond. Res. 20, 643–653.

Khairy, H.M., El-Shafay, S.M., 2013. Seasonal variations in the biochemical composition of

some common seaweed species from the coast of Abu Qir Bay, Alexandria, Egypt. Oceanologia

55, 435–452.

Kim, Y.H., Choi, Y.K., Park, J., Lee, S., Yang, Y.H., Kim, H.J., Park, T.J., Hwan Kim, Y.,

Lee, S.H., 2012. Ionic liquid-mediated extraction of lipids from algal biomass. Bioresour. Technol.

109, 312–315.

Kitts, D., Weiler, K., 2003. Bioactive proteins and peptides from food sources. Applications

of bioprocesses used in isolation and recovery. Curr. Pharm. Des. 9, 1309–1323.

Knill, C.J., Kennedy, J.F., 2003. Degradation of cellulose under alkaline conditions.

Carbohydr. Polym. 51, 281–300.

Korhonen, H., Pihlanto, A., 2006. Bioactive peptides: Production and functionality. Int. Dairy

J. 16, 945–960.

Krimpen, M.M. Van, Bikker, P., Van Der Meer, I.M., van der Peet-Schwering, C.M.C.,

Vereijken, J.M., 2013. Cultivation, processing and nutritional aspects for pigs and poultry of

European protein sources as alternatives for imported soybean products.

Krisp, H., 2011. Sea Lettuce, Ulva lactuca [WWW Document]. URL

https://commons.wikimedia.org/wiki/File:Meersalat-Ulva-lactuca.jpg (accessed 9.21.17).

Kumar, V., Kaladharan, P., 2007. Amino acids in the seaweeds as an alternate source of

protein for animal feed. J. Mar. Biol. Ass. India 49, 35–40.

Kuzmina, O., 2016. Methods of IL Recovery and Deconstruction, in: Kuzmina, O., Hallett,

J. (Eds.), Application, Purification, and Recovery of Ionic Liquids. Elsevier B. V., pp. 205–248.

Lahaye, M., Robic, A., 2007. Structure and function properties of Ulvan, a polysaccharide

from green seaweeds. Biomacromolecules 8, 1765–1774.

78

Lakmal, H.H.C., Samarakoon, K.W., Lee, W., Lee, J., Abeytunga, D.T.U., Lee, H., Jeon,

Y., 2014. Anticancer and antioxidant effects of selected Sri Lankan marine algae. J. Natl. Sci.

Found. Sri Lanka 42, 315–323.

Lawton, R.J., Mata, L., de Nys, R., Paul, N.A., 2013. Algal Bioremediation of Waste Waters

from Land-Based Aquaculture Using Ulva: Selecting Target Species and Strains. PLoS One 8.

Lee, J.M., You, S.G., Kim, S.M., 2005. Functional Activities of Low Molecular Weight

Peptides Purified from Enzymatic Hydrolysates of Seaweeds. J. Korean Soc. Food Sci. Nutr. 8,

1124–1129.

Legler, G., Müller-Platz, C.M., Mentges-Hettkamp, M., Pflieger, G., Jülich, E., 1985. On the

chemical basis of the Lowry protein determination. Anal. Biochem. 150, 278–287.

Leip, A., Billen, G., Garnier, J., Grizzetti, B., Lassaletta, L., Reis, S., Simpson, D., Sutton,

M.A., de Vries, W., Weiss, F., Westhoek, H., 2015. Impacts of European livestock production:

nitrogen, sulphur, phosphorus and greenhouse gas emissions, land-use, water eutrophication

and biodiversity. Environ. Res. Lett. 10.

Liao, W.R., Lin, J.Y., Shieh, W.Y., Jeng, W.L., Huang, R., 2003. Antibiotic activity of lectins

from marine algae against marine vibrios. J. Ind. Microbiol. Biotechnol. 30, 433–439.

Liu, C., Wu, X., 1998. Optimization of operation parameters in ultrafiltration process. J.

Biotechnol. 66, 195–202.

Liu, S., Abrahamson, L.P., Scott, G.M., 2012. Biorefinery: Ensuring biomass as a

sustainable renewable source of chemicals, materials, and energy. Biomass and Bioenergy 39,

1–4.

Lobban, C.S., Wynne, M.J., 1983. The Biology of Seaweeds, First. ed, Blackwell Scientific

Publications.

Lourenço, S.O., Barbarino, E., De-Paula, J.C., Pereira, L.O.D.S., Lanfer Marquez, U.M.,

2002. Amino acid composition, protein content and calculation of nitrogen-to-protein conversion

factors for 19 tropical seaweeds. Phycol. Res. 50, 233–241.

MacFarlane, D., Tan, S., 2010. Ionic liquids in biomass processing. Top. Curr. Chem. 290,

311–339.

Mai, N.L., Ahn, K., Koo, Y., 2014. Methods for recovery of ionic liquids — A review. Process

Biochem. 49, 872–881.

Malihan, L.B., Nisola, G.M., Mittal, N., Seo, J.G., Chung, W.J., 2014. Blended ionic liquid

systems for macroalgae pretreatment. Renew. Energy 66, 596–604.

Marambe, H.K., Wanasundara, J.P.D., 2017. Protein From Flaxseed (Linum usitatissimum

L.), in: Nadathur, S., Wanasundara, J.P.D., Scanlin, L. (Eds.), Sustainable Protein Sources.

Academic Press, pp. 133–144.

Marinho, G., Nunes, C., Sousa-Pinto, I., Pereira, R., Rema, P., 2013. The IMTA-cultivated

Chlorophyta Ulva spp. as a sustainable ingredient in Nile tilapia (Oreochromis niloticus) diets. J.

Appl. Phycol. 25, 1359–1367.

79

Marinova, K.G., Basheva, E.S., Nenova, B., Temelska, M., Mirarefi, A.Y., Campbell, B.,

Ivanov, I.B., 2009. Physico-chemical factors controlling the foamability and foam stability of milk

proteins: Sodium caseinate and whey protein concentrates. Food Hydrocoll. 23, 1864–1876.

Marshall, T., Williams, K.M., 2004. Interference in the Coomassie Brilliant Blue and

Pyrogallol Red protein dye-binding assays is increased by the addition of sodium dodecyl sulfate

to the dye reagents. Anal. Biochem.

Marsham, S., Scott, G.W., Tobin, M.L., 2007. Comparison of nutritive chemistry of a range

of temperate seaweeds. Food Chem. 100, 1331–1336.

Matanjun, P., Mohamed, S., Mustapha, N.M., Muhammad, K., 2009. Nutrient content of

tropical edible seaweeds, Eucheuma cottonii, Caulerpa lentillifera and Sargassum polycystum. J.

Appl. Phycol. 21, 75–80.

Matsubara, K., Sumi, H., Hori, K., 1996. Platelet aggregation is inhibited by phycolectins.

Experientia 52, 540–543.

Matzke, M., Aming, J., Ranke, J., Jastorff, B., Stolte, S., 2010. Design of Inherently Safer

Ionic Liquids: Toxicology and Biodegradation, in: Wassercheid, P., Stark, A. (Eds.), Handbook of

Green Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp. 235–298.

Mazzola, P.G., Lopes, A.M., Hasmann, F.A., Jozala, A.F., Penna, T.C.V., Magalhaes, P.O.,

Rangel-Yagui, C.O., Pessoa, A., 2008. Liquid-liquid extraction of biomolecules: An overview and

update of the main techniques. J. Chem. Technol. Biotechnol. 83, 143–157.

McHugh, D.J., 2003. A Guide to the Seaweed Industry, FAO Fisheries Technical Paper.

Food and Agriculture Organization of the United States, Rome, Italy.

McKee, T., McKee, J., 1999. Biochemistry: An Introduction, 2nd ed. WCB/McGraw-Hill,

Boston.

Merchuk, J.C., Andrews, B.A., Asenjo, J.A., 1998. Aqueous two-phase systems for protein

separation: Studies on phase inversion. J. Chromatogr. B Biomed. Sci. Appl. 711, 285–293.

Merck Millipore, 2015. User Guide - Amicon® Stirred Cells. USA.

Millward, D.J., Layman, D.K., Tomé, D., Schaafsma, G., 2008. Protein quality assessment:

impact of expanding understanding of protein and amino acid needs for optimal health. Am. Soc.

Clin. Nutr. 87, 1576–1581.

Mintel, 2016. Seaweed-flavoured food and drink launches increased by 147% in europe

between 2011 and 2015 [WWW Document]. URL http://www.mintel.com/press-centre/food-and-

drink/seaweed-flavoured-food-and-drink-launches-increased-by-147-in-europe-between-2011-

and-2015 (accessed 11.9.17).

Mohd, N., Draman, S.F.S., Salleh, M.S.N., Yusof, N.B., 2017. Dissolution of cellulose in

ionic liquid: A review, in: AIP Conference Proceedings. AIP Publishing, p. 20035.

Mori, T., O’Keefe, B.R., Sowder, R.C., Bringans, S., Gardella, R., Berg, S., Cochran, P.,

Turpin, J.A., Buckheit, R.W., McMahon, J.B., Boyd, M.R., 2005. Isolation and characterization of

Griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. J. Biol. Chem. 280,

9345–9353.

80

Naeem, A., Saleemuddin, M., Khan, R.H., 2007. Glycoprotein targeting and other

applications of lectins in biotechnology. Curr. Protein Pept. Sci. 8, 261–71.

Naushad, M., Abdullah, Z., Bashar, A., Ali, M., 2012. Effect of ionic liquid on activity, stability

, and structure of enzymes : A review. Int. J. Biol. Macromol. 51, 555–560.

Necas, J., Bartosikova, L., 2013. Carrageenan: a review. Vet. Med. (Praha). 58, 187–205.

Nelson, D.L., Cox, M.M., 2008. Lehninger principles of biochemistry, 5th ed. W. H. Freeman

and Company, New York.

Notoya, M., 2010. Production of Biofuel by Macroalgae with Preservation of Marine

Resources and Environment, in: Cellular Origin, Life in Extreme Habitats and Astrobiology. pp.

217–228.

Oliferenko, A.A., Oliferenko, P. V., Huddleston, J.G., Rogers, R.D., Palyulin, V.A., Zefirov,

N.S., Katritzky, A.R., 2004. Theoretical scales of hydrogen bond acidity and basicity for

application in QSAR/ QSPR studies and drug design. Partitioning of aliphatic compounds. J.

Chem. Inf. Comput. Sci. 44, 1042–1055.

Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernández, J., Bozzo, C., Navarrete, E.,

Osorio, A., Rios, A., 2006. Dietary fiber, amino acid, fatty acid and tocopherol contents of the

edible seaweeds Ulva lactuca and Durvillaea antarctica. Food Chem. 99, 98–104.

Passos, H., Freire, M.G., Coutinho, J.A.P., 2014. Ionic liquid solutions as extractive

solvents for value-added compounds from biomass. Green Chem. 16, 4786–4815.

Pei, Y., Li, Z., Liu, L., Wang, J., Wang, H., 2010. Selective separation of protein and

saccharides by ionic liquids aqueous two-phase systems. Sci. China Chem. 53, 1554–1560.

Perez Espitia, P.J., de Fátima Ferreira Soares, N., dos Reis Coimbra, J.S., de Andrade,

N.J., Souza Cruz, R., Alves Medeiros, E.A., 2012. Bioactive Peptides: Synthesis, Properties, and

Applications in the Packaging and Preservation of Food. Compr. Rev. Food Sci. Food Saf. 11,

187–204.

Pezoa-Conte, R., Leyton, A., Anugwom, I., von Schoultz, S., Paranko, J., Mäki-Arvela, P.,

Willför, S., Muszyński, M., Nowicki, J., Lienqueo, M.E., Mikkola, J.P., 2015. Deconstruction of the

green alga Ulva rigida in ionic liquids: Closing the mass balance. Algal Res. 12, 262–273.

Pimentel, M., Pimentel, D., 2003. Sustainability of meat-based and plant based diets and

the environment. Am. J. Clin. Nutr. 78, 660S–663S.

Pinker, A., Marsh, K.N., Pang, S., Staiger, M.P., 2009. Ionic Liquids and Their Interaction

with Cellulose. Chem. Rev. 109, 6712–6728.

Plaza, M., Cifuentes, A., Ibáñez, E., 2008. In the search of new functional food ingredients

from algae. Trends Food Sci. Technol. 19, 31–39.

Plaza, M., Santoyo, S., Jaime, L., García-Blairsy Reina, G., Herrero, M., Senoráns, F.,

Ibánez, E., 2010. Screening for bioactive compounds from algae. J. Pharm. Biomed. Anal. 51,

450–455.

Raja, S., Murty, V.R., Thivaharan, V., Rajasekar, V., Ramesh, V., 2012. Aqueous Two

Phase Systems for the Recovery of Biomolecules – A Review. Sci. Technol. 1, 7–16.

81

Ranke, J., Stolte, S., Störmann, R., Arning, J., Jastorff, B., 2007. Design of sustainable

chemical products-the example of ionic liquids. Chem. Rev. 107, 2183–2206.

Ratanapongleka, K., 2010. Recovery of Biological Products in Aqueous Two Phase

Systems. Int. J. Chem. Eng. Appl. 1, 191–198.

Robertson-Andersson, D. V., Potgieter, M., Hansen, J., Bolton, J.J., Troell, M., Anderson,

R.J., Halling, C., Probyn, T., 2008. Integrated seaweed cultivation on an abalone farm in South

Africa. J. Appl. Phycol. 20, 579–595.

Ross, A.B., Anastasakis, K., Kubacki, M., Jones, J.M., 2009. Investigation of the pyrolysis

behaviour of brown algae before and after pre-treatment using PY-GC/MS and TGA. J. Anal.

Appl. Pyrolysis 85, 3–10.

Ross, A.B., Jones, J.M., Kubacki, M.L., Bridgeman, T., 2008. Classification of macroalgae

as fuel and its thermochemical behaviour. Bioresour. Technol. 99, 6494–6504.

Rouxel, C., Daniel, A., Jérôme, M., Etienne, M., Fleurence, J., 2001. Species identification

by SDS-PAGE of red algae used as seafood or a food ingredient. Food Chem. 74, 349–353.

Saito, M., Hagino, H., 2005. Antihypertensive effect of oligopeptides derived from nori

(Porphyra yezoensis) and Ala-Lys-Tyr-Ser-Tyr in rats. J. Japan Soc. Nutr. Food Sci. 58, 177–184.

Samarakoon, K., Jeon, Y.J., 2012. Bio-functionalities of proteins derived from marine algae

- A review. Food Res. Int. 48, 948–960.

Sambusiti, C., Bellucci, M., Zabaniotou, A., Beneduce, L., Monlau, F., 2015. Algae as

promising feedstocks for fermentative biohydrogen production according to a biorefinery

approach: A comprehensive review. Renew. Sustain. Energy Rev. 44, 20–36.

Santa Cruz Biotechnology, 2017. 1-Butyl-3-methylimidazolium dibutyl phosphate [WWW

Document]. URL https://www.scbt.com/scbt/product/1-butyl-3-methylimidazolium-dibutyl-

phosphate-663199-28-8 (accessed 10.1.17).

Santelices, B., 1991. Production ecology of Gelidium. Hydrobiologia 221, 31–44.

Saqib, A., Tabassum, M.R., Rashid, U., Ibrahim, M., Gill, S.S., Mehmood, M.A., 2013.

Marine macro algae ulva: a potential feed-stock for bio-ethanol and biogas production. Asian J.

Agric. Biol. 1, 155–163.

Sato, M., Hosokawa, T., Yamaguchi, T., Nakano, T.T., Muramoto, K., Kahara, T.,

Funayama, K., Kobayashi, A., 2002. Angiotensin I-Converting Enzyme Inhibitory Peptides

Derived from Wakame (Undaria pinnatifida) and Their Antihypertensive Effect in Spontaneously

Hypertensive Rats. J. Agric. Food Chem. 50, 6245–6252.

Schenk, P.M., Thomas-Hall, S.R., Stephens, E., Marx, U.C., Mussgnug, J.H., Posten, C.,

Kruse, O., Hankamer, B., 2008. Second Generation Biofuels: High-Efficiency Microalgae for

Biodiesel Production. BioEnergy Res. 1, 20–43.

Schneider, C.P., Shukla, D., Trout, B.L., 2011. Arginine and the Hofmeister Series: The

Role of Iom-Ion Interactions in Protein Aggregation Suppression. J. Phys. Chem. B 115, 7447–

7458.

Seddon, K.R., 1997. Ionic Liquids for Clean Technology. J. Chem. Technol. Biotechnol. 68,

351–356.

82

Sekar, S., Chandramohan, M., 2008. Phycobiliproteins as a commodity: Trends in applied

research, patents and commercialization. J. Appl. Phycol.

Sharma, A., Gupta, M.N., 2002. Three phase partitioning of carbohydrate polymers:

Separation and purification of alginates. Carbohydr. Polym. 48, 391–395.

Shuuluka, D., Bolton, J.J., Anderson, R.J., 2013. Protein content, amino acid composition

and nitrogen-to-protein conversion factors of Ulva rigida and Ulva capensis from natural

populations and Ulva lactuca from an aquaculture system, in South Africa. J. Appl. Phycol. 25,

677–685.

Sigma Aldrich, 2017a. Choline Chloride [WWW Document]. URL

http://www.sigmaaldrich.com/catalog/product/sigma/c1879?lang=pt&region=PT (accessed

10.1.17).

Sigma Aldrich, 2017b. 1-Butyl-3-methylimidazolium acetate ≥95% [WWW Document]. URL

http://www.sigmaaldrich.com/catalog/product/aldrich/39952?lang=pt&region=PT (accessed

10.1.17).

Sigma Aldrich, 2017c. 1-Butyl-3-methylimidazolium chloride ≥98 [WWW Document]. URL


10.1.17).

Sigma Aldrich, 2017d. Trihexyltetradecylphosphonium chloride [WWW Document]. URL

http://www.sigmaaldrich.com/catalog/product/aldrich/89744?lang=en&region=GB (accessed

10.1.17).

Sigma Aldrich, 2017e. Trihexyl(tetradecyl)phosphonium dicyanamide 99.5% [WWW

Document]. URL http://en.solvionic.com/products/trihexyl-tetra-decyl-phosphonium-

dicyanamide-99.5 (accessed 10.1.17).

Sigma Aldrich, 2017f. Choline acetate ≥95 [WWW Document]. URL


10.1.17).

Sigma Aldrich, 2017g. 1-Ethyl-3-methylimidazolium dibutyl phosphate ≥97 [WWW

Document]. URL


10.1.17).

Sigma Aldrich, 2017h. 1-Butyl-3-methylimidazolium dicyanamide ≥97 [WWW Document].

URL http://www.sigmaaldrich.com/catalog/product/aldrich/55220?lang=pt&region=PT

Siró, I., Kápolna, E., Kápolna, B., Lugasi, A., 2008. Functional food. Product development,

marketing and consumer acceptance-A review. Appetite 51, 456–467.

Smit, A., 2004. Medicinal and pharmaceutical uses of seaweed natural products : a review.

J. Appl. Phycol. 16, 245–262.

Snyder, S.M., Cole, K.D., Sziag, D.C., 1992. Phase Compositions, Viscosities, and

Densities for Aqueous Two-Phase Systems Composed of Polyethylene Glycol and Various Salts

at 25 °C. J. Chem. Eng. Data 37, 268–274.

83

Socha, A.M., Parthasarathi, R., Shi, J., Pattathil, S., Whyte, D., Bergeron, M., George, A.,

Tran, K., Stavila, V., Venkatachalam, S., Hahn, M.G., Simmons, B.A., Singh, S., 2014. Efficient

biomass pretreatment using ionic liquids derived from lignin and hemicellulose. Proc. Natl. Acad.

Sci. 111, E3587–E3595.

Somerville, C., 2006. Cellulose Synthesis in Higher Plants. Annu. Rev. Cell Dev. Biol. 22,

53–78.

Stark, A., 2011. Ionic liquids in the biorefinery: a critical assessment of their potential.

Energy Environ. Sci. 4, 19–32.

Stark, A., Sellin, M., Ondruschka, B., Massonne, K., 2012. The effect of hydrogen bond

acceptor properties of ionic liquids on their cellulose solubility. Sci. China Chem. 55, 1663–1670.

Suetsuna, K., 1998a. Separation and identification of angiotensin I-converting enzyme

inhibitory peptides from peptic digest of Hizikia fusiformis protein. Nippon Suisan Gakkaishi 64,

862–866.

Suetsuna, K., 1998b. Purification and identification of angiotensin I-converting enzyme

inhibitors from the red alga Porphyra yezoensis. J. Mar. Biotechnol. 6, 163–167.

Sugahara, T., Ohama, Y., Fukuda, A., Hayashi, M., Kawakubo, A., Kako, K., 2001. The

cytotoxic effect ofEucheuma serraagglutinin (ESA) on cancer cells and its application to molecular

probe for drug delivery system using lipid vesicles. Cytotechnology 36, 93–99.

Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., 2002. Dissolution of Cellose with

Ionic Liquids. J. Am. Chem. Soc. 124, 4974–4975.

Tabarsa, M., Rezaei, M., Ramezanpour, Z., Waaland, J.R., 2012. Chemical compositions

of the marine algae Gracilaria salicornia (Rhodophyta) and Ulva lactuca (Chlorophyta) as a

potential food source. J. Sci. Food Agric. 92, 2500–2506.

Tan, Z. jian, Li, F. fang, Xu, X. lei, Xing, J. min, 2012. Simultaneous extraction and

purification of aloe polysaccharides and proteins using ionic liquid based aqueous two-phase

system coupled with dialysis membrane. Desalination 286, 389–393.

Tan, Z.J., Wang, C.Y., Yi, Y.J., Wang, H.Y., Zhou, W.L., Tan, S.Y., Li, F.F., 2015. Three

phase partitioning for simultaneous purification of aloe polysaccharide and protein using a single-

step extraction. Process Biochem. 50, 482–486.

The World Bank, 2013. Fish to 2030: Prospects for fisheries and aquaculture.

Toledo Hijo, A.A.C., Maximo, G.J., Costa, M.C., Batista, E.A.C., Meirelles, A.J.A., 2016.

Applications of Ionic Liquids in the Food and Bioproducts Industries. ACS Sustain. Chem. Eng.

4, 5347–5369.

Vaclavik, V.A., Christian, E.W., 2008. Introduction to Food Components, in: Heldman, D.R.

(Ed.), Essentials of Food Science. Springer Science + Business Media, LLC, New York, p. 27.

Valenzuela, C., Abugoch, L., Tapia, C., Gamboa, A., 2013. Effect of alkaline extraction on

the structure of the protein of quinoa (Chenopodium quinoa Willd.) and its influence on film

formation. Int. J. Food Sci. Technol. 48, 843–849.

84

Van Der Bruggen, B., Vandecasteele, C., Van Gestel, T., Doyen, W., Leysen, R., 2003. A

review of pressure-driven membrane processes in wastewater treatment and drinking water

production. Environ. Prog. 22, 46–56.

Van Hal, J.W., Huijgen, W.J.J., López-Contreras, A.M., 2014. Opportunities and challenges

for seaweed in the biobased economy. Trends Biotechnol. 32, 231–233.

Van Wychen, S., Laurens, L.M., 2013. Determination of Total Solids and Ash in Algal

Biomas: Laboratory Analytical Procedure (LAP).

Vanderlei, E.S.O., Patoilo, K.K.N.R., Lima, N.A., Lima, A.P.S., Rodrigues, J.A.G., Silva,

L.M.C.M., Lima, M.E.P., Lima, V., Benevides, N.M.B., 2010. Antinociceptive and anti-

inflammatory activities of lectin from the marine green alga Caulerpa cupressoides. Int.

Immunopharmacol. 10, 1113–1118.

Ventura, S.P.M., e Silva, F.A., Quental, M. V., Mondal, D., Freire, M.G., Coutinho, J.A.P.,

2017. Ionic-Liquid-Mediated Extraction and Separation Processes for Bioactive Compounds:

Past, Present, and Future Trends. Chem. Rev. 117, 6984–7052.

Ventura, S.P.M., Marques, C.S., Rosatella, A.A., Afonso, C.A.M., Gonçalves, F., Coutinho,

J.A.P., 2012. Toxicity assessment of various ionic liquid families towards Vibrio fischeri marine

bacteria. Ecotoxicol. Environ. Saf. 76, 162–168.

Vijayaraghavan, R., Izgorodin, A., Ganesh, V., Surianarayanan, M., MaxFarlane, D.R.,

2010. Long-Term Structural and Chemical Stability of DNA in Hydrated Ionic Liquids. Angew.

Chemie Int. Ed. 49, 1631–1633.

Viskari, P.J., Colyer, C.L., 2003. Rapid extraction of phycobiliproteins from cultured

cyanobacteria samples. Anal. Biochem. 319, 263–271.

Weis, W.I., Drickamer, K., 1996. Structural basis of lectin-carbohydrate recognition. Annu.

Rev. Biochem. 65, 441–473.

Wells, M.L., Potin, P., Craigie, J.S., Raven, J.A., Merchant, S.S., Helliwell, K.E., Smith,

A.G., Camire, M.E., Brawley, S.H., 2017. Algae as nutritional and functional food sources:

revisiting our understanding. J. Appl. Phycol. 29, 949–982.

Wessman, P., Håkansson, S., Leifer, K., Rubino, S., 2013. Formulations for Freeze-drying

of Bacteria and Their Influence on Cell Survival. J. Vis. Exp.

Westhoek, H., Rood, T., van den Berg, M., Janse, J., Nijdam, D., Reudink, M., Stehfest,

E., 2011. The Protein Puzzle, The protein puzzle: The consumption and production of meat, dairy

and fish in the European Union.

WHO/FAO/UNU, 2007. Protein and amino acid requirements in human nutrition., in: Report

of a Joint WHO/FAO/UNU Expert Consultation. WHO Press, Geneva.

Wijffels, R.H., Barbosa, M.J., 2010. An Outlook on Microalgal Biofuels. Science (80-. ). 330,

796–799.

Wong, K.H., Cheung, P.C.K., 2000. Nutritional evaluation of some subtropical red and

green seaweeds. Food Chem. 71, 475–482.

Wu, B., Liu, W., Zhang, Y., Wang, H., 2009. Do we understand the recyclability of ionic

liquids? Chem. - A Eur. J. 15, 1804–1810.

85

Wu, B., Zhang, Y.M., Wang, H.P., 2008. Phase behavior for ternary systems composed of

ionic liquid plus saccharides plus water. J. Phys. Chem. B 112, 6426–6429.

Xu, W.J., Liao, X.J., Xu, S.H., Diao, J.Z., Du, B., Zhou, X.L., Pan, S.S., 2008. Isolation,

structure determination, and synthesis of galaxamide, a rare cytotoxic cyclic pentapeptide from a

marine algae Galaxaura filamentosa. Org. Lett. 10, 4569–4572.

Yaich, H., Garna, H., Bchir, B., Besbes, S., Paquot, M., Richel, A., Blecker, C., Attia, H.,

2015. Chemical composition and functional properties of dietary fibre extracted by Englyst and

Prosky methods from the alga Ulva lactuca collected in Tunisia. Algal Res. 9, 65–73.

Yan, J.K., Ma, H. Le, Pei, J.J., Wang, Z. Bin, Wu, J.Y., 2014. Facile and effective separation

of polysaccharides and proteins from Cordyceps sinensis mycelia by ionic liquid aqueous two-

phase system. Sep. Purif. Technol. 135, 278–284.

Zhang, C., Sanders, J.P.M., Xiao, T.T., Bruins, M.E., 2015. How does alkali aid protein

extraction in green tea leaf residue: A basis for integrated biorefinery of leaves. PLoS One 10, 1–

14.

Zhang, J., Wu, J., Yu, J., Zhang, X., He, J., Zhang, J., 2017. Application of ionic liquids for

dissolving cellulose and fabricating cellulose-based materials: state of the art and future trends.

Mater. Chem. Front. 1, 1273–1290.

Zhang, Y., Cremer, P.S., 2006. Interactions between macromolecules and ions: the

Hofmeister series. Curr. Opin. Chem. Biol. 10, 658–663.

Zhao, H., Xia, S., Ma, P., 2005. Use of ionic liquids as “green” solvents for extractions. J.

Chem. Technol. Biotechnol. 80, 1089–1096.

Ziółkowska, N.E., Wlodawer, A., 2006. Structural studies of algal lectins with anti-HIV

activity. Acta Biochim. Pol. 53, 617–626.

A-1

A Appendix A Protein,

carbohydrate and ionic liquid quantification

A-3

A.1. Protein Quantification

The calibration curves used in protein quantification were prepared with BSA for a working

range of 0–1.4 mg.mL-1 for Lowry’s method and 0–2 mg.mL-1 for Bradford assay. Equation A. 1

to Equation A. 8 describe the calibration curves, and respective correlation factors, used in each

experiment for protein determination.

Total protein content

Lowry’s method

Abs750𝑛𝑚 = 0.20[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 − 1.47 × 10−2 (𝑟2 = 0.9938)

Equation A. 1

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = 1.50𝐴𝑏𝑠595𝑛𝑚2 + 0.61𝐴𝑏𝑠595𝑛𝑚 + 0.04 (𝑟2 = 0.9870)

Equation A. 2

Aqueous and alkaline extraction

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = 1.83𝐴𝑏𝑠595𝑛𝑚2 − 1.05𝐴𝑏𝑠595𝑛𝑚 + 0.16 (𝑟2 = 0.9898)

Equation A. 3

Extraction by high-shear force

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = 1.83𝐴𝑏𝑠595𝑛𝑚2 − 1.05𝐴𝑏𝑠595𝑛𝑚 + 0.16 (𝑟2 = 0.9898)

Equation A. 4

Aqueous biphasic system PEG/Na2CO3

Lowry’s method

Abs750𝑛𝑚 = 0.17[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 + 0.14 (𝑟2 = 0.9831)

Equation A. 5

Ionic liquid selection

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = 1.50𝐴𝑏𝑠595𝑛𝑚2 + 0.61𝐴𝑏𝑠595𝑛𝑚 + 0.04 (𝑟2 = 0.9870)

Equation A. 6

Aqueous three-phase partitioning system [Emim][dbp]/K2HPO4

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = −0.20𝐴𝑏𝑠595𝑛𝑚2 + 0.84𝐴𝑏𝑠595𝑛𝑚 + 0.02 (𝑟2 = 0.9966)

Equation A. 7

A-4

Ultrafiltration

Bradford assay

[𝑃𝑟𝑜𝑡𝑒𝑖𝑛]𝑚𝑔. 𝑚𝐿−1 = 1.82 𝐴𝑏𝑠595𝑛𝑚2 − 0.39𝐴𝑏𝑠595𝑛𝑚 + 0.04 (𝑟2 = 0.9873)

Equation A. 8

A.2. Carbohydrates Quantification

The calibration curves used in carbohydrate quantification were prepared with glucose for

a working range of 0–0.1 mg.mL-1. Equation A. 9 to Equation A. 12 describe the calibration curves,

and respective correlation factors, used in each experiment for carbohydrate determination.

Total carbohydrate content

Abs483𝑛𝑚 = 4.37[𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒]𝑚𝑔. 𝑚𝐿−1 + 2.48 × 10−3 (𝑟2 = 0.9703)

Equation A. 9

Aqueous biphasic system PEG/Na2CO3


Equation A. 10

Aqueous three-phase partitioning system [Emim][dbp]/K2HPO4


Equation A. 11

Ultrafiltration


Equation A. 12

A.3. Ionic liquid Quantification

The calibration curve and respective correlation factor used for ionic liquid quantification,

in ultrafiltration experiment, was prepared with [Emim][dbp] for a working range of 100–400

mg.mL-1 (Equation A. 13).

𝐴𝑟𝑒𝑎𝑝𝑒𝑎𝑘 = 3.54 × 102[𝐼𝐿]𝑚𝑔. 𝑚𝐿−1 − 1.56 × 103 (𝑟2 = 0.9977)

Equation A. 13

B-1

B Appendix B Binodal Curve

B-3

B.1. Binodal curve correlation

The binodal experimental data obtained for the ATPS composed by [Emim][dbp] an

K2HPO4 and respective correlation factor are described in Equation B. 1. The r-squared statistical

measure is quite far from 1, suggesting that the model does not fit the experimental data so as

one should not use it for the determination of component concentrations. Due to time concerns

the experimental procedure was not repeated and the concentrations of K2HPO4 and IL used for

the ATPS formation were estimated by visual observation of the Figure 5.7.

[𝐸𝑚𝑖𝑚][𝑑𝑏𝑝] = 420.25 × exp(−1.32 × [𝐾2𝐻𝑃𝑂4]3) (𝑟2 = 0.7964)

Equation B. 1

screening of ionic liquids for the extraction of proteins from the … · o 1-etil-3-metilimidazol...

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