Ângelo Paggi Matos

CULTIVO, CARACTERIZAÇÃO E TÉCNICAS DE

PROCESSAMENTO DE ALGAS

Tese submetida ao Programa de Pós-

Graduação em Ciência dos Alimentos
da Universidade Federal de Santa
Catarina para a obtenção do Grau de
Doutor em Ciência dos Alimentos.

Orientador: Prof. Dr. Ernani Sebastião

Sant‘Anna
Co-orientadora: Profa. Dra. Elisa
Helena Siegel Moecke

Florianópolis, Brasil
2017
 Ficha de identificação da obra elaborada pelo autor, através do
Programa de Geração Automática da Biblioteca Universitária da UFSC.

Matos, Ângelo Paggi

Cultivo, Caracterização e Técnicas de Processamento de Algas / Ângelo
Paggi Matos ; orientador, Ernani Sebastião Sant‘Anna ; coorientadora, Elisa Helena
Siegel Moecke. – Florianópolis, SC, 2017.
220 p.
Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de
Ciências Agrárias. Programa de Pós-Graduação em Ciência dos Alimentos.
Inclui referências
1. Ciência dos Alimentos. 2. Microalgas. 3. Composição química. 4.
Biotecnologia. 5. Alimentos. I. Sant‘Anna, Ernani Sebastião. II. Moecke Elisa
Helena Siegel. III. Universidade Federal de Santa Catarina. Programa de Pós-
Graduação em Ciência dos Alimentos. IV. Titulo.
 Ângelo Paggi Matos

CULTIVO, CARACTERIZAÇÃO E TÉCNICAS DE

PROCESSAMENTO DE ALGAS

Esta tese foi julgada adequada para obtenção do Título de ―Doutor em

Ciência dos Alimentos‖ e aprovada em sua forma final pelo Programa
de Pós-Graduação em Ciência dos Alimentos da UFSC.

Florianópolis, 21 de Setembro de 2017.

________________________
Profa. Dra. Ana Carolina de Oliveira Costa.
Coordenadora do Curso de Pós-Graduação em Ciência dos Alimentos

Banca Examinadora:
________________________
Prof. Dr. Ernani Sebastião Sant‘Anna
PGCAL/CCA/UFSC - Presidente
________________________
Prof.ª Dr.ª Elisa Helena Siegel Moecke
Co-orientadora - UNISUL
________________________
Prof.a Dr.a Flávia Marisa Prado Saldanha Corrêa
Universidade de São Paulo (USP)
________________________
Prof.a Dr.a Anelise Leal Vieira Cubas
Universidade do Sul de Santa Catarina (UNISUL)
________________________
Prof.a Dr.a Itaciara Larroza Nunes
PGCAL/CCA/UFSC
________________________
Prof.a Dr.a Jane Mara Block
PGCAL/CCA/UFSC
 I dedicate this thesis to the
great professor and friend Ernani
Sant’Anna, for his continuous
positive approach, neat work
example and kind loyalty. God
bless you abundantly.
 AGRADECIMENTOS

Aos que contribuíram para a realização deste trabalho, meu sincero

reconhecimento e agradecimento.
Ao professor orientador Ernani Sebastião Sant’Anna e a professora
co-orientadora Elisa Helena Siegel Moecke, vocês são geniais e muito
generosos.
Ao grande colega de trabalho e amigo nas horas ‘vagas’ Rafael
Feller.
Especial agradecimento à Flávia Saldanha-Corrêa do Instituto
Oceanográfico da Universidade de São Paulo (IOUSP) pelo
fornecimento da microalga Nannochloropsis gaditana e contribuições
na versão final desta tese; você é muito gentil.
A minha aluna de iniciação científica Monnik Gandin Cavanholi.
Ao amigo Carlos Roberto dos Santos (Carlão) por sua dedicação em
manter ligado o compressor, necessário para comprimir ar-CO2 nos
cultivos de algas, ininterruptamente durante oito anos de pesquisa.
Ao amigo Sérgio de Souza, secretário do Programa de Pós-
Graduação em Ciência dos Alimentos.
Aos colegas do laboratório de Análises Físico-Químicas – LABCAL
da UFSC, em especial Gisele Olivo e Danilo Fernando Fantini.
As colegas do Núcleo de Microscopia de Alimentos – NUMIC da
UFSC, em especial a Marina Silva Teixeira (Nina).
Aos colegas do Laboratório de Biologia Molecular da UFSC.
Ao grupo de pesquisa do Laboratório de Plasma da UNISUL.
To adored Fatima Matos and Hans Hollema for dedication to revise
and contribute in this thesis work.
A toda a minha família e em especial minha esposa Daniela Bidone.
Agradecimentos ao Programa de Pós-Graduação em Ciência dos
Alimentos e a CAPES pelo auxílio na bolsa de estudos.
A UFSC, o maior universo que tenho assistido.
 RESUMO

Nos últimos 10 anos, houve um interesse substancial na utilização de

microalgas para produzir biomassa, bioprodutos e biocombustíveis. Esse
interesse na biotecnologia das microalgas é baseado no fato de que as
microalgas crescem muito rapidamente podendo duplicar o número de
células em menos de um dia. Além disso, muitas espécies de algas são
capazes de armazenar energia na forma de lipídeos, que têm o dobro da
densidade energética de carboidratos e proteínas. A conversão da
biomassa de algas em bioprodutos inclui co-produtos que podem ser
destinado para o consumo humano e/ou ração animal, indústria química,
farmacêutica e alimentos, entre outros. Esta tese tem como título
―Cultivo, Caracterização e Técnicas de Processamento de Algas‖. Os
principais objetivos deste trabalho foram: i) apresentar uma revisão do
estado da arte da biotecnologia de microalgas com foco na ciência e
tecnologia de alimentos (Capítulos 1 e 2); ii) estudar a viabilidade do
uso do concentrado de dessalinização (CD) como potencial substrato
para o cultivo de microalgas (Capítulos 3, 4 e 5); iii) determinar a
composição química de seis microalgas (C. vulgaris, S. platensis, N.
gaditana, N. oculata, P. tricornutum e P. cruentum) para aplicação na
indústria de alimentos (Capítulos 6 e 7); e iv) estudar a aplicação de
plasma a frio na biomassa de N. gaditana, e o efeito do plasma na
composição lipídica/ácidos graxos (Capítulo 8). O concentrado de
dessalinização (CD) é rico em minerais inorgânicos, tais como Cl-, Na+
e Ca2+, contém outros nutrientes (N e P) e microelementos necessários
para o crescimento de microalgas, incluindo Si, K+, Mg+2 e Fe+3. As
estirpes de microalgas (C. vulgaris, S. platensis e N. gaditana) são
capazes de crescer em CD, mas requerem diferentes concentrações. Por
exemplo, os resultados indicaram que o CD representa um bom meio de
cultura para a espécie marinha N. gaditana, pois é capaz de tolerar alta
concentração (~75% CD), com uma concentração de biomassa de 0,96 g
L-1 e teor de lipídios de 12,6%. Em geral, quando N. gaditana é exposta
à alta concentração de CD, que é uma condição de ‗stress‘, a alga
diminui a produção de proteína (de 41,6% para 27,0%) e aumenta a
síntese de lipídeos intracelulares (de 5,0% a 12,6%) e ácidos graxos
saturados (de 39,0% a 57,0%), especialmente o ácido palmítico (C16:0,
de 29,4% para 48,7%), sugerindo que a produção de lipídeos em N.
gaditana, funciona melhor quando exposta uma salinidade subótima.
Com relação ao efeito das condições tróficas, isto é, cultivo autotrófico,
mixotrófico e heterotrófico e alteração nos ciclos de luz:escuro (L/D),
24L:00D, 16L:08D, 12L:12D e 08L:16D no cultivo de N. gaditana para
a produção de biomassa e lipídeos, foi observado que para a máxima
produtividade lipídica (PL = 15,9 mg L-1 dia-1) em condições
autotróficas, N. gaditana requer um período de luz mais longo, i.e., um
ciclo 16L:08D. O cultivo mixotrófico, por sua vez, requer um período
de luz mais curto, i.e., um ciclo de 08L:16D, para atingir a máxima PL
(15,3 mg L-1 dia-1). Além disso, verificou-se que a alteração no
fotoperíodo teve um impacto crucial na produtividade de N. gaditana,
que está diretamente correlacionado com a produtividade lipídica,
indicando que o ajuste do fotoperíodo é um fator importante no cultivo
de algas. Em relação à composição química de seis microalgas
estudadas (C. vulgaris, S. platensis, N. gaditana, N. oculata, P.
tricornutum e P. cruentum), a biomassa dessas espécies contém em
média 40 g de proteína, 18 g de carboidratos, 12 g de fibra e 10 g de
lipídeos por 100 g de biomassa seca. As espécies C. vulgaris e S.
platensis são ricas em ácido α-linolênico (2,8 g/100 g) e ɣ-linolênico
(1,9 g/100 g), respectivamente. As algas marinhas P. tricornutum e N.
oculata contêm 42% e 37% de ácidos graxos poli-insaturados e são ricas
em ácidos eicosapentaenóico e docosaexaenoico. A alga P. cruentum
contém elevado teor de ácido araquidônico (3,7 g/100 g). Os resultados
indicaram que a Spirulina é excelente fonte de proteína, a Chlorella tem
alto teor de carboidratos e baixa teor de fibra, e as espécies marinhas N.
oculata e P. tricornutum contêm alto teor de ômega-3. Quando o plasma
a frio foi aplicado na biomassa de N. gaditana, foi observado que 10
min. de plasma a frio promove uma ruptura da parede celular algal,
permitindo uma maior eficiência da extração de lipídeos (18,5%) em
relação ao tratamento controle (9,5%). Os lipídeos das células originais
são ricos em ácidos graxos poli-insaturados (31,0%), contendo
principalmente o ácido eicosapentaenóico (C20:5ω3), enquanto que as
células rompidas foram predominantemente constituídas por ácidos
graxos saturados, principalmente o ácido palmítico (C16:0), o que pode
estar associado ao desencadeando de uma possível hidrólise e ou
oxidação das ligações duplas dos ácidos graxos insaturados. Esta nova
técnica de ruptura da parede celular de microalgas ainda requer estudos
mais aprofundados.

Palavras-chave: Microalgas; concentrado de dessalinização; biomassa;

composição química; plasma a frio; ruptura da parede celular;
aplicações em alimentos e/ou biodiesel.
 ABSTRACT

Over the last 10 years there has been substantially renewed interest in
the potential of microalgae to produce biomass, biofuels and
bioproducts. The potential interesting in microalgae biotechnology is
based on the fact that microalgae are among the fastest growing
organism and can potentially double their cell numbers in less than a
day. In addition, many algal species facultatively store energy reserves
as hydrocarbons, which have twice the energy density of carbohydrates
and proteins. The conversion of algal biomass into bioproducts include
high-value co-products and animal feed, their ability to capture CO2,
recycle nutrient-rich wastewaters. This thesis is an original research
work that is addressed to following title ―Algal Cultivation,
Characterization and Processing Techniques‖. The main aims of this
work were: i) to review the current state-of-the-art of microalgae
biotechnology with a focus on food science and technology (Chapters 1
and 2); ii) to evaluate the feasibility of using desalination concentrate
(DC) as a potential substrate for microalgae cultivation (Chapters 3, 4
and 5); iii) to determine the chemical composition (moisture, ash, fiber,
carbohydrate, protein, lipid and fatty acid composition) of six
microalgae (C. vulgaris, S. platensis, N. gaditana, N. oculata, P.
tricornutum and P. cruentum) biomass for food application (Chapters 6
and 7); and iv) to study the effect of non-thermal plasma (NTP) on N.
gaditana biomass as well as to determine the effect of NTP on lipid and
fatty acid compositions (Chapter 8). Desalination concentrate is rich in
inorganic minerals, such as Cl-, Na+ and Ca2+, contains other nutrients
(N and P) and trace elements necessary for microalgae growth,
including Si, K+, Mg+2, Fe+3. The microalgae strains (C. vulgaris, S.
platensis and N. gaditana) are able to grow in DC, but require different
optimum DC concentrations. The results suggest that DC represents a
good medium for marine N. gaditana, because it has the ability to
tolerate high extent of DC concentration (~75% DC concentration), with
a biomass concentration of 0.96 g L-1 and lipid content of 12.6%. In
general, when N. gaditana is exposed to optimum DC concentration,
which is a ―stress‖ condition, the alga decrease the production of protein
(from 41.6% to 27.0%) and increase the synthesis of intracellular lipids
(from 5.0% to 12.6%) and saturated fatty acids (from 39.0% to 57.0%),
especially of palmitic acid (C16:0; from 29.4% to 48.7%), suggesting
that marine N. gaditana production for lipids work well when exposed
to suboptimal salinity.
Considering the effect of trophic conditions (autotrophic, mixotrophic
and heterotrophic) and light:dark (L/D) cycles, 24L:00D, 16L:08D,
12L:12D, and 08L:16D in N. gaditana biomass/lipid productivities, it
was shown that under autotrophic conditions, N. gaditana requires a
longer light period, i.e. a 16L:08D cycle for a high lipid productivity
(15.9 mg L-1 day-1). With a shorter light period (08L:16D cycle), high
lipid productivity (15.3 mg L-1 day-1) requires mixotrophic cultivation.
In addition, the light/dark cycle was found to have a crucial impact on
biomass prod. of N. gaditana, which is directly correlated with lipid
prod., indicating that the illumination photoperiod is an important factor
that need to be taken into consideration. With regard to the chemical
composition of six microalgae (C. vulgaris, S. platensis, N. gaditana, N.
oculata, P. tricornutum and P. cruentum), algal biomass from these
species contain on average 40 g protein, 18 g carbohydrate, 12 g fiber
and 10 g lipid per 100 g of biomass dry mass. The species C. vulgaris
and S. platensis are rich in ALA (2.8 g/100 g) and GLA (1.9 g/100g),
respectively. The marine algae P. tricornutum and N. oculata contain
42% and 37% PUFA (EPA and DHA) respectively, with a favorable
ɷ3/ɷ6 ratio of around 6.5. The alga P. cruentum contains high
concentration of AA (3.7 g/100 g). Taken together, the results show that
microalgae are excellent candidates as sources of high protein
(Spirulina), high carbohydrate/low fiber (Chlorella) and high LC-
PUFA-ɷ3 (N. oculata and P. tricornutum) contents with a high
nutritional value, similar to sardine fish oil. When non-thermal plasma
(NTP) was applied on N. gaditana biomass, it shows that 10 min. of
NTP-assisted promotes a disruption of rigid cell wall, with greater lipid
recovery 18.5% than control treatment (9.5%, without any
pretreatment). Lipids from unruptured cells were rich in polyunsaturated
fatty acids (31.0%), mainly composed by eicosapentaenoic acid (EPA,
C20:5ɷ-3) while the ruptured microalgal cells were predominantly
composed in saturated fatty acids, mainly palmitic acid (C16:0). This is
may be associated to the high-voltage from NTP and possible hydrolysis
of the double bonds of the unsaturated fatty acids into simple bonds.
This new approach towards microalgal cell disruption prior to lipid
recovery is still in its infancy and requires further, in-depth studies for
biodiesel production.

Keywords: Microalgae; desalination concentrate; biomass productivity;

chemical composition; non-thermal plasma; cell wall disruption; food,
feed, pharmaceutical and biofuel applications.
 LIST OF FIGURES

Fig. 1-1 - Work plan flowchart.............................................................. 23

Fig. 1-2 Microalgae production flow sheet ........................................... 29
Fig. 1-3 Examples of cultures of microalgae in different
photobioreactors systems. ..................................................................... 30
Fig. 1-4 Example of algal cultivation in open raceway ponds. ............. 31
Fig. 1-5 Overview of the community of Uruçu located in São João do
Cariri, Paraíba State, Brazil.. ................................................................. 36
Fig. 1-6 Fluxogram of Spirulina platensis production in an algal-farm
facility located in São João do Cariri – Paraíba, Brazil. ........................ 36
Fig. 1-7 Plasma constitutes.................................................................... 39
Fig. 2-1 - Products synthesized by microalgae and potential area of
application. ............................................................................................ 47
Fig. 2-2 - Chemical structures of the most commonly polyunsaturated
fatty acids in microalgae. ...................................................................... 56
Fig. 2-3 - Chemical structures of some microalgal pigments. ............... 61
Fig.4-1 - Flow diagram for the cultivation of marine N. gaditana in
desalination concentrate (DC) and subsequent studies using the DC
percentage selected................................................................................ 94
Fig. 4-2 - Scheme showing the procedure to test the potential for the
reuse of the water medium with a DC concentration of 75% in
successive N. gaditana cultivation cycles. ............................................ 97
Fig. 4-3 - Effect of concentration of desalination concentrate (DC) on
biomass concentration (g L-1) and lipid content (%). .......................... 100
Fig. 4-4 - Images of N. gaditana cultured in medium with optimum DC
concentration of 75%. ......................................................................... 102
Fig. 4-5 - Results for N. gaditana cultured in medium with the optimum
DC concentration (75%) applied in three cultivation cycles. .............. 105
Fig. 5-1 – N. gaditana growth under different trophic conditions and
light regimes of 24L:00D, 16L:08D, 12L:12D and 08L:16D. ............ 122
Fig. 5-2 - Summary scheme showing how to enhance lipid productivity
(LP, mg L-1 day-1) in marine N. gaditana through the manipulation of
growth parameters. .............................................................................. 125
Fig. 5-3 - Principal component analysis (PCA) of the biomass, protein,
lipid and main fatty acids composition under variable trophic and light
photoperiod conditions, based on a culture medium with optimal
desalination concentrate (DC) of 75%. ............................................... 129
Fig. 7-1 - Chart showing fatty acids composition from six microalgae
biomass ................................................................................................ 154
Fig. 7-2 - Principal component analysis (PCA) projection and
distribution of the six microalgae species and their major fatty acid
component. .......................................................................................... 155
Fig. 8-1 - Schematic diagram and a photograph of the non-thermal
plasma (NTP) reactor. ......................................................................... 161
Fig. 8-2 - Flow diagram for the usage of sonication and NTP technology
as a tool for microalgal lipid extraction. ............................................. 165
Fig. 8-3 - Comparison of lipid content of N. gaditana biomass after
different time of application of NTP-assisted and sonication treatment.
............................................................................................................ 166
Fig. 8-4 - Scanning electron micrographs (SEM) of N. gaditana cells
that were exposed to non-thermal plasma ........................................... 170
Fig. 8-5 - Laser scanning confocal micrographs (LSCM) of N. gaditana
cells. .................................................................................................... 171
 LIST OF TABLES

Table 1-1 - Main microalgae species tested for medium- to large-scale

biomass production. .............................................................................. 28
Table 1-2 - Concentrations of N and P as well as their molar ratios in
different types of wastewaters. .............................................................. 34
Table 1-3 - Chemical compositions of different microalgae species. ... 37
Table 1-4 - Particularities of fatty acids from microalgae. .................... 38
Table 1-5 - Non-thermal plasma application in diverse segments. ....... 41
Table 2-1 - Microalgal fatty acids useful in food application. .............. 57
Table 2-2 - Microalgal pigments and potential fields of application..... 62
Table 2-3 - Recommended dosages of pigments useful for human
consumption mainly due to antioxidant activity. .................................. 59
Table 2-4 - Chemical products synthesized by engineered cyanobacteria.
............................................................................................................... 70
Table 2-5 - Safety aspect of relevant microalgae for food application.. 74
Table 2-6 - Some quality standards for microalgae............................... 77
Table 3-1 - Typical compositions of groundwater and desalination
concentrate (DC) obtained from the Community of Uruçu (semiarid
region of Paraíba state, Brazil). ............................................................. 84
Table 3-2 - Optimal physicochemical parameters of culture media based
on desalination concentration for three different algae species. ............ 85
Table 3-3 - Biomass concentration and lipid productivity of microalgae
grown in different proportion of desalination concentrate (DC). .......... 88
Table 4-1 - Compositions of desalination concentrate (DC) and F/2
medium. ................................................................................................. 95
Table 4-2 - Biomass and lipid productivities of N. gaditana cultured in
medium with optimum DC concentration reused in successive cycles.
............................................................................................................. 103
Table 4-3 - Comparison of biomass and lipid productivities of N.
gaditana cultured in medium with optimum DC concentration, with
additional carbon source in different concentrations with 7 days of
mixotrophic cultivation. ...................................................................... 106
Table 4-4 - Main fatty acids found in N. gaditana under different
experimental conditions. ..................................................................... 109
Table 5-1 - Effect of desalination concentrate (DC) concentrations on
biomass productivity (BP) and lipid productivity (LP) of marine N.
gaditana............................................................................................... 120
Table 5-2 - Growth parameters of N. gaditana cultured with optimal DC
concentration of 75% under different experimental conditions. ......... 123
Table 5-3 - Protein and lipid productivities of N. gaditana cultured with
optimal DC concentration of 75% under different experimental
conditions. ........................................................................................... 126
Table 5-4 - Fatty acids composition (% of total fatty acid content) of N.
gaditana cultured with optimal DC concentration of 75% under different
experimental conditions. ..................................................................... 130
Table 6-1 - Chemical composition of six microalgal biomass (%). .... 141
Table 6-2 - Fatty acids composition of biomass of six microalgae
(mg/100g)............................................................................................ 145
Table 6-3 - Nutritional quality indexes of the lipid fraction in the
biomass of six microalgae. .................................................................. 149
Table 7-1 - Lipid content (%), (saturated + monounsaturated
/polyunsaturated fatty acids) and ɷ-3/ɷ-6 ratios from six microalgae
species ................................................................................................. 154
Table 8-1 - Main fatty acids methyl esters (FAMEs) presented in N.
gaditana biomass (%). ........................................................................ 168
Table 8-2 - Comparison of physical properties of biodiesel produced
from N. gaditana with vehicular biodiesel standards (ASTM D6751 and
EN 14214), and plant based biodiesel (Jatropha oil methyl esters (JME)
and palm oil methyl esters (PME)). .................................................... 173
 LIST OF ABBREVIATIONS

AA/ARA – Arachidonic Acid

AI – Atherogenicity Index
ALA – -Linolenic Acid
AOAC – Association of Official Analytical Chemists
ANVISA – Agência Nacional de Vigilância Sanitária
BBM – Bold Basal Medium
DM – Dry Matter
DW – Dry Weight
DHA – Docosahexaenoic Acid
EPS - Extracellular Polysaccharides Sulphated
EPA – Eicosapentaenoic Acid
FAME – Fatty Acid Methyl Ester
FDA – Food and Drug Administration
FAO – Food and Agriculture Organization
GC – Gas Chromatography
GLA – -Linolenic Acid
GRAS – Generally Recognized as Safe
H/H – Hypocholesterolemic/Hypercholesterolemic ratio
IQN – Nutritional Quality Indexes
IAL – Instituto Adolfo Lutz
LABCAL – Laboratório de Análises Químicas – Ciência e Tecnologia
de Alimentos
LC-PUFA – Long Chain Polyunsaturated Fatty Acid
LDL – Low-Density Lipoproteins
MUFA – Monounsaturated Fatty Acid
NPN – Non-Protein Nitrogen
NTP – Non-thermal Plasma
PSM – Paoleti Synthetic Medium
P/S – Polyunsaturated and Saturated Fatty Acids ratios
PUFAs – Polyunsaturated Fatty Acids
RO – Reverse Osmosis
ROS – Reactive Oxygen Species
SCO – Single Cell Oil
SFA – Saturated Fatty Acid
SCP – Single Cell Protein
TAGs – Triglycerides
TI – Thrombogenicity Index
WHO – World Health Organization
 SUMMARY

1 INTRODUCTION ......................................................................... 21

2 STRATEGY AND AIMS OF THE STUDY ................................ 23

3 STRUCTURE OF THIS THESIS ................................................ 25

CHAPTER 1 ........................................................................................ 27

1. LITERATURE REVIEW ............................................................. 27

1.1. Microalgae Technology .............................................................. 27

1.2. General Growth Conditions Affecting Lipid Metabolism .......... 31
1.2.1. Temperature ............................................................................... 31
1.2.2. Light............................................................................................ 32
1.2.3. Salt concentrations ..................................................................... 32
1.2.4. pH ............................................................................................... 33
1.2.5. Nutrients ..................................................................................... 33
1.3. Wastewater as a Source of Nutrients for Microalgae Biomass
Production ............................................................................................. 34
1.3.1. Desalination concentrate in Brazil ............................................. 35
1.4. Bioproducts from Microalgae in Food Science .......................... 36
1.5. Application of Non-Thermal Plasma Technique for Algal
Bioprocesses.......................................................................................... 39

CHAPTER 2 ........................................................................................ 43
THE IMPACT OF MICROALGAE IN FOOD SCIENCE AND
TECHNOLOGY ............................................................................... 43

CHAPTER 3 ........................................................................................ 81
THE USE OF DESALINATION CONCENTRATE AS A
POTENTIAL SUBSTRATE FOR MICROALGAE
CULTIVATION IN BRAZIL .......................................................... 81
CHAPTER 4 ........................................................................................ 91
BIOMASS, LIPID PRODUCTIVITIES AND FATTY ACIDS
COMPOSITION OF MARINE Nannochloropsis gaditana
CULTURED IN DESALINATION CONCENTRATE ................ 91

CHAPTER 5 ...................................................................................... 111

EFFECTS OF DIFFERENT PHOTOPERIOD AND TROPHIC
CONDITIONS ON BIOMASS, PROTEIN AND LIPID
PRODUCTION BY THE MARINE ALGA Nannochloropsis
gaditana AT OPTIMAL CONCENTRATION OF
DESALINATION CONCENTRATE ........................................... 111

CHAPTER 6 ...................................................................................... 133

CHEMICAL CHARACTERIZATION OF SIX MICROALGAE
WITH POTENTIAL UTILITY FOR FOOD APPLICATION.. 133

CHAPTER 7 ...................................................................................... 151

ESSENTIAL FATTY ACIDS FROM MICROALGAE ................ 151

CHAPTER 8 ...................................................................................... 157

NON-THERMAL PLASMA ASSISTED EXTRACTION OF
INTRACELLULAR LIPIDS FROM Nannochloropsis gaditana
FOR BIODIESEL PRODUCTION .............................................. 157

4 GENERAL DISCUSSION AND CONCLUSIONS ..................... 177

REFERENCES.................................................................................. 179

APPENDIX ........................................................................................ 205

ANNEX .............................................................................................. 209

 21

1 INTRODUCTION

Brazilian country has been occupied a privileged position in terms

of using biomasses for agricultural purposes, due to its solar intensive
radiation during all the year, abundant water, climatic diversity, and
pioneering in the production of large-scale biofuels, especially from
sugar cane, the ethanol industry. In this sense, photosynthetic
microorganism such as microalgae, emerge as a potential feedstock and
renewable source for a wide range of applications, such as biomass,
bioproducts and biofuels (Carioca 2010).
Microalgae cultivation has an advancing role in solving some of
the future limitations of traditional biomass production and markets (i.e.,
food, feed, energy, emission mitigation, chemicals, biological material,
etc.). In addition, microalgae are considered to be one of the most
promising feedstock materials for developing a sustainable supply of
commodities, including food and non-food products (Batista et al.
2013). They also produce natural compounds that could be used as
functional food ingredients to enhance the nutritional value of foods
(Borowitzka 2013).
A key challenge in mass culturing of microalgae is to find strains
that not only produce marketable products or biomass for energy and
alternative agriculture, but also grow well under industrially relevant
outdoor conditions. These may include the need for growth under i) high
(or low) temperatures due to local climatic conditions or bubbling of hot
flue gases, ii) wide temperature variations over a diurnal cycle typically
seen in desert conditions, iii) high light and UV radiation when using
solar radiation to drive photoautotrophic growth, iv) high CO2 while
bubbling flue gases or limiting CO2 while growing with ambient CO2 if
no source of CO2 supplementation is available, iv) local water
conditions such as high salt content (e.g., seawater), alkaline or acidic
pH and metal and organic carbon content originating either from local
water bodies or from industrial wastewater that needs to be used for
algal growth (Varshney et al. 2015).
Several microalgae species can effectively grow in wastewater
conditions through their ability to utilize abundant organic carbon and
inorganic N and P. A wide range of studies have analyzed the growth of
microalgae under a variety of wastewater conditions, in particular
growth in multiple municipal, industrial and agricultural wastewaters
(Pittman et al. 2011). In Brazil, desalination concentrate (DC) as a
source of water and nutrients is of special interest for utilizing and
recycling inland desalination wastewater, which is loaded with
22

microelements useful for algal biomass production (Sánchez et al.

2015). Experimental studies on the use of DC as an alternative medium
for microalgae, particularly studies that also assess variables that
determine maximal algal biomass production and biochemical
composition, will have significant benefits for the evaluation of
wastewater-grown microalgae in Brazil.
Microalgae have been identified by many researchers as a
promising feedstock for biofuel production (Chisti 2007; Moheimani et
al. 2015). In fact, several species of microalgae are able to accumulate
carbohydrates and lipids which can be chemically converted into
biofuel. Among them, the ones belonging to the Eustigmatophyceae
genus is Nannochloropsis gaditana species which is particularly
interesting because of its high growth rate and lipid productivity as well
as the ability to synthesize high value chemicals such as fatty acids with
medium (C16 and C18) and long (C20:5ɷ3) chain carbons, which are ideal
lipid sources for the production of the nutraceutically valuable
eicosapentaenoic acid (EPA) and biodiesel (Mitra et al. 2015).
Production and extraction of intracellular metabolites from
microalgae involve extensive processing steps starting with microalgal
cultivation, microalgal biomass recovery or harvesting through
separation of the biomass from the carrier media, followed by further
processing such as dewatering, drying, cell disruption, extraction and
product purification (Show et al. 2015). The complete disruption of the
microalgal cell wall prior to product recovery has been shown to be
critical for successful process-scale lipid extraction from wet microalgal
biomass. In this context, non-thermal plasma has been proposed as a
potential method to disrupt the algal cell wall.
Non-thermal plasma has been also investigated in the area of
biofuels, such as bioethanol from cellulose (Benoit et al. 2012) and
biodiesel from waste cook oil (Cubas et al. 2015). As a result, it could
be an excellent pretreatment to disrupt algal cell wall, and can
eventually convert the algal-oil into biodiesel. There have no studies
during the last 10 years in the literature on the use and effect of non-
thermal plasma on algal biomass application, given an opportunity and
potential area of research.
 23

2 STRATEGY AND AIMS OF THE STUDY

This thesis is an investigation of algal cultivation, biomass

characterization and processing techniques. In order to guide this study,
the flowchart below (Fig. 1.1) was designed based on the title of this
thesis.

Fig. 1-1 - Work plan flowchart.

Algal Cultivation Characterization Processing Tecniques

Related Project: Related Project: Related Project:

Desalination concentrate Biomass composition Non-thermal plasma
Algal physiology Algal chemistry Algal Bioprocess

Highlights: Highlights: Highlights:

Salinity/Conductivity Protein/Carbohydrates High-electric-voltage
N:P ratio Lipid/Fatty acids Disrupt algal cell wall
Lipid enhanced ω-3/ω-6 ratio Electroporation

Based on the flowchart, the main objectives of this work were:

(1) Desalination concentrate project: To maximize the biomass and

lipid productivities of marine microalgae N. gaditana cultured in
desalination concentrate which affect algae through manipulation
of physiologic parameters such as salinity, conductivity, light:dark
cycles and different photoperiods of the culture medium.
(2) Biomass composition project: To determine the chemical
composition and nutritional quality indexes of six microalgae for
food application.
(3) Non-thermal project: To study the effect of non-thermal plasma on
algal biomass, lipids and fatty acids composition.

In order to achieve these objectives, specific objectives and some

hypotheses are proposed:
24

(1) Desalination concentrate project:

 To determine the best desalination concentrate (DC) dilution for N.
gaditana growth;
 To evaluate the effect of DC concentration on biomass and lipid
content of N. gaditana;
 To evaluate the effect of the medium reuse on biomass and lipid
productivities of the N. gaditana;
 To manipulate the growth parameters based on DC medium for
maximum lipid production of N. gaditana;
 To study the effect of trophic conditions and photoperiod regimes on
N. gaditana growth and biomass;
 To determine fatty acids composition of N. gaditana;

(2) Biomass composition project:

 To characterize the biochemical composition of six microalgae
(Chlorella vulgaris, Spirulina platensis, Nannochloropsis gaditana,
Nannochloropsis oculata, Phaeodactylum tricornutum and
Porphyridium cruentum);
 To determine the biomass composition in terms of moisture, ash,
fiber, carbohydrate, protein, lipid and fatty acids composition of the
six algal biomass;
 To evaluate the nutritional quality indexes in terms of
polyunsaturated/saturated fatty acids (P/S), ω-3/ω-6 ratio,
hypocholesterolemic fatty acids/hypercholesterolemic fatty acids
(H/H), atherogenicity index (AI) and thrombogenicity index (TI)
based on their fatty acid compositions.

(3) Non-thermal plasma project:

 To apply the non-thermal plasma (NTP) on N. gaditana biomass;
 To observe the algal biomass after NTP-assisted through microscope
techniques (scanning electron microscopy and laser scanning
confocal microscopy);
 To determine the lipid content as well as the fatty acid composition
of the N. gaditana after NTP application;
 To compare the NTP and sonication as pretreatment methods.
 25

Hypotheses: Since NTP is an innovative method and novel technique

applied on algal biomass processing, and has no similar work in the
literature, three hypotheses are proposed:

 NTP may disrupt the algal cell wall and release the intracellular
target molecules, such as lipids in the aqueous phase;
 NTP may be compared to traditional method (sonication) as a
potential pretreatment method;
 NTP-assisted promotes higher lipid recovery than without any
pretreatment.

3 STRUCTURE OF THIS THESIS

This original research work on microalgae science brings new
insights on algal cultivation, characterization and processing techniques.
This thesis begins (Chapter 1) with an introduction based on literature
review of microalgae biotechnology. Chapter 2 reviews the main
existing and potential high-value products which can be derived from
microalgae and considers their commercial development with a
particular focus in food science and technology. It also describes the
gross and fine chemical composition of various algal species and details
the nutritive importance of selected constituents. This chapter was
published in Journal of the American Oil Chemists’ Society (volume 94,
issue 11, pages 1333-1350, October 2017). Chapter 3 reviews the
current state-of-art of combining desalination concentrate and algal
cultivation in Brazil. This chapter was published in Algal Research
(volume 24, Part B, pages 505-508, June 2017).
One of the key attractions of microalgae is the high lipid content
and fatty acid composition through the manipulation of the metabolism
which are covered by Chapter 4 and Chapter 5. These chapters were
published in Bioresource Technology (Chapter 4 - volume 197, pages
48-55, December 2015 and Chapter 5 - volume 224, pages 490-497,
January 2017), and have shown the manipulation of marine algae N.
gaditana cultured in desalination concentrate in order to maximize lipid
content and productivity.
Chapter 6 was developed in collaboration with Dr. Rafael Feller
from the Department of Chemical Engineering (UFSC) and has the main
aim to evaluate the biochemical composition and nutritional quality
indexes of six microalgae (Chlorella vulgaris, Spirulina platensis,
Nannochloropsis gaditana, Nannochloropsis oculata, Phaeodactylum
tricornutum and Porphyridium cruentum) for food application. This
26

chapter was published as an original research paper in Journal of the

American Oil Chemists’ Society (volume 93, pages 963-972, issue 27,
May 2016). In complement with previous work, Chapter 7 describes
additional analysis and correlation through principal component analysis
of the six microalgae studied. These results were published in Inform
(Champaign): International News on Fats, Oils and Materials (AOCS
Magazine, volume 27, pages 22-26, November-December 2016).
The complete disruption of the microalgal cell wall prior to product
recovery has been shown to be critical for successful process-scale
based on lipid extraction from wet microalgal biomass. As such, the
effect of non-thermal plasma (NTP) on algal biomass and lipid recovery
has been discussed and covered in Chapter 8. This chapter was
submitted for publication in Energy Conversion and Management
(Elsevier).
General discussions and future directions are described in the final
of the document (see Topic 4), in order to sum up the research work.
Suggestions for further work are also presented.
 27

CHAPTER 1

1. LITERATURE REVIEW

1.1. Microalgae Technology

There has recently been extensive research focus on biology,

physiology, engineering and their integration for microalgae cultivation
to produce sustainable products such as biofuels, food, feed, and high-
value products. Algae belong to many different and unrelated taxonomic
groups that all contain chlorophyll a and are able to utilize solar energy
to fix CO2 to produce organic compounds (Moheimani et al. 2015).
More than a dozen algal species have been mentioned in the
literature as potential candidates for large-scale production. However,
conclusive information obtained through commercial trials is not yet
available to assess suitability of most of these species. The ideal
microalga must be able to grow very well even under high biomass
concentration and varying environmental conditions. It must be able to
produce high concentration of products of interest (i.e. high-value
products, lipids and hydrocarbons) (Borowitzka 2013). However, it is
unknown how many species of algae exist, with estimates ranging
between several hundred thousand and several million different species
– with new types identified all of the time (Lourenço 2006; Moheimani
et al. 2015). Only a small portion of microalgal species can be kept alive
in culture, and only a handful of them have been successfully grown
commercially (Mata et al. 2010). Table 1.1 summarizes the main
microalgae species tested for medium- to large-scale production
(especially for feed, high-value products and biofuel).
Commercial large-scale production of microalgae for bioproducts
began in 1960s and 1970s with Chlorella and Spirulina and followed in
the 1980s with production of β-carotene from Dunaliella salina
(Varshney et al. 2015). All three species were successfully grown in
mixed or unmixed open ponds (Craggs et al. 2013). The ability to grow
at high selective environments is the main reason for the successful
growth of these species (Spirulina = high pH and high HCO3-, D. salina
= high salinity and Chlorella = high nutrients) (Borowitzka &
Borowitzka 1990; Lourenço 2006; Henrikson 2009). Moheimani and
Borowitzka (2006) also showed that Chrysotila cartarae reliable long-
term culture in raceway pond is successful due to the ability of this alga
to grow at very high pH.
28

In the late 1990s commercial production of the freshwater green

alga Haematococcus pluvialis as a source of the carotenoid astaxanthin
started at Cyanotech in Hawaii (Moheimani et al. 2015). The culture
system is a combination of ‗closed‘ tower reactors and raceway ponds.
Haematococcus production by several other small producers
commenced in Hawaii in subsequent years using closed culture systems
(e.g. Olaizola 2000). The astaxanthin from Haematococcus is mainly
sold as a nutraceutical, antioxidant and as a colouring agent in the
farming of salmonids (Haque et al. 2016; Panis & Carreon 2016).
Other species that do not have this selective advantage may need to
be grown in closed photobioreactors. The selection of growth
technologies or production systems for microalgae will need to be based
to a large extent on the microalga of choice and cultivation systems
(Moheimani et al. 2015).

Table 1-1 - Main microalgae species tested for medium- to large-scale

biomass production.
Chlorophyceae Neochloris oleoabundans; Scenedesmus
dimorphus; Botryococcus braunii; Dunaliella
tertiolecta; Nannochloris sp.; Chlorella
vulgaris
Euglenophyceae Euglena gracilis
Prasinophyceae Tetraselmis spp. (i.e. T. chuii and T. suecica)
Haptophyceae Chrysotila carotene; Isochrysis galbana
Eustigmatophyceae Nannochloropsis spp. (e.g. N. salina, N.
oculata, N. gaditana)
Bacillariophyceae Cyclotella cryptica; Chaetoceros sp.;
(diatoms) Skeletonema sp.
Cyanobacteria (blue- Arthrospira (Spirulina) platensis
green algae)
Source: (Moheimani et al. 2015)

A conventional microalgae production system consists of (A)

growth and cultivation of microalgae, (B) biomass harvesting and
dewatering and (C) extraction/conversion of the biomass to the product
of interest. It is to be noted that for this process to be energetically,
environmentally, and economically sustainable, it is critical to recycle
medium (water and fertilizers) in harvest and extraction stages of
production (Fig. 1.2).
 29

Fig. 1-2 Microalgae production flow sheet (modified and redrawn from
Moheimani et al. 2015)

Light/Water/Nutrients/CO2

(A) Grow
•Ponds
•Photobioreactor
•Hybrids
(B) Harvest
•Rate & Frequency
Recyle water De-watering
•Semi-dry
•Dry

(C) Extraction Oil Biodiesel

•Wet
•Dry Sugar Bio-ethanol

Remaining-biomass High-value products

Recyle water & nutrients Anaerobic digestion Pigments

(Bio-metane) High value oils
Animal Feed EPA, DHA, AA

There are two main types of microalgae cultivation systems: closed

photobioreactors (Fig. 1.3) and open ponds (Fig. 1.4).
Closed algal cultures (photobioreactors) are not exposed to the
atmosphere and covered with a transparent material or contained within
transparent tubing. Photobioreactors have the distinct advantage of
preventing evaporation. Culturing microalgae in these kinds of systems
have the added benefit of reducing the contaminants risks, limiting the
CO2 losses, creating reproducible cultivation conditions, ad flexibility in
technical design (Moheimani et al. 2015). Closed and semi-closed
photobioreactors are mainly used for producing high-value products
such as pigments, protein, and single cell oils (EPA + DHA + AA) for
human consumption (Borowitzka 2013). The main disadvantages of
closed systems are the high costs of construction, operation for pumping
and cooling (related to energy consumption) and maintenance such as
cleaning and sterilization (Kumar et al. 2015). However, if these
difficulties can be overcome, these controlled closed systems may allow
commercial mass production of an increased number of microalgal
species at a wider number of locations.
30

Fig. 1-3 Examples of cultures of microalgae in different

photobioreactors systems. A) Blue-green algae S. platensis cultivation in
inverted conical photobioreactor (working volume 3.5-L); B) Marine
algae N. gaditana culture in balloon reactor (working volume 5.0-L) and
C) Green algae C. vulgaris cultivation in controlled photobioreactor
system (working volume 5.5L). Cultures developed at Laboratory of
Food Biotechnology (UFSC).

A B C

Open ponds are the most usual setting for large-scale outdoor
microalgae cultivation. The major commercial production of algae is
today based on open channels (raceway) which are less expensive and
easier to build and operate compared with closed photobioreactors
(Moheimani et al. 2015). In addition, the growth of microalgae meets is
less challenging in open than closed cultivation systems; however, just a
few species of microalgae (e.g. Chlorella, D. salina, Spirulina sp.,
Chlorella sp. and P. carterae) have been successfully grown in open
ponds (Lourenço 2006; Borowitzka 2013). Profitable production of
microalgae, at present, are limited to a comparatively few small-scale
(<10 ha) plants producing high-value health foods, most located in
south-east Asia, Australia and the USA (Chisti 2007). Relatively low
cost of construction and operation are the main reasons for culturing
algae in open ponds. However, the high contamination risks and low
productivity, induced mainly by poor mixing regime and light
penetration, are the main disadvantages of open systems (Rogers et al.
2014).
 31

Fig. 1-4 Example of algal cultivation in open raceway ponds (5.0 m

length, 1.6 m width and 0.5 m depth, total area of 8 m2, and total
capacity of 4000 m3 each pond). Cultivation of C. vulgaris
supplemented with desalination concentrate. Ponds located in an algal-
farm facility in São João do Cariri – Paraíba state, Brazil.

SANT’ANNA

MATOS

1.2. General Growth Conditions Affecting Lipid Metabolism

1.2.1.Temperature

Light and temperature are probably most important and well-

studied factors influencing the lipid and fatty acid composition of algae.
Changes in the lipids of photosynthetic tissues (and other organisms) as
a response to different temperatures and/or light conditions have been
recently reviewed (Simionato et al. 2011). It is believed that many of the
lipid changes alter the physical properties of membranes which allows
their unimpaired functioning in important physiological processes
including photosynthesis, respiration and membrane transport (Guschina
& Harwood 2013).
In general, lower growth temperatures lead to increased levels of
unsaturated fatty acids in algae although the details of these alterations
may vary from species to species.
32

1.2.2. Light

Light intensity also influences algal lipid metabolism and,

therefore, lipid composition. In general, high light intensities usually
lead to oxidative damage of polyunsaturated fatty acids. In
Nannochloropsis sp., the degree of unsaturation of fatty acids decreased
with increasing irradiance, especially the percentage of total ω-3 fatty
acids (from 29 to 8% of total fatty acids) mainly due to a decrease of
eicosapentaenoic acid (Simionato et al. 2011). In general, light will
stimulate the algal biomass production. Moreover, stimulation of fatty
acid and membrane lipid, mainly chloroplast, synthesis are normally
expected as a result of an increase in light intensity.

1.2.3.Salt concentrations

Salinity is usually growth-limiting at the extremes of salt tolerance

in most microalgal species, and every microalga has an optimum salinity
range. The effect of salinity on microalgal growth relates to
osmoregulation, which in microalgae is achieved through diverse
strategies (Moheimani et al. 2015). Osmoregulatory metabolites are
organic substances produced by microalgae that, when the latter are
exposed to water stress conditions, respond appropriately to the changes
in extracellular water activity (Kumar et al. 2015). Microalgae main
osmoregulators (function as intracellular osmotic regulators) are as
follows: (a) polyhydric alcohols (i.e. glycerol, mannitol or sorbitol), (b)
variety of glycosides (i.e. galactosyl glycerides, floridoside and
isofloridoside) and (c) amino acids (i.e. glutamic acid and proline)
(Lawton et al. 2015).
Some algae exhibit an excellent ability to tolerate high salt
concentrations. The genus Dunaliella, growing in the wide range of
salinities, is a good example as well as an useful model in studying
mechanisms of such resistance. In these algal cells, a considerably
higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty
acids has been noted (Davis et al. 2015).
An increase in the initial salt concentration from 0.5 M NaCl to 1.0
M resulted in an increase (from 60 to 67%) of intracellular lipid content
in Dunaliella tertiolecta (Takagi et al. 2006). The further increase in
lipid content up to 70% has been achieved when 0.5 or 1.0 M NaCl were
added at mid-log phase or the end of log phase during cultivation with
the initial NaCl concentration of 1.0 M (Takagi et al. 2006).
 33

1.2.4.pH

Lipids are also affected during growth of algae at extreme pH.

Thus, it has been shown that alkaline pH stress increased the TAG
percentage accumulation and decreased the relative level of membrane
lipids in Chlorella sp. (Lourenço 2006). Fatty acids in the polar lipids
were more saturated in the unidentified Chlamydomonas sp. than those
in C. reinhardtii when grown under the same conditions. The TAG
content (as % of total lipids) was also higher in Chlamydomonas sp.
grown at pH 1.0 than that in the cells cultivated at higher pH>8.0. The
increase in saturation of fatty acids in membrane lipids of
Chlamydomonas was proposed to be an adaptive reaction to low pH in
order to decrease membrane lipid fluidity (Guschina & Harwood 2013;
Tatsuzawa et al. 1996).

1.2.5.Nutrients

Nutrient availability affects significantly the lipid composition of

algae, and a number of broad effects of nutrient limitation have been
reported as important modulators of algal lipid biosynthesis. It is
accepted, that when algal growth slows down as a result of nutrient
deficiency, and there is no requirement for the synthesis of new
membrane compounds, the cells can transfer fatty acids into their
storage lipids before conditions improve. There are many examples
showing that algal species deficient in nutrients can more than double
their lipid and TAG content (Isleten-Hosoglu et al. 2012).
Nutrient-deficiency has been shown to affect markedly the lipid
composition of microalgae under silicon, nitrogen, or phosphorus
limitation (Cheirsilp & Torpee 2012) An increase in TAG accumulation
and a decrease of polar lipids (as % of total lipids) was noticed in all of
the nutrient-limited cultures (Guschina & Harwood 2013). An increase
in TAG percentages (from 69 to 75% of total lipids) together with
phospholipids (from 6 to 8%) was reported for the microalga P.
tricornutum as a result of reduced nitrogen concentration (Alonso et al.
2000).
34

1.3. Wastewater as a Source of Nutrients for Microalgae Biomass

Production

Many industries and human activities generate wastewater, and as a

consequence, there are many different types of wastewaters, each with a
different chemical composition and volumetric production over time. In
this regard, microalgae play an important role in many wastewater
treatment facilities around the world.
Production of microalgal biomass requires large amounts of
nitrogen (N) and phosphorus (P) and consequently the sustainability and
economic viability of microalgae production could be significantly
improved if N and P are not supplied by synthetic fertilizers but with
wastewater. It is important to note that microalgae have low content of
structural carbohydrates such as lignin or cellulose, and as a
consequence microalgal biomass has a high content of nitrogen (N) and
phosphorus (P): about 10 % N and 1 % P per unit dry weight. Table 1.2
gives an overview of different types of wastewaters and their content of
N and P.

Table 1-2 - Concentrations of N and P as well as their molar ratios in

different types of wastewaters.
Wastewater N (mg L-1) P (mg L-1) N:P ratio
source (molar)
Domestic 20-85 5-20 12
Animal manure
Pigs 800-2300 50-320 14
Beef cattle 63 14 10
Dairy cattle 185 30 4
Poultry 800 50 32
Industrial
Coke production 757 1 3000
Tannery 273 21 29
Paper mill 11 1 40
Textile 90 18 11
Winery 110 52 5
Olive mill 530 182 3
Desalination 30 1 30
Source: Based on Muylaert et al. 2015
 35

1.3.1.Desalination concentrate in Brazil

Many arid zones of South America have aquifers that are naturally
saline or have been salinized due to irrigation. The northeast region of
Brazil, for example, which has a semiarid climate and frequent droughts,
is a vast region with brackish groundwater. This semiarid region of
Brazil is known as the ―Polígono das Secas‖. It is one of the largest
semiarid areas of the Americas, covering 969,589.4 km2 and a
population of 23.5 million. This part of Brazil is an exceptional example
of inland desalination and brine management, partially because of a
massive governmental program launched in 2004 named ―Água Doce‖
(Fresh Water) that has benefitted 500,000 inhabitants of the region
(Ministério do Meio Ambiente 2004, Sánchez et al. 2015).
To face the scarcity of good quality water in some parts of the
semiarid region of Brazil, brackish groundwater has been desalinized to
make it appropriate for use. Desalination through reverse osmosis is the
most attractive solution for water supply. However, this process
produces large amounts of desalination wastewater, with a high salt
concentration, and is associated with problems related to brine disposal
(Greenlee et al. 2009; Menezes et al. 2011). Several small reverse
osmosis plants were built to serve rural communities or to reinforce the
supply of water in already existing networks that link several medium
sized municipalities. Equipment for the desalination of water based on
reverse osmosis has been installed in the community of Uruçu, located
in São João do Cariri in Paraíba State. Currently, the local community
has access to potable water obtained from the brackish groundwater
through a small well via a tubular pump coupled with a reverse osmosis
plant. In general, the piped water has a flow intake of around 2-3 m3/h,
operating with an average recovery rate of 90%. As a result, a waste
stream of brine with a high ionic concentration is produced.
The desalination concentrate (DC) is basically comprised of
mineral salts which include Na+, Ca+2 and Mg+2. With a growing need
for the inland desalination of brackish water in Brazil, the suggested
green route for the reuse and valorization of this DC is via aquaculture,
the irrigation of halophyte plants, hydroponic crops and, algaculture
(Fig. 1.5) (Soares et al. 2006; Dias et al. 2010; Matos et al. 2015).
Furthermore, Sánchez et al. (2015) have been suggested an integrated
scheme using DC for agricultural purposes (Tilapia + Spirulina +
Atriplex) toward the production of food and feed with high protein
content for humans and livestock (Fig. 1.6).
36

Fig. 1-5 Overview of the community of Uruçu located in São João do

Cariri, Paraíba State, Brazil. A – Desalination equipment based on
reverse osmosis (average production of around 2-3 m3/h); B – Utilizing
desalination concentrate (DC) for fish cultivation (tilapia species); C –
Cultures of Chlorella vulgaris combined with DC in open raceway
ponds (working volume 3,500 L each pond); D – Hydroponic crops
(lettuce, chili, tomatoes) using DC as mineral nutrient.

Fig. 1-6 Fluxogram of Arthrospira (Spirulina) platensis production in an

algal-farm facility located in São João do Cariri – Paraíba, Brazil.

Spirulina Inoculum Raceway pond Harvesting by Drying Biomass

(100-L) (4000-L) filter

1.4. Bioproducts from Microalgae in Food Science

Microalgae are a group of eukaryotic organisms and photosynthetic

cyanobacteria able to accumulate sugars, carbohydrates, proteins, lipids
and other valuable organic substances by the efficient use of solar
energy, CO2, and nutrients. These microorganisms convert inorganic
 37

substances such as carbon, nitrogen, phosphorus, sulfur, iron, and trace

elements into organic matter (green, blue-green, red, brown, and other
colored biomass) (Batista et al. 2015). Table 1.3 shows the main
chemical compositions of different microalgae species.

Table 1-3 - Chemical compositions of different microalgae species.

Algal species Nucleic
Protein Carbohydrates Lipids
acid
Scenedesmus
50-56 10-17 12-14 3-6
Obliquus
Scenedesmus
47 - 1,9 -
quadricauda
Scenedesmus
8-18 21-52 16-40 -
dimorphus
Chlorella
51-58 12-17 14-22 4-5
vulgaris
Chlorella
57 26 2 -
pyrenoidosa
Spirogyra 6-20 33-64 11-21 -
Nannochloropsis
40 32 12 -
gaditana
Dunalliella
57 32 6 -
salina
Euglena gracilis 39-61 14-18 14-20 -
Prymnesium
28-45 25-33 22-38 1-2
parvum
Tetraselmis
52 15 3 -
maculata
Porphyridium
28-39 40-57 9-14 -
cruentum
Arthrospira
46-63 8-14 4-9 2-5
platensis
Arthrospira
60-71 13-16 6-7 3-4,5
maxima
Synechoccus sp. 63 15 11 5
Anabaena
43-56 25-30 4-7 -
cylindrica
Source: Lourenço 2006
38

Algal intracellular lipids as the target molecules have gained great

attention by several researcher experts, because of algae ability to
synthesize different classes of fatty acids, i.e. from medium- to long-
chain fatty acids. Algal lipids can be divided into two main groups: the
non-polar lipids (acylglycerols, sterols, free (non-esterified) fatty acids,
hydrocarbons, wax and steryl esters) and polar lipids
(phosphoglycerides, glycosylglycerides). They are essential constituents
of all living cells where they perform important functions (Guschina &
Harwood 2013).
Many algae accumulate substantial amounts of non-polar lipids,
mostly in the form of TAG or hydrocarbons, and these levels may reach
up to 20–50% of dry cell weight. These oleaginous algal species have
been considered as promising sources of oil for biofuels, such as
surrogates of gasoline, kerosene and diesel, being both renewable and
carbon neutral (Moheimani et al. 2015). In addition, algae can produce
valuable bio-products including carotenoids (β-carotene, astaxanthin,
canthaxanthin and lutein), other pigments (phycocyanin and
phycoerythrin), ω-3 fatty acids (eicosapentaenoic and docosahexaenoic
acids), vitamins (tocopherols, vitamin B12 and provitamin A),
polysaccharides and proteins (Draaisma et al. 2013). Table 1.4 shows
the fatty acids extracted from microalgae and potential application as
food for humans and feed for aquaculture.

Table 1-4 - Particularities of fatty acids from microalgae.

PUFA Molecule Application Specie producer
γ-linolenic acid Infant/food
C18:3 ω6 Arthrospira
(GLA) formulation
Arachidonic acid Infant/food
C20:4 ω6 Porphyridium
(ARA) formulation
Infant/food
Eicosapentaenoic Nannochloropsis,
C20:5 ω3 supplement
acid (EPA) Phaeodactylum
Aquaculture
Docoxahexaenoic Food supplement Crypthecodinium,
C22:6 ω3
acid (DHA) Aquaculture Schizochytrium
Sources: Spolaore et al. (2006); Draaisma et al. (2013)
 39

1.5. Application of Non-Thermal Plasma Technique for Algal

Bioprocesses

Non-thermal plasma (NTP) is a partially ionized gas that is created

by flowing gas through a relatively high electric field. This electric field
accelerates the few naturally occurring free electrons which then ionizes
the gas atoms and molecules creating more electrons and hence creating
an ‗electron avalanche‘, and if conditions are right, sustaining a plasma.
The energetic electrons will also undergo excitation and dissociative
collisions with the gas particles, with the final result being a mixture of
electrons, negative and positive ions, excited gas species, free radicals
and electromagnetic radiation (Fig. 1.7) (Alkawareek et al. 2014).
The use of NTP has been emerging as a potential alternative
approach into diverse applications, including industrial sterilization
(Alkawareek et al. 2014), pollution control (Kim 2004), polymer science
(Pandiyaraj et al. 2015), food safety (Ma et al. 2015), biomedicine
(Zhang et al. 2014) and nanoscience (Graham and Stalder 2011) (Table
1.5).

Fig. 1-7 Plasma constitutes according to Nehra et al. (2008).

Plasma

Positive ions Electrons Atons Photons

Negative ions Metaestability Free radicals

Among of the field application of NTP, it has been investigated in

the area of biofuels, such as bioethanol (Benoit et al. 2012) and
biodiesel (Cubas et al. 2015). In this regard, NTP based on corona
discharge technology was applied on oil (free fatty acids) from food
industry waste to convert into biodiesel (Cubas et al. 2015). According
to the same authors, the major advantage of applying NTP for biodiesel
purposes is that the methyl ester can be obtained in the absence of
chemical catalysts and without the formation of the co-product
(glycerin). In addition, NTP based on corona discharge offers
advantages on faster reaction time and easy separation of the final
product (Istadi et al. 2009).
40

Preliminary tests at Laboratory of Environmental Engineering

(Laboratório de Engenharia Ambiental - LEA) of the South University
of Santa Catarina (Universidade do Sul de Santa Catarina - UNISUL,
Palhoça, SC) have shown that the use of NTP in algal biomass promotes
rupture of the algal cell wall, with possible release of the intracellular
lipids into aqueous phase and consequently, greater lipid recovery is
obtained.
Further analysis and potential application of NTP for algal
bioprocess can be found in Chapter 8.
Table 1-5 - Non-thermal plasma application in diverse segments.
Type of plasma Application Highlights and advantages Reference
Discharge corona Biofuels (Biodiesel) Applied on residual fatty acids from soy- Cubas et al. (2015)
oil. No-glycerin is produced.
Plasma jet Biofuels (Bio-ethanol) Depolymerization of cellulose in low Benoit et al. (2012)
molecular weight.
Plasma jet Food science Antimicrobial. Inactivation of strawberries Ma et al. (2015)
contaminated with S. aureus. 10-20 min.
of application.
Dielectric barrier Food science Inactivation of Salmonella from egg Ragni et al. (2010)
surface.
Discharge corona Plant science Favoring the germination of rape seeds. Ling et al. (2015)
Dielectric barrier Biologic control Inactivation of Microcystis aeruginosa Li et al. (2015)
species which promotes algal blooms.
Plasma jet Medicine Restoration of teeth. Improve the Zhang et al. (2014)
penetration of resin adhesive.
Discharge corona Polymer/Biomedicine Improve the properties of the Pandiyaraj et al. (2015)
polypropylene-adhesive used in blood
coagulation.
Plasma jet Environmental control Control of air pollution. Sterilizer and air EPA (2005)
sanitizer.
Dielectric barrier Environmental control Degradation of pharmaceutical Magureanu et al. (2015)
compounds detected in water bodies.
41
 43

CHAPTER 2

THE IMPACT OF MICROALGAE IN FOOD SCIENCE AND

TECHNOLOGY

Abstract

Microalgae (including cyanobacteria) are promising organisms for

sustainable products for use as feedstocks for food, feed, fine chemicals
and biofuels. They are able to synthesize a broad range of products with
medium- to high-value market price such as β-1,3-glucan
polysaccharide, single-cell-protein, carotenoids and phycobilin pigments
and long-chain polyunsaturated fatty acids that are commercialized in
the food industry as dietary supplements and functional foods, in the
pharmaceutical and chemical industries as cosmaceuticals and
flavorants, and in the therapeutic field as nutraceutical compounds.
These microorganisms are also exceptional producers of omega-3 and
omega-6 fatty acids such as eicosapentaenoic, docosahexaenoic and
arachidonic acids that have been linked to several human health
benefits. This paper briefly reviews the main existing and potential high-
value products which can be derived from microalgae and considers
their commercial development with a particular focus in food science
and technology. It also describes the gross and fine chemical
composition of various algal species and details the nutritive importance
of selected constituents. Finally, nutritional quality standards and
legislative provisions to ensure food safety in the use of algal biomass
are presented.

Keywords: Microalgae; chemical composition; polyunsaturated fatty

acids; pigments; cosmaceuticals; nutraceuticals; functional foods, vegan
food; regulations; GRAS.
44

Useful definitions:

Nutraceutical: can be defined as a food or food product that reportedly

provides health and medical benefits, including the prevention and
treatment disease. Carotenoids and long-chain polyunsaturated fatty
acids are good examples.
Functional foods: can be broadly defined as a food that have either
enriched or fortified, a process called nitrification, which has been
satisfactorily demonstrated to improved state of health and well-being.
An example of microalgal ―functional food‖ are biscuits enriched with
ω-3 fatty acids by the inclusion of Isochrysis galbana biomass.
Vegan foods: can be ethical defined as avoidance of animal food
products and their derivates. In this context, food products from algae
are considering alternative food source.
Cosmaceuticals: are cosmetics products with bioactive ingredients
purported to have medical or drug-like benefits. For example,
phycobilin pigments, algae-derived carotenoids and extracts of algae
such as Spirulina, Chlorella and Dunaliella, Haematococcus.
Dietary supplement: is a product taken by mouth that contains a
―dietary ingredient‖ intended to supplement the diet. For examples, pills
of β-carotene (pro-vitamin A), softgels of EPA + DHA ω-3, and
capsules/tablets of single cell protein (SCP).
Single cell protein (SCP): refers to edible unicellular microorganisms
containing protein-derived from algae or yeast or fungi or bacteria
source.

Introduction

With predictions that the world population will have increased by

another 2 billion by 2050, a major challenge for the planet is to provide
enough food for its population. Current estimates indicate that sufficient
water and arable land is not available to support such a demand
(Smithers 2016). Microalgae, therefore, have several advantages over
traditional land plants that include: i) the high growth rate and high area
(or volumetric) productivity; ii) efficient CO2 conversion via
photosynthesis, thereby mitigating greenhouse gas emission; iii) being
able to grow on salt or wastewater streams (saline/coastal water,
brackish seawater, agricultural/domestic/municipal/industrial
wastewater), thereby reducing potable water use; iv) being able to grow
in areas unsuitable for agricultural purposes (e.g. desert and seashore
lands), and thus there is no competition with arable land for food
 45

production; v) having a very short harvesting cycle (1-10 days)

compared with other feedstocks (which are harvested once or twice a
year); vi) compatibility with integrated production of biofuels and co-
products within biorefineries (Chen et al. 2013; Varshney et al. 2015).
These advantages allow microalgae to be an excellent candidates for
supply of commodities, including both food and non-food products
(Draaisma et al. 2013; Tibbetts et al. 2015).
The microalgae (i.e. the prokaryotic cyanobacteria and the
eukaryotic microalgae) are an extremely diverse collection of
microorganisms able to accumulate macromolecules such as protein,
carbohydrates and lipids through the efficient use o solar energy, CO2
and nutrients (Borowitzka 2013; Matos et al. 2015; Wijffels et al. 2013).
In addition, microalgae are considered as perfect candidates for
contemporary fashionable ―nutraceutical‖ or ―functional food‖ due to
their ability to synthesize valuable products like carotenoids, long-chain
fatty acids, sugar, essential and non-essential amino acids, enzymes,
vitamins and minerals useful for human nutrition (Koller et al. 2014;
Henrikson 2009; Armenta & Valentine 2013; Parniakov et al. 2015).
Some of the most biotechnologically relevant microalgae are the
green algae (Chlorophyceae) Chlorella vulgaris, Haematococcus
pluvialis, Dunaliella salina and the cyanobacterium (also known as
blue-green algae) Arthrospira platensis (Spirulina), which are already
widely commercialized and used mainly as nutritional supplements for
humans and as animal feed additives (Becker 2013). Algae also act as
chemical platform for cosmetic purposes (e.g. coloring pigments and
especially skin supplements: extracts from Chlorella vulgaris are
reported to support collagen repair mechanisms), pharmaceutical and
therapeutic applications (Koller et al. 2014). The chlorophyceae
Haematococcus and Dunaliella shall be highlighted as powerful
pigment factories, and Spirulina platensis with outstanding capacity for
protein accumulation (Borowitzka 2013; Koller et al. 2014; Henrikson
2009).
Many algae accumulate substantial amounts of long-chain fatty
acids (LC-PUFAs) in the form of triacylglycerols (TAG), such as
omega-3 (ω-3): α-linolenic acid (ALA), eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA) and omega-6 (ω-6): linoleic acid
(LA), γ-linolenic acid (GLA) and arachidonic acid (AA) that have been
linked to human health effects (i.e., mitigation effects of hypertension,
anti-inflammatory effects, macular degeneration, rheumatoid arthritis,
osteoporosis, etc.) (Armenta & Valentine 2013). Some of the algae
reported to produce edible oil, normally commercialized as single cell
46

oil (SCO) containing LC-PUFAs, include Chlorella vulgaris (ALA-

producer), Spirulina platensis (GLA-producer), Isochrysis galbana,
Nannochloropsis oculata and Phaeodactylum tricornutum (EPA-
producers), Crypthecodinium cohnii and Schizochytrium limacinum
(DHA-producers) and Porphyridium cruentum (AA-producer)
(Borowitzka 2013; Draaisma et al. 2013; Koller et al. 2014). Thus, SCO
have attracted attention for their commercial development to provide
superior nutritional supplement through consumption of LC-PUFAs
(Armenta & Valentine 2013).
Microalgae-derived compounds and products are shown in Fig. 2.1,
where it is possible to visualize the schematic overview of potential
microalgal ―cell factory‖ production like lipids, pigments,
polysaccharides, protein and bioactive compounds correlated to its areas
of final application, such as agriculture, food technology, fine-
chemicals, pharmaceutical and nutritional purposes.
Products from algae have been reviewed in several papers (e.g.
[Borowitzka 2013; Koller et al. 2014; Pulz & Gross 2004; Spolaore et
al. 2006]). To underline the significance of the products synthesize and
extracted from microalgae, this paper aims to provide up-to-date and
comprehensive information on the potential commercial high-value
products from microalgae, with a particular focus on the use of these
high-value products in food science and technology. In addition,
toxicological aspects in terms of nucleic acids, heavy metals and algal
toxins as well as the nutritional quality standards in algae for human
consumption are discussed.
Fig. 2-1 - Products synthesized by microalgae and potential area of application.

Microalgal “cell factory”

Polysaccharides Lipids Bioactive compounds Biopolyesters Proteins Pigments

β-1,3-Glucan, Polyunsaturated, Terpenes, Poly Amino acids, Chlorophylls,
gelling agents, fatty acids, various antibacterial, (hydroxybutrate polypeptides, carotenoids,
starch, cellulose hydrocarbons anti-viral, anti-fungal, & lactic acid) enzymes phycobilins

 Pharmaceutical  Pharmaceuticals  Fine chemicals  Biopolymers  Human nutrition  Cosmetics

 Cosmetics  Nutraceutics Antiobiotics Biodegradable  Supplements Human nutrition
 Human nutrition  Food technology  Vaccines plastics  Recombinant  Food technology
 Functional foods &  Energy creation  Cosmetics proteins  Functional foods
therapeutical  Agrochemicals  Vaccines Pharmaceutical/
application  Therapeutic cosmetics
antibodies  Flavorants

Source: Modified from Koller et al. (2014)

47
48

Market potential of microalgal products

Important considerations while investing in algae products business

include: the size of the potential market, possible competing non algae
sources, the time and cost of achieving approval for new products and
their acceptance by the consumer (Oilgae 2016). Market prices for algal
biomass and its valued components are strongly fluctuating globally
area where the production site is located, the actual market situation, and
especially the production purity (Koller et al. 2014). According to
Transparency Market Research (accessed on
www.algaeindustrymagazine), a new market report entitled ―Algae
market, by application, by cultivation technology, and geography –
global industry analysis, size, share, growth, trends, and forecast –
2016-2024‖, has reported that the global algae market was value at US$
608.0 million in 2015 and is projected to reach US$ 1.143 billion with a
volume of 27,552 tons by 2024 (Algae Industry Magazine 2016).
Traditionally, microalgae such as Spirulina and Chlorella are
directly sold as dietary supplements. Spirulina production is
concentrated in Asia and the USA, Chlorella mostly in Asia, although
both are produced in a small number of other countries with warm
climates (Voort et al. 2015). China has become the biggest country in
Spirulina production in the world, with a total production of about 3,500
t (dw) in 2009, and a special attempt is given to Spirulina (Arthrospira)
industry in Inner Mongolia of China, most of are located at Wulan Town
in the Ordos Plateau, where the Spirulina production costs is estimated
of 3-4 US-$/Kg, with total annual production of 700 t (dw) in 2009 (Lu
et al. 2011).
In general, the market price of algal product (e.g. astaxanthin or
algal oil) is measured according to the market size of synthetic or
traditional fish oil alternatives. Spirulina and Chlorella still have by far
the largest production volumes, but also point to the large potential
markets for high-value products such as long-chain fatty acids
(DHA/EPA) and carotenoids (β-carotene, astaxanthin and lutein). Algae
products such as Spirulina and Chlorella have significant benefits as
potential single cell protein (SCP) sources; Spirulina was sold at a price
of US$ 20/Kg and Chlorella at a price of US$ 44/Kg in 2010 (Oilgae
2016).
The hydrocolloids, which are considered as medium-high value
product from microalgae, are mostly used in the food industry as gelling
and thickening agents. Some relevant algal carbohydrates that are
important for technical applications are soluble fiber β-1,3-glucan
 49

(market value of US $ 20-25/Kg), agar (prices between US$ 20-23/Kg),

carageenan (prices between US$ 10-12Kg) and emulsifiers command
between US$ 3-8/Kg, while gelling agents are worth US$ 5-22/Kg in
2012 (Oilgae 2016).
The market for carotenoids in 2010 was about US$ 1.2 billion with
the bulk of the carotenoids being produced by chemical synthesis
(Borowitzka 2013). In the pigment sector, the market value for β-
carotene, based on Dunaliella salina, is estimated at US$ 300 to
3000/Kg depending upon the product purity, and its global market size
of US$ 285 million and production volume of 1,200 ton per year (Koller
et al. 2014). The astaxanthin carotenoid extracted from the fresh alga
Haematococcus pluvialis, which contains the highest amount of
astaxanthin of any natural source, the average market price of
astaxanthin is US$ 2500/Kg and current global market size of natural
pigment for the human market is estimated at annually US$ 200 million
(Voort et al. 2015). For the high-prized phycobiliproteins from Spirulina
and Porphyridium (phycoerythrin, phycocyanin and allo-phycocyanin),
US$ 3000 to up to 25,000/Kg is projected (Koller et al. 2014), while the
C-phycocyanin sells between US$ 500 to 100,000/Kg depending on
purity, with the purity usually determined by the A620/A280 absorbance
ratio with food grade having a ratio of ≥ 0.7, reagent grade ~3.9 and
analytical grade ≥ 4.0 (Borowitzka 2013; Rito-Polamares et al. 2001);
the current total market value for phycobiliprotein products (including
fluorescent agents) is estimated to be greater than US$ 60 million
(Borowitzka 2013).
Algal oil represents one of the significant segments within the
omega-3 polyunsaturated fatty acid (PUFA) ingredients market. The
current wholesale market price for algae omega-3 oil is about US$
140/Kg (range, US$ 80-160/Kg) mainly produced by Crypthecodinium
cohnii and Schizochytrium sp. In 2012, the global market for microalgae
based DHA + 30% oil was estimated to be nearly US$ 350 million, and
about 4,614 metric tons. Globally, the infant formula application
represented about 48.9% of microalgae based DHA + 30% oils sold,
followed by dietary supplements with about 28.4%, and food and
beverages was 19.4% of the volumes (Shanahan et al. 2014).

Chemical composition of microalgae

The chemical composition of algae is frequently determined with

the objective of providing the necessary nutritional information for the
consumer, and also determining variations with respect to the conditions
50

under which the microalgae are been cultivated (Becker 2013). There is
a general agreement that chemical composition of algae varies from
species to species, from strain to strain, and from batch to batch culture
growth. To obtain an algal biomass with a desired pattern of
constituents, their proportion can be modified to a certain extent by
varying the culture conditions, for example provide nitrogen limitation
for lipid accumulation (Franz et al. 2013), and excess temperature or
light intensity for carotenoid synthesis (Panis & Carreon 2016).
Many analyses of gross chemical composition have been published
in the literature (e.g. [Tibbetts et al. 2015; Matos et al. 2016; Tibbetts et
al. 2014; Batista et al. 2013]). Although there are marked differences in
the compositions of the microalgal classes and species and strains,
protein is always the major organic component, followed by
carbohydrate + fiber or by lipid depending upon growth culture
conditions (Matos et al. 2016). It is not surprising that varying culture
conditions for the same strain, different compositions is achieved.
For food applications, the harvested and concentrated algal
biomass to be further utilized, a product with a water content of less
than 10% is required. Moisture affects spoilage of the dried algal
product by supporting the growth of bacteria, mould, and fungi (Becker
2013).

Protein

The high protein content of several microalgal species was one of

the main reasons for considering these organisms as a source of food,
which is reflected by the protein content reported in some algae, for
example Spirulina platensis (60-65%) and Chlorella vulgaris (51-58%)
of dry matter (Henrikson 2009). It has to be considered that those
estimations of the so-called crude protein, which is obtained by
measuring the total nitrogen and multiplying this value by the factor N x
6.25, which has been historically applied for microalgae, and this
calculation involves certain errors, because algae contain additional
nitrogen-bearing constituents, for example, glucosamides, nucleic acids,
amines, and cell-wall materials. For a correct purpose of N-to-P factor
for algal protein content, a conversion factor of N x 4.78 is
recommended (Lourenço et al. 2002).
Since protein is one of the most valuable algal components, four
important parameters of protein quality are used to determine the
appropriate nutritive value of algal protein, i.e. protein efficiency ratio
(PER), biological value (BV), digestibility coefficient (DC) or true
 51

digestibility and net protein utilization (NPU) (Becker 2013). Largely,

the quality and nutritive value of the alga depend on the type of post-
harvesting process, since it is vital importance for the utilization of the
algal biomass as proteinaceous matter. With the exception of the
cyanobacteria Spirulina platensis and Aphanizomenon flos-aquae, most
of the other types of microalgae posses a relatively thick cellulosic cell
wall, which makes improperly treated algae indigestible for humans and
most animals. Hence, to make the algal protein nutritionally accessible a
variety of disruption methods (mechanical or non-mechanical) have
been currently available for algal cell wall disruption. For instance,
mechanical forces such as solid-shear forces (e.g., high speed
homogenization, bead mill), liquid-shear forces (e.g., microfluidization,
high pressure homogenization), energy transfer through waves (e.g.,
microwave, ultrasonication), currents (e.g., pulsed electric field) or heat
(e.g., autoclaving, thermolysis), and non-mechanical methods such as
enzymatic cell lysis and chemical cell disruption (Gunerken et al. 2015).
The growing world population and consequential deficiency in
protein supply for human nutrition lead to increased activities for
exploring novel and alternative protein sources like single cell proteins
(SCP). Many microorganisms (algae, bacteria, fungi yeast/filamentous)
can be used as a source of SCP, but due to their low nucleic acid content
and high level of essential amino acids, algae are preferred over fungi
and bacteria as a source of SCP for human consumption (Anupama &
Ravindra 2000).
An interesting algal manufacturer, i.e. TerraViaTM has recently
announced the Health Canada has issued and classified as Generally
Recognized as Safe (GRAS) in compliance with US FDA, a regulatory
approval for its portfolio of AlgaVia® Whole Algae Ingredients,
including AlgaVia Whole Algal Protein that contains approximately
65% vegan protein plus amino acids, fiber, and microelements and
AlgaVia Whole Algal Flour making it an option for fortifying food due
to its neutral flavor profile and texture (Algae Industry Magazine 2017).
With regard to a protein-cosmetic product, a relevant cosmaceutical
derived from a protein-rich Spirulina extracts is ‗Protulines® - trade
name‘ by Exsymol S.A.M (Monaco) that has been used in cosmetic
industry with skin properties, such as anti aging, forming face and body
benefits (Enzing et al. 2014).
52

Amino acids

The subunits that make up proteins are amino acids and therefore
the nutritional quality of a protein is primarily determined by the
content, proportion, and availability of these compounds. Humans and
animals are limited to the biosynthesis of certain amino acids only (non-
essential amino acids); the remaining (essential) ones have to be
acquired through food.
The amino acids pattern of almost all algae compares favorably
with that of the FAO (Food and Agriculture Organization) reference,
with minor deficiencies among the sulfur-containing amino acids
methionine and cystein. During prolonged storage or excessive heat
treatment, lysine tends to form compounds with reducing carbohydrates,
resulting in the non-availability of lysine. This so-called Maillard
reaction has to be considered in connection with the heat treatments
applied during the processing of algal biomass (Becker 2013).
Two methods are common for estimating the quality of a given
protein by its amino acid composition, that is, the chemical score (CS)
and the essential amino acid index (EAAI). Deficiencies in certain
amino acids can be compensated by supplementing the protein directly
either with the limiting amino acid or with proteins from other sources
rich in the limiting amino acid (Becker 2013).
According to Henrikson (2009) Spirulina platensis offers rich
protein, about 60% by weight, which contains all of the essential amino
acids, and 10 of the 12 non-essential amino acids, and has been
extensively used as a source of single-cell-protein by astronauts during
space travel. In addition, Spirulina foods provide superior nutrition and
have decades of history with chefs and household cooks that deliver
high nutralence with colorful and tasty forms.
As microalgae are able to synthesize a wide range of amino acids,
an interesting cosmetic active product based on algal-derived amino acid
is ‗Exsy-Algine® - trade name‘(Exsymol, Monaco) that has been
marketed with the allegation to have skin care properties due to a
biobetter polar alga peptide (derivative of arginine)
(www.exsymol.com).

Carbohydrates

Carbohydrates perform numerous roles in living organisms and

served as storage of energy (e.g., starch and glycogen) and as structural
components (e.g., cellulose in plants/algae and chitin in arthropods).
 53

Special carbohydrates produced by microalgae are of increasing interest

due to their potential therapeutic application (Koller et al. 2014;
Sadovskaya et al. 2014; Turu et al. 2016). Here, β-1,3-glucan, a natural
soluble fiber active as immune-stimulator, antioxidant and reducer of
blood cholesterol has to be mentioned, which is accessible from the
cultivation of Chlorella strains (Koller et al. 2014), Isochrysis galbana
(Sadovskaya et al. 2014) and Euglena (Shibakani et al. 2016). In
addition to the therapeutic use, β-1,3-glucan can be also used in food
industry, mainly as fat substitute for texturing food products such as
functional bread, ready-to-serve soups, functional snacks and a variety
of other products (Koller et al. 2014; Shibakani et al. 2016).
Extracellular polysaccharide (exopolysaccharides – EPS) is another
carbohydrate-derived by-product extract from microalgae. Botryococcus
braunii which has a unique colonial organization that individual cells of
the colony are embedded in an extracellular matrix composed of liquid
hydrocarbons and exopolysaccharides. A recently paper Arad & Moppes
(2013) has swhon the extraction of EPS-derived from B. braunii in order
to prepare a cryogel structure helping in capture nutrients that can act as
anti-corrosive agent or bioflocculant in the food industry.
An exceptional attention is given to sulfated polysaccharides of red
microalgae extracted from Porphyridium cruentum. The cells of the red
microalgae are encapsulated within sulfated polysaccharides, and during
growth of the algae in a liquid medium, the viscosity of the medium
increases due to dissolution of the polysaccharide from the cell surface
into the medium (soluble polysaccharide) (Atobe et al. 2015). Hence, on
of the main characteristic of the red microalgal polysaccharides that
makes them suitable for industrial areas, for example cosmetics,
pharmaceuticals and nutrition, is their dynamic behavior, that is,
aqueous solutions are highly viscous at relatively low polysaccharide
concentrations, yielding rheological properties comparable with those of
industrial polysaccharides (e.g., xanthan gum) (Koller et al. 2014).
Sulfated polysaccharide form thermally reversible gels similar to agar
and carrageenan, which are used in food technology as thickeners,
stabilizers, and emulsifiers. Furthermore, sulfated polysaccharides
produced by microalgae can be even applied in anti-adhesive therapies
against bacterial infections and as coadjuvant in vaccines where the
polysaccharide protect protein-antibodies (Atobe et al. 2015).
Microalgae extracts are already used as sources of cosmaceuticals –
cosmetic products with biologically active ingredients purporting to
have medical or drug-like benefits. This is the case of a marketed
cosmaceutical product ‗Alguronic Acid‘ (trade name for a undetermined
54

mix of polysaccharides produced by microalgae clogging filters in algae

cultures) sold by Algenist/Solazyme (USA, California) (Enzing et al.
2014). This joins several other microalgae-derived cosmetic ingredients
such as polysaccharides from Porphyridium and extracts of Chlorella,
Spirulina and Aphanizomenon (Borowitzka 2013).

Lipids and fatty acids

Lipids and fatty acids are constituents of all plant cells, where they
function as membrane components, storage products, metabolites, and
sources of energy. The average lipid content of microalgae varies
between 1% to 40% and, under certain conditions, it may be as high as
85% of the dry weight. In the case of Botryococcus braunii, one of the
best scrutinized microalgal lipid producers, a total of 75% (w/w) of
hydrocarbons in cell mass were reported (Keng et al. 2009; Tasic et al.
2016). A large number of monounsaturated, polyunsaturated, and even
branched hydrocarbons are produced by B. braunii. These compounds
can be chemically converted into biodiesel by the well-established
alkaline transesterification with alcohols like methanol (Tasic et al.
2016). Glycerol phase is the main by-product of this transesterification
process, and can be commercialized for manufacturing cosmetic
products, or can be applied in food industry as humectant (E422) (Koller
et al. 2014).
Algal lipids are typically composed of fatty acids having carbon
numbers in the range of C12-C22. In general, saturated fatty acids (SFAs)
in algae are mainly composed of myristic (C14:0) and palmitic (C16:0)
acids, this last one is also naturally present in butter, cheese, milk, and
meat, as well as cocoa butter, soybean oil, and sunflower oil. Palmitic
acid is used to incorporate soaps, cosmetics and industrial mold release
agents (Keng et al. 2009). Oleic acid (C18:1) is the major
monounsaturated fatty acid (MUFA) usually found in algae, and is
frequently included in daily human diet as a part of animal fats and
vegetables oils. It is also used as emollient in cosmetical preparations,
emulsificant in food and solubilizant agent in aerosol products. A good
example of commercial culinary algae oil is AlgaWise® Ultra Omega-9
Algae Oil (cooking and high stability oils) from Solazyme and Bunge
Limited Company with operational facility in São Paulo state, Brazil.
With high level of MUFA (>90%), this unique oil is a clean-tasting oil
ideal for healthier alternatives to saturated fat oils with excellent
culinary performance (AIM 2015).
 55

Many algal oils contain special, often polyunsaturated, fatty acids

with high market values, such as omega-3 (ω-3) including α-linolenic
(ALA, C18:3), eicosapentaenoic (EPA, C20:5) and docosahexaenoic
(DHA, C22:6) acids and omega-6 (ω-6) including linoleic (LA, C18:2), ɣ-
linolenic (GLA, C18:3) and arachidonic (ARA, C20:4) acids. These
PUFAs can be commercialized for pharmaceutical and therapeutic
applications, and yield much higher prices than after converting the
lipids to biofuels (Koller et al. 2014). The proportion of PUFA differs
greatly depending on the species. For example, Crypthecodinium cohnii
microalgae possess only DHA and no practically other PUFA;
Nannochloropsis oculata and Phaeodactylum tricornutum contain
predominantly EPA, whereas Porphyridium cruentum contains
predominantly ARA.
PUFAs encompass ω-3 and ω-6 fatty acids that are well-known
components in fish oil. However, fish do not produce PUFAs
themselves; few fish actually directly consume plankton. Most fish get
their PUFA by consuming small fish that have consumed zooplankton
than have consumed phytoplankton. Consequently, algae are the
primarily source of these essential nutritional components that display
important functions in the human metabolism. For human health, the
PUFA ω-3 and ω-6 are entirely derived from the diet. Since Western
diet contains massive quantities of ω-6, ω-3/ω-6 fatty acids ratio of up
1:25 have been reported in the literature (Kleiner et al. 2014; Martin et
al. 2006) and nutrition experts have recommended that an ω-3/ω-6 fatty
acids of ≤ 1:5 is desirable (FAO 2008). Therefore, nutritionists
emphasize the need to consume seafood (notably fish) and oleaginous
seeds (linseed, Brazil nut, walnut, hazelnut, among others) in order to
supply PUFAs (Lemahieu et al. 2013). Since global fish stocks are
declining due to over-fishing and the derived oils are contaminated with
heavy metals and toxins, the production of PUFAs in the form of single-
cell-oil (SCO) from microalgal biotechnology is an alternative approach
(Armenta & Valentine (2013).
The subsequent paragraphs, together with Table 2.1, provide an
insight into potential applications of high-value microalgal fatty acids.
Fig. 2.2 shows the chemical structures of selected high-value fatty acids.
 56

Fig. 2-2 - Chemical structures of the most commonly polyunsaturated fatty acids in microalgae.
Table 2-1 - Microalgal long chain fatty acids useful in food application.
Fatty acid fraction Microalgae Application of the fatty Daily intake References
source acid recommendation
for human*
Omega-3
Alpha-linolenic acid Chlorella Nutritional supplement 1000-2000 mg (Guedes et al. 2011)
(ALA) vulgaris (single cell oil)
Eicosapentaenoic Nannochloropsis Nutritional supplement, 250-500 mg Rezenka et al. 2014;
acid (EPA) oculata, psycotherapeutic Guedes et al. 2011;
Phaeodactylum medication, brain Nelson et al. 2017)
tricornutum, development for children
Docosahexaenoic Schizochytrium Food supplement, 250-500 mg (Koller et al. 2014;
acid (DHA) limacinum, important for brain and eye Raposo et al. 2014;
Crypthecodiniu development at fetus and Sun et al. 2017)
m cohnii for children.
Omega-6
Gamma-linolenic Arthrospira Nutritional supplements, 500-750 mg (Henrikson 2009;
acid (GLA) platensis anti-inflammatory, auto- Turak 2017)
immune diseases
Arachidonic acid Porphyridium Nutritional supplements, 50-250 mg (Lewis et al. 2016;
(ARA) cruentum, anti-inflammatory, muscle Carlson & Colombo
Mortieriella anabolic formulations 2016)
alpina (body buider)
* ANVISA (2014) recommendation intake for human.
57
58

Omega-3 fatty acids

The ω-3 PUFAs ingredients market include EPA and DHA omega-
3s from marine oils, such as fish oil, krill oils, squid oils, and algal oils.
The end use application markets include dietary supplements, food and
beverages, pet aquaculture nutrition, infant formulation, pharmaceutical
and clinical nutrition. Traditionally, fish oils are the main source for ω-3
PUFAs, but its usage has been limited due to unpleasant odor and taste,
as well as poor oxidative stability (less shelf-life). Consumption
worldwide of ω-3 PUFAs was estimated at 134.7 thousand metric tons
valued at US$ 2.5 billion in 2014 and is projected that the demand for
ω-3 PUFAs globally will reach 241 thousand metric tons with a value of
US$ 4.96 billion in 2020 (AIM 2014).
The human body converts α-linolenic acid (ALA) to
eicosapentaenoic acid (EPA), a ω-3 fatty acid that plays an important
role in human body because act as a precursor for prostaglandins,
thromboxane and leukotriene eicosanoids which is succession are
responsible for various functions in immune system, blood clotting,
vascular pressure, and cancer prevention (Nelson et al. 2017). This is
based on various positive health effects related to this compound as
stated by the FAO, which recommend a daily intake of 250-500 mg for
human nutrition. Eustigmatophyceae strains of Nannochloropsis sp. and
diatom Phaeodactylum tricornutum are the most prominent microalgal
EPA-producers (Koller et al. 2014). EPA-rich oils are mainly used in
combination with DHA for infant formulation or dietary supplements
(single cell oils). Additionally, EPA is used in aquaculture for fish-
farming (Salmon farming) that plays a crucial role in their juvenile
development (Becker 2013; Carlson & Colombo 2016).
Functionally, EPA is a precursor to docosahexaenoic acid (DHA)
in human body, which is a primary structural component of the human
brain, cerebral cortex, skin, and retina. It is important for infantile brain
and eye development. Anti-inflammatory effects of DHA (Kleiner et al.
2014), and its importance for the development human fetus and healthy
breast milk are also evidenced by recent scientific studies (Pangestuti &
Kim 2011). The popularity of DHA allows including it in infant formula
fortifications, which is recommended by various health and nutrition
organizations for example, FAO (2008).
Microalgae such as Crypthecodinium cohnii and Schizochytrium
limacinum are exceptional producers of DHA and became the major
source alternative for production of ω-3 PUFAs. The former Martek
Biosciences Corporation based in Columbia, Maryland USA, a part of
 59

Royal DSM Dutch Multinational as of the end of 2010, has been the
most successful microalgae company to develop a patented process to
produce DHA-rich oil from for food, feed and pharmaceutical
applications. In addition to algal-based nutraceutical formulations, there
is an increasing demand for so called ―vegan health food‖ rich in
PUFAs for which microalgal biomass could act as the raw material of
choice (Borowitzka 2013; Koller et al. 2014).

Omega-6 fatty acids

Spirulina platensis is one of the best sources of ɣ-linolenic acid

(GLA), an ω-6 fatty acid (Henrikson 2009). GLA is as a precursor of
C20 eicosanoids to synthesize prostaglandin and has been associated
with beneficial health effects, anti-inflammatory effects and for auto-
immune diseases. A dairy intake dosage of 500-750 mg/GLA (Table 1)
is recommended by FAO (2008). In addition, GLA-rich supplements are
promoted to help people suffering from diabetes, obesity, rheumatoid
arthritis, heart disease, multiple sclerosis, and neurological problems
related to diabetes (Koller et al. 2014). Exsymol S.A.M (Monaco) is a
good example of manufacturer that sells anti aging skin cosmetic
product based on GLA-lipids from S. platensis (www.exsymol.com).
Arachidonic acid (ARA), a four-fold-unsaturated ω-6 fatty acid, is
an essential component of membrane phospholipids and is abundant in
the brain, muscles, and liver. It also acts as a vasodilator, shows anti-
inflammatory affects, and is therefore used for nutrient supplements. A
commercial source of ARA has been mainly derived by Mortierella
alpina fungus, although marine microalgae Porphyridium cruentum is
also ARA-producer (Borowitzka 2013). ARA is necessary for the repair
and growth of skeletal muscle tissue. This role makes ARA an important
dietary component in support of the muscle anabolic formulations, and
supplementation of 50-250 mg daily for healthy adults has been
recommended (Kleiner et al. 2014). DSM Corporation, for example,
produces an infant milk food supplement with a combination of ARA ω-
6 + DHA ω-3 long-chain polyunsaturated fatty acids (LC-PUFAs) with
trade name ‗DHASCO‘. Besides this food product, an example of a
commercial cosmetic active product from microalgae is ARCT‘ALG®
(Exsymol, Monaco) that formulates an extract from red alga containing
ARA-ω-6-rich offering energetic strength for human body with a series
skin benefits such as increases skin‘s resistance against extreme
conditions, increases ATP level, and improves cutaneous metabolism
through cytostimulation and lipolysis breakdown (www.exsymol.com).
60

Pigments

Algal pigments are responsible for light harvesting, CO2 fixation,

protection of algal cells against damage by excessive illumination and,
macroscopically, the coloration of the algal culture. Three major groups
of pigments are found in microalgae, namely chlorophylls (green
coloration), carotenoids (among them, carotenes provide an orange
coloration, whereas xanthophylls are responsible for yellowish shade),
and phycobilins (blue and red coloration) (Koller et al. 2014).
Novel extraction and separation techniques, such as supercritical
CO2 extraction, centrifugal partition chromatography and pressurized
liquid method have recently been employed in the development of
pigments from algae (Pangestuti & Kim 2011; Panis & Carreon 2016).
Due to the low productivities and high product recovery costs, the
market penetration of microalgal pigments is still in a very early stage if
compared to the chemosynthetic production of the same compounds, or
their isolation from other natural sources (Borowitzka 2013). In the case
of β-carotene, the portion produced by Dunaliella salina-β-carotene
amounts to about a quarter of the entire β-carotene market, because
there was also an established market for synthetic β-carotene, which
meant an easier and quicker path to the market for the algal product.
This situation is even more tremendous in the case of astaxanthin, where
the development of the ‗natural‘ astaxanthin market was more difficult
than for β-carotene, where less than 1% of the commercialized quantity
is allotted to the algal-derived pigment (Koller et al. 2014).
Various pigments are isolated from algae and thus have attracted
much attention in the fields of food, cosmetic and pharmacology. The
pigment fraction of algae can be applied as nutrient supply due to their
high contents of pro-vitamin A (E160a) and vitamin E (E306, E307,
E308) (Koller et al. 2014), and for other pharmaceutical, veterinary and
medical purposes (antioxidant activity, anti-inflammatory effects,
neuroprotective, cancer prevention (Pangestuti & Kim 2011), as well as
in cosmetic industry and food technology. In addition, canthaxanthin
pigment (E161g) is recommended on animal nutrition in feedstuffs for
salmon and trout, lying hens and other poultry due to colouring matter
(European Commision 2002). These general facts are collected in Table
2.2, whereas Fig. 2.3 presents the chemical structures of some of the
most important algal pigments.
Fig. 2-3 - Chemical structures of some microalgal pigments.
Chlorophylls Carotenoids Phycobilins

Carotenes

β-Carotene Bixin

Xanthophylls Phycocyanin
(linked to protein)
“Phycocyanobilin”

Astaxanthin Canthaxanthin

Violaxanthin Fucoxanthin
Phycoerythrin

Chlorophyll a

Zeaxanthin Lutein

Source: Koller et al. 2014

61
 62

Table 2-2 - Microalgal pigments and potential fields of application.

Pigment Color of Microalgal Application of the pigment References
pigment representative
Chlorophylls Green All phototrophic Pharmaceutical and cosmetics (Koller et al. 2014)
oxygenic algae (deodorant)
Carotenoids (Carotenes)
β-Carotene Yellow Dunaliella salina, D. Pro-vitamin A, antioxidant food, (Koller et al. 2014;
bardawil additive E160a, coloration of Enzing et al. 2014)
egg yolk
Bixin Yellowish to Dunaliella salina Food additive E160b (colorant), (Koller et al. 2014)
peach-color cosmetics
Carotenoids (Xanthophylls)
Astaxanthin Reddish- Haematococcus Food additive E161j, (Panis & Carreon
salmon pluvialis, antioxidant, farming of salmon 2016)
Botryococcus braunii and trout
Canthaxanthin Golden-orange Haematococcus Food additive E161g, farming of (EC 2002;
pluvialis, Chlorella salmonids and chicken, tanning Esatbeyoglu &
zofingiensis pills Rimbach 2016)
Fucoxanthin Brown to olive P. tricornutum Anti-adipositas (Loredo et al. 2016)
Lutein Yellow-orange Chlorella salina, C. Food additive E161b, yellow (Gayathri et al. 2016;
zofingiensis, D. salina coloration of egg yolk (feed Nwachukwu et al.
additive), pharmaceutical (anti- 2016)
macular degeneration)
Violaxanthin Orange Botryococcus braunii, Food additive E161e (approved (Koller et al. 2014)
Dunaliella tetriolecta in Australia & New Zealand)
Zeaxanthin Orange-yellow Phaeodactylum Food additive E161h, animal (Nwachukwu et al.
tricornutum feed, pharmaceutical (anti-colon 2016; Eilers et al.
cancer, eye health) 2016)
Phycobilins
Phycocyanin Blue-green Arthrospira, Spirulina Food colorant (beverages, ice (Henrikson 2009; Vo
(―cyano‖) (cyanobacteria) cream, sweets), cosmetics, et al. 2017)
fluorescent marker in
histochemistry, antibody labels
Phycoerythrin Red Porphyridium, Immunofluorescence (Tang et al. 2016; Han
cyanobacteria techniques, labels for antibodies et al. 2013)

Table 2-3 - Recommended dosages of pigments useful for human consumption mainly due to antioxidant activity.
β-carotene Astaxanthin Phycobilins (phycocyanin, phycoerythrin)
(carotenoid) (carotenoid)
Daily intake recommendation 2-7 mg 6-12 mg 200-400 mg
Source: ANVISA (2014) recommendation intake for human.
63
64

Chlorophylls

Chlorophylls are greenish lipid-soluble pigment which contain a

porphyrin ring and found in all algae, higher plants and cyanobacteria.
Structurally, chlorophylls are substituted tetrapyrrole with a centrally
coordinated magnesium atom. Chlorophylls are modest absorbers of
green and near-green portions of the spectrum, and this causes the
typical and well-known green coloration of the green algae
(Chlorophyta) that harbor chlorophylls as the predominant pigments.
Chlorophyll is registered and approved as a colorant additive
(E140) and mostly used in the food pigmentation and dietary
supplement industries. Famous ―Chef de cuisine‖ use chlorophyll to
provide a green coring of foodstuffs and beverages, such as pasta, pesto
or absinthe (Koller et al. 2014). Most chlorophyll available on the
market is actually in the form of the derivative sodium copper
chlorophyllin. These structural changes convert fat-soluble chlorophyll
into a water-soluble compound and this derivative molecule
(chlorophyllin) has shown antimutagenic effects to various polycyclic
procarcinogens such as aflatoxin-B1, polycyclic aromatic hydrocarbons
and some heterocyclic amines, demonstrating potential chemo-
preventive agent (Coates et al. 2013). Due to its high deodorant
capacity, chlorophyll a is also used as an ingredient in items of personal
hygiene products, such as deodorants or as pastilles; it is
commercialized in formulations against bad breath (Koller et al. 2014).

Carotenoids

Carotenoids are lipophilic compounds and are usually colored

yellow, orange or red and are the most diverse and wide spread
pigments found in nature (Gong & Bassi 2016). Most carotenoids share
a common C40 backbone structure of isoprene units (termed terpenoid),
and are divided into two groups: carotenes, which are unsaturated
hydrocarbons; and xanthophylls, which present one or more functional
groups containing oxygen. Carotenoids display so-called ―secondary
light harvesting pigments‖, supporting the ―primary pigment‖
chlorophyll in capturing light energy. They also act as antioxidants that
inactivate reactive oxygen species (ROS) formed by exposure to
excessive solar radiation. Due to the fact that antioxidants are also
required by the human metabolism to prevent the manifold negative
impacts caused by free radicals, this outstanding property explains the
 65

significance of carotenoids for nutraceutical or functional food products

(Koller et al. 2014; Gong & Bassi 2016; Pangestuti & Kim 2011).
At the present time, the major carotenoids of market interest are β-
carotene, astaxanthin, lutein (with zeaxanthin), canthaxanthin and
fucoxanthin. Astaxanthin and β-carotene are the two most recognized
carotenoids in the global market, and make-up almost half of the
carotenoid market (Business Communications Company (2015).
β-carotene was the first high-value product commercialized from a
microalga (Dunaliella salina – sometimes also called Dunaliella
bardawil), with production starting in the 1980s mainly cultivated in
Israel and Australia (Borowitzka 2013). With reported intracellular β-
carotene contents of up to 14%, this green alga (Chlorophyceae) is
characterized by its preference for extremely saline habitats like salt
evaporation ponds, a fact that facilitates its cultivation in open raceway
ponds without risking microbial contamination (Varshney et al. 2015).
In humans, one molecule of β-carotene can be cleaved by the intestinal
enzyme β,β-carotene 15,15‘-monooxygenase into two molecules of
vitamin A, and thus β-carotene has been used for human vitamin
supplement with a daily intake of 2-7 mg/day (Table 2.3) and for food
coloring (E160a). In addition, β-carotene is of outstanding importance in
human metabolism for retinal synthesis needed for generation of
rhodopsin. Besides this, it plays a certain role for the prevention of toxin
build-up in the liver, potentially improves the immune system, and may
have a preventive role in eye diseases like night blindness and cataract
(Gong & Bassi 2016).
The second carotenoid from algae traditionally commercialized is
astaxanthin (E161j) extracted from the freshwater green alga
Haematococcus pluvialis, which is characterized to synthesize up to
3.0% by dry mass of astaxanthin (Koller et al. 2014). Algatechnologies®
in Israel is a good example of established industry for Haematococcus
cultivation and astaxanthin production, which is produced in closed
system of glass photobioreactors energized by the abundant natural
sunlight of the Arava desert in Israel (Borowitzka 2013). Furthermore,
Israel-based Algatechnologies Ltd. (Algatech) has received Brazilian
Health Regulatory Agency (Anvisa) approval for its all-natural
AstaPure® astaxanthin to be used as a food ingredient. This approval
makes Algatech the first supplier to start marketing its clean-label
astaxanthin in Brazil. Astaxanthin is best recognized for the pinkish
color in aquatic fish and shrimps. Being the strongest anti-oxidant in
carotenoids, astaxanthin exhibits several-fold stronger anti-oxidant
activity than vitamin E and β-carotene (Gong & Bassi 2016; Mao et al.
66

2017), and it is recommended a daily intake of 6-12 mg/day (Table 2.3).

In human metabolism, it is has the potential to enhance antibody
production, anti-aging, alleviating sports fatigue, promoting hair growth,
anti-tumor therapy, and sun-proofing in sunscreen creams due to its UV-
protecting activity that by far surmounts the activity of vitamin E
(Borowitzka 2013; Koller et al. 2014; Panis & Carreon 2016; Vo et al.
2012).
Two other carotenoids xanthophylls by-products, i.e., lutein and
zeaxanthin are also becoming increasingly important in the nutraceutical
market since they are now understood to play a significant role in eye
heath (Nwachukwu et al. 2016; Vo et al. 2012). As the predominant
pigments in the macula, lutein is clinical proven to prevent cataract and
macular degeneration. Lutein is traditionally used in chicken feed in
order to fortify yellow egg yolk and food additive (E161b) approved for
use in the EU, Australia and New Zealand (European Commision 2002).
In general, a daily intake of 20 mg/day of these two carotenoids is
relatively safe for human consumption.
Fucoxanthin is another xanthophylls carotenoid that can be
produced by microalgae, for example the diatom Phaeodactylum
tricornutum. It has been marketed as an anti-obesity functional food,
and for anti-cancer and potential anti-inflammatory activities (Foo et al.
2017; Gong & Bassi 2016). For many dairy products such as cheese,
butter or margarine, the food additive bixin (E160b), a further example
of the carotene group of carotenoids, provides a yellowish to peach-
color shade, and also used as colorant in cosmetics (Koller et al. 2014).
Violaxanthin and canthaxanthin are additional members of the
xanthophylls. Violaxanthin has an orange coloration that makes it
another food colorant (E161e) approved in Australia and New Zealand.
Potential producers of this pigment are Dunaliella tertiolecta and
Botryococcus braunii. Canthaxanthin is approved in United States and
EU for use as a food coloring agent (E161g), for example Strasbourg
sausages (maximum concentration of 15 mg/Kg), as a colouring agent
for medical products (tanning pills especially in Canada) and as colour
additive in animal feed for poultry and fish feeds (European Commision
2002). Typical production strains of canthaxanthin are the
Eustigmatophyceae species Nannochlorospsis sp. like N. salina, N.
gaditana or N. oculata (Enzing et al. 2014; Koller et al. 2014).
 67

Phycobilins

Phycobilins (phycocyanin and phycoerythrin) are light-capturing

bilins found in the stroma of chloroplasts of cyanobacteria, rhodophyta
(red algae), Cryptophyta and Glaucophyta. Chemically, they constitute
open-chain tetrapyrroles molecules that typically act as chromophores
and structurally related to the pigments of mammalian bile and pigments
of hemoglobin and chlorophyll, which makes them colored.
Phycocyanin is a blue pigment primarily found in cyanobacteria, for
example Spirulina platensis and Aphanizomenon flos-aquae species,
while phycoerythrin is a pigment occurring predominantly in red algae,
for example Porphyridium cruentum and Gracilaria gracilis species,
and responsible for its characteristic red coloration (Borowitzka 2013;
Koller et al. 2014). Phycobilins are unique among all known
photosynthetic pigments because they are bonded to certain water-
soluble protein, building a pigment-protein complex known as
phycobiliproteins. Similar to carotenoids, they serve as ―secondary light
harvesting pigments‖ and therefore forward the energy of the harvested
light to chlorophylls for photosynthesis (Koller et al. 2014).
Phycobilins, in the form of C-phycocyanin and R-phycoerythrin,
are useful in a wide range of applications including fluorescent tag for
use in flow cytometry and immunology, as photosensitisers in
photodynamic therapy for treatment of cancers and fluorescent dyes in
microscopy techniques (Tang et al. 2016). Besides these highly
sophisticated applications as chemical-pigment tag; such
phycobiliproteins are the algal-derived products revealing the by far
highest market values (Koller et al. 2014). Furthermore, phycobilins are
also used as natural coloring agent in food industry acting as a
component of functional foods (sweets, ice cream, and beverages), as
possible antioxidant in personal care skin products (e.g., phycocyanin
cosmetic ‗Line Blue‘ sold by DIC Lifetec Corp., Japan) and hydrating
skin product (e.g., phycoerythrin from Porphyridium cruentum sold by
Soliance®, France) (Borowitzka 2013).

Vitamins

Microalgae represent a valuable source of nearly all important

vitamins, which improve the nutritional value of algal biomass. In
general, the concentrations of the various vitamins are comparable
between the different algae as well as in higher plants. An important
exception seems to be vitamin B12 and various vitamins B complex such
68

as cobalamin and cyanocobalamin. In addition to the vitamin B12, other

vitamins such as vitamin K, vitamin A, isomers of tocopherol (vitamin
E), as well as metabolic intermediates can be found in almost all algae.
The usual dietary sources of vitamin B12 are meat, milk, egg, fish, and
shellfish (Becker 2013). Especially Spirulina sp. is sold as health food,
expecting to contain a large amount vitamin B12. Watanabe et al. (1999)
found that commercially available Arthrospira tablets contained 127-
244 μg vitamin B12 per 100 g weight. In humans, the intrinsic factor-
mediated intestinal absorption system is estimated to be saturated at
about 1.5-2.0 μg per meal under physiologic conditions (Becker 2013).
Thus, Spirulina is far good source of vitamin B12 and it could be an
excellent source of vitamin B complex for vegetarian consumers.

Other valuable compounds

Aside from compounds essential to basic physical structure, energy

metabolism, and reproductive mechanisms, algae produce a myriad of
secondary metabolites that assist in the survival of the organism. The
ecological function of secondary metabolites in microalgae or
cyanobacteria may play some roles from defense against predation to
warding off infection by various microorganisms to communication. For
example, the brevetoxins produced by marine dinoflagellates, which are
responsible for neurotoxic shellfish poisoning (NSP), are thought to
have an ecological role as a feeding deterrent (Coates et al. 2013).
Lyngbyatoxin is an indole alkaloid and a cyanotoxin isolated from the
cyanobacterium Moorea producens, responsible for ―swimmer‘s itch‖,
is also thought to have an ecological role as a defense against grazing
(Mao et al. 2017).
It is this rich chemical and biological diversity that gives the
secondary metabolites of algae and cyanobacteria potential value as
commercial products. Some of these molecules retain their natural
functions when used for anthropogenic purposes; for example,
polyunsaturated fatty acids found in many algae are used in the
nutraceutical industry for their health benefits (Guedes et al. 2011), as
discussed later in this paper. A number of human sun-screen products
contain the mycosporine-like amino acids (MAAs) produced by many
microalgae through metabolic engineering; these compounds have
antioxidant and UV protective properties in both the natural as well as
human product contexts. Several metabolites isolated from microalgae,
such as noscomin from the cyanobacterium Nostoc commune, are
 69

promising antibacterial compounds, and may serve similar functions in

nature as well (Han et al. 2013).
There are a variety of algal- and cyanobacterial-derived
biopolymers that have been used for commercial purposes, including the
polysaccharides agarose, alginate, and carrageenan, as well as
polyhydroxyalkanoates (PHAs). Of these biopolymers, PHAs are the
only ones that have been applied to plastic applications, and this
chemical class has been shown to be produced by a number of
cyanobacterial genera including Spirulina platensis and Spirulina
maxima, Synechococcus sp. MA19 and Synechocystis sp. 6803
(Borowitzka 2013; Coates et al. 2013; Koller et al. 2014). As microbial
storage compounds with plastic-like properties,
poly(hydroxyalkanoates) (PHAs) are of increasing significance to
replace well-established plastics of fossil origin on the market (Koller et
al. 2014).
Many secondary metabolites of microalgae are terpenoids,
molecules that are derived from isoprene units, which have the chemical
formula C5H8. A common example of a terpene-derived metabolite is
the plant steroid β-sitosterol, where microalgae are able to synthesize a
wide range of phytoesterols including brassicasterol, sitosterol, and
stigmasterol depending on the taxonomic affiliation of the alga
(Borowitzka 2013). These algal phytoesterols may have pharmaceutical
applications or in functional foods, but the potential of microalgae as
sources of phytoesterols remains to be fully explored.
The hydrocarbons produced by the microalga Botryococcus braunii
are one class of terpenoid currently being developed for their biofuel
potential. Squalene, a linear triterpene natural product is traditionally
isolated from the liver oils of deed-sea shark, but it is also synthesized
and produced by the microalga Botryococcus braunii, although in
relatively low amounts. Aurantiochytrium strains (thaurostrochytrid) are
the most promising microalgal source of squalene, which have been
shown to accumulate a level of 198 mg g-1 squalene (Borowitzka 2013).
Squalene has found many applications in medicine, food, and cosmetic
industries due to its antioxidant, antistatic and anti-carcinogenic
properties (Mao et al. 2017), and special attempts have been given for
the production of squalene by metabolic engineering of cyanobacterium
Synechocystis sp. PCC 6803 (Englund et al. 2014).
Microbial production of isoprene has been recently demonstrated
by an engineered Synechocystis sp. PCC 6803 strain (Lindberg et al.
2010). In cyanobacteria, isoprene is important for synthesis of
terpenoid-like molecules required for many cell functions included
70

photosynthesis, membrane stability and cellular production of

carotenoids. To produce isoprene in Synechocystis, the isoprene
synthase gene from kudzu vine (Pueraria montana) was expressed
under regulation of the photosystem component psbA2 promoter
(Machado & Atsumi 2012). According to the same authors, isoprene is
highly volatile hydrocarbon that microorganisms such as cyanobacteria
offers the advantages to be cultivated in enclosed bioreactors, which
permits collection and sequestration of the volatile isoprene.

Table 2-4 - Chemical products synthesized by engineered

cyanobacteria.
Product Potential applications in food Host
science and technology organism
1 – Butanol Used as an ingredient in S. elongatus
processed and artificial PCC7942
flavorings, for the extraction of
lipid-free protein from egg yolk,
and for the manufacture of hop
extract in beermaking.
Isobutanol Flavoring agent in food industry, S. elongatus
ink ingredient. PCC7942
Isobutyraldehyde Used primarily as a chemical S. elongatus
intermediate to produce glycols, PCC7942
essential amino acids, flavor and
fragrance, polymers, insecticides
and isobutanol.
Isoprene Used as a synthetic version of Synechocystis
natural rubber. Isoprene-derived sp. PCC6803
several biological molecules such
as phytol, retinol (vitamin A),
tocopherol (vitamin E), and
squalene.
Source: Modified from Machado & Atsumi (2012)

The advancement of synthetic biology and genetic manipulation

has permitted engineering of cyanobacteria to produce non-natural
chemicals typically not produced by these organisms in nature (Atsumi
 71

et al. 2009). This is the case of engineering cyanobacteria for production

of high-value chemicals including higher alcohols (Table 2.4). The
production of isobutyraldehyde by the cyanobacterium Synechpcoccus
elongatus PCC7942 has been reported (Atsumi et al. 2009) where the 2-
ketoacid pathway was selected for engineering and expression in a
mutant S. elongatus strain to synthesize isobutyraldehyde.
Isobutyraldehyde is used primarily as a chemical intermediate to
produce plasticizers, glycols, flavor and fragrance and isobutanol
(Machado & Atsumi 2012). Isobutanol is a another higher alcohol used
as a solvent in the manufacture of flavor/fragrances and flavoring agent
in food industry, and has been synthesized by engineered S. elongatus
PCC7942 using a keto acid pathway (Atsumi et al. 2008).
Further higher-chain alcohol of interest as a chemical feedstock is
1-butanol that has been produced by engineered S. elongatus using a
modified CoA-dependent pathway (Lan et al. 2011). 1-Butanol is a
primary alcohol produced industrially from the petrochemical feedstock
propylene and used as an intermediate in the production of butyl esters.
Butyl esters also known as butyl ethanoate has impart flavor
characteristic and associated with banana or apple smell. It is used as a
synthetic fruit flavoring in foods such as candy, ice cream, cheeses, and
baked goods. 1-Butanol can also be produced as a by-product of
microbial fermentation processes using Clostridium acetobutylicum
(Lee et al. 2008).

Toxicological aspects

Unconventional food items such as microalgae have to undergo a

series of toxicological tests to prove their harmlessness.
Recommendations for the performance of such evaluations have been
published by different international organizations, but it has to be
assumed that additional national regulations exist, which specify the
recommended evaluation.
As part of the toxicological characterization, the algal material has
to be analyzed for the presence of toxic compounds, either synthesized
by the alga itself (biogenic toxins) or accumulated from the environment
(non-biogenic toxins). The biogenic toxins include nucleic acids and
algal toxins, whereas non-biogenic products comprise environmental
contaminants, such as heavy metals (Becker 2013).
Nucleic acids
72

The nucleic acids RNA and DNA belong to the constituents that
are present in all living organisms. Since these acids are sources of
purines, they are sometimes considered as limitation in the use of alga
and other forms of single-cell-protein as food and feed.
The nucleic acid content in algae varies between 4% and 6% (8-
12% for yeast and up to 20% for bacteria) in dry matter (Becker et al.
2013). For humans, the ingestion of purines may lead to an increase of
plasma uric acid concentration and urinary excretion; elevated serum
levels of uric acid in humans may increase the risk of gout, while
elevated urinary levels may result in the formation of uric acid stones in
the kidney and nephropathy (Kelley & Anderson 2014; Liu et al. 2017).
The Protein Advisory Group of the United Nations (Nutrition Bulletin)
has recommended that a daily intake of nucleic acid from
unconventional sources should not exceed 2.0 g, with total nucleic acids
from all sources not exceeding 4.0 g d-1. As mentioned early, single-
cell-protein from algae source are preferred over fungi and bacteria
sources due to their low nucleic acid content (Anupama & Ravindra
2000). However, the safe level should be at about 20 g of algae per day
or 0.3 g of algae per kg of body weight (Becker 2013).

Algal toxins

Poisoning of humans and animals by algal toxins, resulting from

blooms of algae, occurs with considerable frequency – sometimes
unpredictably – in regions of South America, Europe, Asia, South
Africa, and Australia. Certain marine algae (Anabaena sp., Mycrocystis
sp., Dynophysis sp. and Pseudo-nitzschia produce potent toxins
(saxitoxins, anatoxin-a, domoic and okadaic acids) that have an impact
on human health through consumption of contaminated shellfish, clams,
mussels, oysters and scallops, which filtered these toxins from the water
accumulating to levels that can be lethal to consumers, including
humans (Proença et al. 2011). Algal toxins can give rise to a number of
different poisoning syndromes (Enzing et al. 2014):

NSP – neurotoxic shellfish poisoning;

PSP – paralytic shellfish poisoning;
ASP – amnesic shellfish poisoning;
DSP – diarrhoeic shellfish poisoning;
 73

Ciguatera fish poisoning.

Algae such as Isochrysis, Chlorella sp, Tetraselmis sp., Spirulina

(Arthrospira), Phaeodactylum tricornutum, Porphyridium cruentum,
among others that are commercially used in aquaculture and food
supplements do not produce toxin. Even within the same species, large
differences exist between toxic and non-toxic algae. Dinoflagellates and
diatoms are best known for their production of toxins that can affect
humans, but for instance the Dinoflagellate strain Crypthecodinium
cohnii has a GRAS (Generally Recognized as Safe) status by FDA for
ω-3 DHA human food consumption (Enzing et al. 2014) (Table 2.5).
Consequently, in view of application of algae for food or feed, it is very
important to know their safety at strain level. Fortunately, no such cases
have ever been reported in connection with mass-cultured algae.
However, there is continuous concern about the possibility that
commercially sold algae may contain contaminants from inadequate
algal cultivation management (Becker et al. 2013).

Heavy metals

Almost all microorganisms, primarily those living in an aquatic

biotop, are capable of accumulating heavy metal ions by adsorption and
absorption at concentrations that are several orders of magnitude higher
than those present in the surroundings. According to WHO/FAO
guidelines, an adult person of 60 kg body weight should not incorporate
more than 3 mg of lead, 0.5 mg of cadmium, 20 mg of arsenic, and 0.3
mg of mercury per week through food and beverages. At present, no
official standards exist for the heavy metal content of microalgal
products. On a voluntary basis, some algae manufactures have
established internal guidelines for metal levels in their products (Becker
et al. 2013).
 74

Table 2-5 - Safety aspect of relevant microalgae for food application.

Organism Species Safety Organism Species Safety
aspect aspect
Chlorophyta Chlamydomonas NT Haptophyta Isochrysis galbana NT
reinhardtii
Chlorella vulgaris GRAS Pavlova sp. NT
Dunaliella salina NT Heterokontophyta Nitzschia dissipata NT
Haematococcus NT Nannochloropsis sp. NT
pluvialis
Scenedesmus sp. NT Phaedactylum NT
tricornutum
Tetraselmis sp. NT Skeletonema sp. NT
Cyanobacteria Spirulina GRAS Schizochytrium GRAS
(Arthrospira)
Synechococcus sp. NT Thalassiosira NT
pseudonoma
Dinophyta Crypthecodinium GRAS Rhodophyta Porphyridium GRAS
cohnii cruentrum
NT = no toxins known; GRAS = Generally Recognized As Safe by Food Drug Administration (FDA)
Source: Enzing et al. (2014)
 75

Nutritional quality standard and regulations

As many of the high-value products from microalgae are intended

for human and animal nutrition or related use, these compounds are
subject to a range of regulations and standards. There exist
internationally testing programs for unconventional foodstuffs such as
single-cell-protein, which have to be or should be performed. There are
only few countries that have so far stipulated legislative standards for
Spirulina. It is not known whether official regulations also exist for
other types of microalgae, that is, Chlorella, Dunaliella, etc. These
official requirements as well as suggested quality criteria found in the
literature and elsewhere are summarized in Table 2.6 (Becker et al.
2013).
According to Becker (2013) several steps need to be necessarily
evaluated for the approval of algal biomass for human and animal
consumption. It seems useful to have the following specifications to
become available:

 proximate chemical composition;

 evaluation of protein quality;
 biogenic toxic substances (phycotoxins, nucleic acids, other
toxicants);
 non-biogenic toxic compounds (heavy metals, residues from
harvesting and processing);
 sanitary analyses (microbial analyses for contamination);
 toxicological and safety evaluations (feeding trials with
experimental animals).

The issues of food safety at the internationals level are considered

by the Codex Alimentarius Commission (CAC), which was created by
the Food and Agriculture Organization (FAO) and the World Health
Organization (WHO) with the intent to develop food standards and
guidelines. Laws and regulations relating to food additives and novel
foods (including nutraceuticals and functional foods) vary from country
to country. For example, in the USA the FDA has the primary
76

responsibility for regulating new food ingredients, whereas the EU

through the EFSA (European Food Safety Authority) has regulations for
food additives, novels foods and genetically modified organisms
(Borowitzka 2013). In January 2011, the FDA Food Safety
Modernization Act was signed into law in the USA. This specifies that
the manufacturer may seek approval for a new ingredient by filing a
food additive petition with the FDA to (a) making a GRAS
determination, or (b) request a formal pre-market review. This petition
needs to be supported by nonclinical and clinical studies supporting the
safety of the product for the use proposed (Borowitzka 2013; Enzing et
al. 2014).
In Australia and New Zealand, the products are most likely to come
under the novel foods and novel food ingredients category regulated
under Standard 1.5.1 – Novel Foods – of the Australian and New
Zealand Food Standards Code administered by Food Standards
Australia New Zealand (Borowitzka 2013). In Brazil, the ANVISA
(Agência Nacional de Vigilância Sanitária) has estimated some limits
for microbial and heavy metal contaminants in tablets/capsules of
Spirulina sold as dietary supplement (Brasil 2001; Brasil 2013; Brasil
2014) (Table 2.6).
For many markets and certifying or regulatory authorities, Good
Manufacturing Practice (GMP) certification and ISO 9001 – 2000 are
essential. For food and related industries, an HACCP (Hazard Analysis
& Critical Control Points) methodology is highly recommended.
Whereas, specific markets may require additional certifications, for
example, Organic certification by a recognized certifying authority, for
example USDA Organic in USA and IBD Accreditation in Brazil.
Table 2-6 - Some quality standards for microalgae.
IUPAC WHO Earthrise Indian Japan France Brazil
(1974) (1972) Algal (1990) (Jassby, (Becker (ANVISA,
Farm 1988) 1994) 2001)
(USA)
Total protein (%) >55.0 >45.0
Total ash (%) <9.0
Moisture (%) <7.0 <9.0 <7.0 <10.0
Standard plate count (x106/g) <0.2 <0.005 <0.1
Mould (number/g) <100 <100
Coliformes (number/g) Absent Absent <10 <10
Salmonella sp. Absent Absent Absent
Staphylococcus sp. Absent <100 <500
(number/g)
Filth (insect fragment) <10 Absent
(number/g)
Pb (ppm) <5.0 <0.1 <0.2 <0.30
Hg (ppm) <0.1 <0.001 <0.025 <0.50
Cd (ppm) <1.0 <0.01 <0.2 <0.10
As (ppm) <2.0 <0.05 <1.00
77
78

Challenges surrounding algal usage

Market introduction of food products using the whole microalgae

organisms (such as Spirulina, or Chlorella) or products that include the
microalgae (like pasta with green algae colour) are subject to food safety
regulations that apply to all food products. Important principles applied
in this regulation are that novel foods and food ingredients must be safe
for consumers (not being dangerous or nutritionally disadvantageous)
and properly labeled so as not to mislead consumers (Enzing et al.
2014).
This is the case, for example, the extraction of polyunsaturated
fatty acids (e.g., EPA, DHA and ARA) from microalgae, where organic
solvents have been widely applied to the extraction of microalgal lipid-
rich for biodiesel production (Cheng et al. 2011; Halim et al. 2012);
however such use for pharmaceutical and nutraceutical sector may lead
to contamination of the final product in the form of residues. Because
the solvents used in extraction or partition will ultimately be removed or
at least reduced by evaporation, solvents with low boiling points should
be chosen to avoid prolonged heating. Thus, the lower boiling fractions
of petroleum ether (35-60°C) could be used instead of the higher boiling
fractions (e.g. hexane 65-69°C) (Amaya et al. 2004). Nevertheless, the
use of petroleum ether for large-scale industrial solvent extraction may
have limited permission in many countries, while n-hexane is ranked on
the list of substances prohibited in cosmetic products (Regulation (EC)
No 1223/1999 of the European Parliament (Rapinel et al. 2016).
When extracting carotenoids from biological samples, such as
microalgae, a water-miscible organic solvent (e.g., acetone, methanol,
ethanol, or mixtures thereof) should be used to allow better solvent
penetration. Acetone has been widely used in carotenoid extraction;
however, the advent of high performance liquid chromatography
(HPLC) has seen tetrahydrofuran (THF) become a popular extraction
solvent (Amaya et al. 2004). Carotenoid-astaxanthin from H. pluvialis is
an example of high-value product that has been habitually extracted with
organic solvent. In such case, supercritical fluid extraction (SFE) based
on carbon dioxide and coupled with ethanol as a cosolvent can greatly
increase the extraction efficiency (Panis & Carreon 2016), and has
emerged as a ‗green‘ technology due to its non-flammable
characteristics, critical temperature and pressure relatively low and
chemically inert (Cheng et al. 2011). In addition to carbon dioxide, other
compressed solvents can be used to extract polyunsaturated fatty acids
and carotenoids from microalgae, including ethane, propane, n-butane,
 79

and dimethyl ether (Capeletto et al. 2016; Goto et al. 2015). In fact, a
recent study (e.g., Feller 2016) has evaluated the efficiency of using
subcritical n-butane in comparison to supercritical CO2 for extraction of
lipids, polyunsaturated ω-3 and ω-6 and carotenoids from three marine
microalgae (Nannochloropsis oculata, Phaedactylum tricornutum and
Porphyridium cruentum) for food and pharmaceutical applications. The
choice of n-butane as alternative solvent was motivated by its gentle
vapor pressure, close chemical structure, low price and its classification
as authorized solvent for foodstuff production without limitation
(Directive 2009/32/EC) (Rapinel et al. 2016). Nevertheless, the
economic viability of these new approaches for extraction of high-value
products from microalgae for food application will have to be
demonstrated.
Another important issue regarding algal usage is the products
containing genetically modified (GM) algae that has two main aspects
related to biosafety: potential adverse environmental consequences and
potential harm to human or animal health in case of food/feed or
pharmaceutical applications (Enzing et al. 2014). In general, all research
– including microalgae – is governed by regulations in the field of good
laboratory practice. In the USA the FDA, and in Europe the EFSA are
responsible for the biosafety evaluation (Enzing et al. 2014). For human
health risks, the biosafety evaluation could refer to the methods applied
in higher plants to guarantee that they are safe and they do not produce
toxic substances or allergens (Song et al. 2012). For the environmental
risks, Henley et al. (2013) have published a comprehensive study on risk
assessment of GM microalgae for commodity-scale biofuel cultivation
which is also relevant for other applications. In particular for food
applications, a history of safe use for a certain algae implies that
production has proven to be safe over a longer period of time (this also
implies some forms of environmental exposure). Nevertheless, the
process of bringing a GM microalgae product to market can therefore be
a long and complicated issue.

Conclusions and outlook

The variety products accessible from the primary and secondary

metabolism of diverse algal species clearly demonstrates the importance
of these versatile microorganisms as cellular factories. Several high-
value microalgae products are already well-established in the
marketplace, and there are clear opportunities for additional new
products. Eukaryotic microalgae are interesting for products for which
80

cellular storage is important such as protein, lipids, starch, and alkanes.

Cyanobacteria are promising host organisms for the production of small
molecules that can be secreted such as isoprene, butanol, fatty acids and
other organic acids. Considering recent progress in the field of genetic
engineering, implementation of improved algal genes is very likely to
understand algal intracellular activities with a broad industrial
application. Besides the genetic engineering, the development of algal
cultivation process from laboratory conditions to large scale production
will require substantial progress towards a cost-efficient algal-based
technology in order to encourage manufactures for new products to be
developed and marketed in the next decade.
 81

CHAPTER 3

THE USE OF DESALINATION CONCENTRATE AS A

POTENTIAL SUBSTRATE FOR MICROALGAE
CULTIVATION IN BRAZIL

Abstract

Desalination concentrate produced by inland desalination plants

can potentially provide water and nutrients for algal growth. Several
studies have been conducted in Brazil to explore the use of desalination
concentrate as an alternative medium for microalgae cultivation. This
review describes examples of microalgae cultured in desalination
concentrate and evaluates the effects of desalination concentrate on
algal biomass productivity, lipid content, lipid productivity, protein, and
fatty acids composition.

Keywords: Microalgae; inland desalination wastewater; biomass

composition; lipid productivity; biofuels.

Introduction

Microalgae are promising sources of biomass, bioproducts, and

biofuel and have become an extensive area of research (Halim et al.
2012; Henkanatte-Gedera et al. 2015; Griffiths & Harrison 2009;
Varshney et al. 2015). One of the advantages of some microalgae
species is their high content of lipids and the composition of their fatty
acids, mainly medium-chain carbon (C16 and C18), triacylglycerols
(TAG), which can be chemically converted into biodiesel (Halim et al.
2015). The majority of commercially available biofuels in Brazil are
currently bioethanol derived from sugar cane and biodiesel derived from
oil crops, including soybean (Costa et al. 2010). It has been proposed to
derive biofuels from the cultivation of algae, because of their ability to
grow well in marginal water resources and in non-agricultural land, and
because of their greater photosynthetic efficiency compared with land
plants. In addition, using algae cultures as a source for biofuels will
reduce the need to compete with resources utilized in conventional
agriculture (Henkanatte-Gedera et al. 2015; Varshney et al. 2015).
82

Several microalgae species can effectively grow in wastewater

conditions through their ability to utilize abundant organic carbon and
inorganic N and P (Pittman et al. 2011). Most of the research on algal
wastewater treatment has come from the analysis of laboratory-based
small scale and pilot pond-scale cultures (Park et al. 2011). A wide
range of studies have analyzed the growth of microalgae under a variety
of wastewater conditions, in particular growth in multiple municipal,
industrial and agricultural wastewaters (Craggs et al. 2011; Henkanatte-
Gedera et al. 2015; Ledda et al. 2015). These studies have mainly been
focused on evaluating the ability of algae to remove N and P, and in
some instances metals, from wastewater. In Brazil, desalination
concentrate (DC) as a source of water and nutrients is of special interest
for utilizing and recycling inland desalination wastewater, which is
loaded with microelements useful for algal biomass production (Matos
et al. 2015). Experimental studies on the use of DC as an alternative
medium for microalgae, particularly studies that also assess variables
that determine maximal algal biomass production and biochemical
composition, will have significant benefits for the evaluation of
wastewater-grown microalgae in Brazil.
This mini-review summarizes the main results of studies in Brazil
on the use of DC as an alternative medium for microalgae cultivation
with a focus on the physicochemical characteristics of DC, the impact
of this medium on algal growth, biomass productivity, and biochemical
composition (protein, lipids and fatty acids composition) are discussed.
In addition, the effects of N/P ratio and salinity parameters in the
optimum DC concentration for three microalgae species (Chlorella
vulgaris, Spirulina (Arthrospira) platensis and Nannochloropsis
gaditana) are also discussed.

Combining desalination concentrate and microalgae cultivation

Desalination of brackish groundwater in Brazil

South America has huge natural inland salts deposits in arid zones.
In Brazil, inland deposits of brackish waters are common in the
Northeast of Brazil that has a semiarid climate and recurrent droughts.
This part of Brazil is an exceptional example of inland desalination and
brine management, partially because of a massive governmental
program launched in 2004 named ‗Água Doce‘ (Fresh Water) that has
benefitted 150,000 inhabitants of the semiarid region of Brazil (Sánchez
et al. 2015).
 83

Desalination through reverse osmosis is the most suitable solution

to provide water supply in the semiarid region of Brazil. It is estimated
that there are 1,500 desalination installations in this part of Brazil
(Menezes et al. 2011). All desalination technologies produce a stream of
fresh water and a highly concentrated stream of minerals and
contaminants (Greenlee et al. 2009; Menezes et al. 2011). In the
semiarid region of Brazil, DC from inland desalination of brackish
groundwater is mainly used for aquaculture (fish farming), for
hydroponic and soil cultivation (lettuce, tomatoes, beetroot, cotton and
sunflower) and for direct irrigation of halophyte forage shrubs (Atriplex
nummularia) (Dias et al. 2010; Sánchez et al. 2015; Soares et al. 2006).
The fact that part of the DC flow can be used for algaculture is an
additional and promising opportunity to exploit the valuable
components in desalination wastewater.
It is well known that microalgal growth is influenced by
environmental parameters, such as salinity (Bartley et al. 2013). A
change in salinity in algal cultivation can have positive effect on the
metabolism of algae (Davis et al. 2015), especially on important cell
membrane constituents, such as lipids and fatty acids (Takagi &
Karseno 2006). The salt concentration influences algae via effects on
osmotic stress, salt stress, and cellular ionic ratios (Kumar et al. 2015).
Each microalgal strain grows optimally at a specific salinity, but some
microalgae can take advantage of different salinities. For example, the
halophilic chlorophyte genus Dunaliella viridis can grow at low (0.3 M
NaCl) and at high (2.3 M NaCl) salinity (Davis et al. 2015).

Desalination concentrate and algal cultivation

Desalination concentrate is rich in inorganic minerals, such as Cl-,

Na and Ca2+, contains other nutrients (N and P) and trace elements
+

necessary for microalgae growth, including Si, K+, Mg+2, Fe+3, and
electrical conductivity normally ranges from 4.0 to 5.5 mS cm-1 (Table
3.1). Since it is known that DC contains abundant amounts of mineral
salts, DC has been tested for stimulating microalgae growth (Matos et
al. 2014; Matos et al. 2015; Volkmann et al. 2007). To be able to assess
the applicability of DC as a culture medium for algal cultivation, the
desalination wastewater needs to be mixed with a conventional medium,
since some microalgae cannot grow in DC due to the high mineral salts
strength. For example, cultivation of freshwater Chlorella vulgaris,
which cannot grow normally in DC, was performed in dilutions of 25%,
35%, 45% and 55% DC mixed with Bold Basal Medium (BBM) (Matos
84

et al. 2014). DC concentrations below 25% had no effect on C. vulgaris

growth compared to cultivation in control medium (BBM), both
yielding a maximum biomass concentration of 0.40 g L-1. However,
incubating C. vulgaris with the higher DC concentrations of 35-55%
(conductivities between 2.0 to 2.8 mS cm-1) resulted in growth-salt
stress, and a proportional decrease in biomass yield (to 0.27 and 0.13 g
L-1). Thus, the optimal DC concentration for C. vulgaris growth is 25%
(~1.6 mS cm-1) and at higher concentrations growth is reduced,
probably due to the high Na+ and Cl- concentrations in DC. In addition,
small quantities of ammonia and sulphate have been detected in DC that
may affect C. vulgaris growth, while DC can contain resides of
pretreatment and cleaning chemicals, and their reaction byproducts
which may also have adverse affects on aquatic organisms (Lattemann
& Hopner 2008).

Table 3-1 - Typical compositions of groundwater and desalination

concentrate (DC) obtained from the Community of Uruçu (semiarid
region of Paraíba state, Brazil).
Parameters Brackish groundwater DC
pH 7.3 ± 0.5 8.5 ± 0.3
Electrical conductivity 3.0 ± 0.8 4.5 ± 1.0
(EC, mS cm-1)
Salinity (M NaCl) ~0.02 ~0.04
TDS (mg L-1) 1823.8 ± 94 2190.5 ± 78
Cl (mg L-1) 720.7 ± 35 1691.3 ± 114
-1
Na (mg L ) 464.0 ± 66 987.5 ± 110
CaCO3 (mg L-1) 506.5 ± 97 985.2 ± 112
-1
Ca (mg L ) 74.0 ± 10 126.5 ± 21
Mg (mg L-1) 77.2 ± 21 80.7 ± 10
-1
SiO2 (mg L ) 48.2 ± 11 82.4 ± 24
SO42- (mg L-1) 15.6 ± 0.6 138.0 ± 14
-1
K (mg L ) 7.0 ± 2 47.0 ± 8
NO3- - N (mg L-1) 8.2 ± 0.1 30.0 ± 5
+ -1
NH4 (mg L ) 0.59 ± 0.3 1.35 ± 0.7
PO43- - P (mg L-1) 0.20 ± 0.1 0.70 ± 0.3
-1
Fe (mg L ) 0.05 ± 0.1 0.13 ± 0.1
 85

When DC was tested in the same way mixed with Paolettic

Synthetic Medium as an alternative culture medium to grow Spirulina
platensis, it was found that the optimal DC concentration for S.
platensis growth is 50% DC (~2.5 mS cm-1) (Volkmann et al. 2007). On
the other hand, marine Nannochloropsis gaditana performed well when
exposed to a high DC concentration of 75% (~8.0 mS cm-1) (Matos et
al. 2015), at which lipid productivity (12.10 mg L-1 day-1) was higher
than lipid productivity of C. vulgaris (7.37 mg L-1 day-1) and S.
platensis (6.97 mg L-1 day-1). These examples of the influence of DC on
algal growth suggest that DC is a good medium for marine algae strains
such as N. gaditana, but that the ionic concentration of DC is too high
for the cultivation of the freshwater C. vulgaris and S. platensis. The
results of studies on the optimal DC concentration for cultivation of
three microalgae species are summarized in Table 3.2.

Table 3-2 - Optimal physicochemical parameters of culture media

based on desalination concentration for three different algae species.
C. vulgaris S. platensis N. gaditana
DC proportion (%) 25 50 75
Culture medium BBM Paoletti F/2
Type of water fresh water fresh water seawater
EC (mS cm-1) 1.0 – 2.2 2.0 – 3.2 8.0 – 10.0
NaCl (g L-1) 0.66 3.21 10.45
Salinity (M NaCl) 0.01 0.05 0.17
pH 8.1 – 9.0 9.4 – 10.5 8.0 – 9.0
N:P ratio 5:1 7:1 18:1
Reference Matos et al. Volkmann et Matos et al.
(2014) al. (2007) (2015)
DC = Desalination concentrate, EC = Electrical conductivity

Different salinities in terms of NaCl concentration in the optimum

DC medium for three algae species have been reported in the literature,
for C. vulgaris (0.01 M NaCl) (Matos et al. 2014), S. platensis (0.05 M
NaCl) (Volkmann et al. 2007) and N. gaditana (0.17 M NaCl) (Matos et
al. 2015). Among the three microalgae species tested in optimum DC
concentration, marine N. gaditana has better ability to cope with
osmotic changes and alterations in the salt ratios than freshwater algae
species, because it has the capability to tolerate a broader range of DC-
salinities with a satisfactory growth. For example, when marine N.
gaditana was cultured in F/2-seawater medium (0.5 M NaCl) and
86

induced to a hypotonic salinity, i.e. to brackish DC medium (0.17 M

NaCl), biomass concentration and lipids enhanced from 0.71 to 0.96 g
L-1 and 4.7 to 12.6%, respectively. These results suggest that marine N.
gaditana production for lipids work well when exposed to suboptimal
salinity.
Taking into consideration that microalgae generally consume the
majority of the nutrients, notably N and P, in the growth medium, Park
et al. (2011) reported that in wastewaters used for algae cultivation the
N:P ratio ranges from 4:1 to almost 40:1. Matos et al. (2015) reported
an initial N:P ratio for marine N. gaditana cultivation of 18:1 (415.0 mg
N/22.5 mg P), which is marginally higher than the theoretical N:P ratio
of 16:1 (7.3 g N/1.0 g P) required for the growth of algae, based on the
assumption that algal biomass has the typical composition
C106H108O45N16P (Craggs et al. 2011; Redfield et al. 1958). In addition,
for N. gaditana growth the N:P ratio in DC (18:1) was almost 3-fold
higher than the N:P ratios in the DC-based culture medium for the
freshwater species C. vulgaris (5:1) and S. platensis (7:1) [Matos et al.
2015; Volkmann et al. 2007 (Table 2.2)]. Given the effect of N:P ratios
on algae growth, we conclude that a culture medium based on DC is
more suitable for marine algae than freshwater algae strains.

Effect of desalination concentrate on algal biomass composition

Effect of desalination concentrate on protein content

Various studies have measured the amount of protein in Chlorella

sp. and C. vulgaris in culture media based on 25% DC under batch,
semi-batch and continuous culture conditions and found that the protein
content ranged from 45.3% to 51.0% dry matter (Matos et al. 2014;
Matos et al. 2014; Matos et al. 2015). In one study (Matos et al. 2014),
the authors observed that intracellular protein content of C. vulgaris
decreased from 51.6% to 15.7% when the DC concentration was
increased in the culture medium from 25% (1.2 mS cm-1) to 55% DC
(3.5 mS cm-1). The reduced protein content in Chlorella cultures at high
DC concentrations is most likely due to a redirection of available energy
towards processes such as osmoregulation rather than towards the
synthesis of proteins (Katz et al. 2007; Lawton et al. 2015).

There is significant interest in using of DC to cultivate Spirulina

platensis for agricultural purposes aimed at the production of algal
biomass with high protein content for human food and animal feed
 87

(Sánchez et al. 2015). Studies carried out in photobioreactors with S.

platensis cultured in optimal 50% DC, showed a protein content of
49.6% which is comparable to the protein content (56.1%) obtained in
cultures of S. platensis developed in conventional Zarrouk medium
(Volkmann et al. 2008). Another advantage of the optimal DC
concentration in culture media for S. platensis is that all essential amino
acids required for human health were found in higher concentrations
than required by the FAO, except lysine and tryptophan.
With regard to the protein content in marine N. gaditana, recent
studies showed that when N. gaditana is cultured in conventional
media, the protein content is 41.6% and the lipid content is 4.9%, but
when grown in optimal 75% DC medium, the protein and lipid contents
were 27.0% and 12.6%, respectively (Matos et al. 2015). Thus, the
protein content decreased 1.5-fold, while the lipid content increased 2.5-
fold compared to cultivation in a conventional medium. The shift from
protein production to accumulation of lipids is a typical response of
algae to stress conditions, such as nutrient depletion or salt-osmotic
stress (e.g. [Karemore et al. 2013; Lawton et al. 2015]).

Effect of desalination concentrate on lipid content and fatty acids

composition

A number of recent laboratory-based studies have examined the

effects of DC on the lipid content of microalgae, grown either in small
batches and semi-continuous cultures or in continuous photobioreactors
(Matos et al. 2014; Matos et al. 2015; Volkmann et al. 2007).
Reasonable lipid accumulation was reported in DC-grown microalgae,
ranging from low (<10% dry matter) to moderate (15-20% dry matter)
lipid content, which in some cases translated to a relatively high lipid
productivity when coupled to high biomass productivity (Matos et al.
2015). Lipid productivities of C. vulgaris, S. platensis, and N. gaditana
cultured in optimal DC concentration are shown in Table 3.3. A recent
study (Matos et al. 2015) found that the lipid content in N. gaditana
cells increased from 5.9% to 12.6% when the DC concentration
increases from 25% to 75% DC in the culture medium. More
importantly, at higher DC concentrations N. gaditana tends to
synthesize more SFAs (C14:0 and C16:0) accompanied with a lower
production of PUFAs (C18:2, C18:3 and C20:5 ɷ3). For example,
increasing the DC concentration from 0% (control test) to 100% DC in
the culture medium for N. gaditana, the percentage of palmitic acid
increased from 29.4% to 53.4%, and the percentage of eicosapentaenoic
88

acid dropped from 12.2% to 0.2% of the total fatty acids. Functionally,
increased intracellular lipid content and higher SFAs in N. gaditana can
be explained by an adaptive osmoregulatory mechanism to cope with
rapid or gradual changes in salinity that are associated with algae-cell-
membrane permeability (Takagi & Karseno 2006).

Table 3-3 - Biomass concentration and lipid productivity of microalgae

grown in different proportion of desalination concentrate (DC).
Species DC Biomass Lipid Lipid Reference
(%) (g L-1) (%) prod. (mg
L-1 day-1)
C. vulgaris 25 0.59 12.5 7.37 Matos et al.
2014a
S. platensis 50 1.55 4.5 6.97 Volkmann et
al. 2007
N. gaditana 75 0.96 12.6 12.10 Matos et al.
2015

The fatty acid profile of C. vulgaris, cultivated at optimal 25% DC

in batch and continuous autotrophic cultures (Matos et al. 2014a; Matos
et al. 2014b), showed that the most abundant fatty acids were (in
percentage of the total fatty acids content) α-linolenic acid (C18:3 ɷ3,
18.0-24.6%), palmitic acid (C16:0, 17.3-20.0%) and linoleic acid
(C18:2 ɷ6, 9.2-15.5%). C. vulgaris cultured in 25% DC supplemented
with glucose (2.0 g L-1) under mixotrophic and heterotrophic conditions
(Matos et al. 2014b), contained more oleic acid (C18:1, 31.5-35.6%)
than cultures developed under autotrophic mode (Matos et al. 2014a),
which is advantageous for biodiesel production (Chiu et al. 2015; Halim
et al. 2012). Volkmann et al. (2008) reported that the main constituents
of the lipids extracted from S. platensis cultivation in 50% DC under
photoautotrophic condition, were palmitic acid (C16:0, 36.1%), oleic
acid (C18:1, 20%) and γ-linolenic acid (C18:3 ɷ3, ~14.0%). When
marine N. gaditana was cultivated in optimum 75% DC medium under
two-stage photoautotrophic-mixotrophic mode utilizing either glucose
(1.0 g L-1) or glycerol (1.0 mL L-1), C16:0 was the most abundant fatty
acid found, ranging from 50.5% to 56.8% of the total fatty acids (Matos
et al. 2015).

In general, the studies on the use of optimal DC as an alternative

culture medium find that freshwater and marine microalgae are virtually
 89

devoid of long-chain fatty acids (e.g. C20 and C22) and show a trend
toward the increased formation of medium-chain (C16 and C18) fatty
acid methyl esters, which is an ideal lipid source for biodiesel
production (Chiu et al. 2015; Halim et al. 2012).

Reuse of desalination concentrate in microalgal cultivation

Water recycling in microalgae cultivation is desirable not only to

reduce the water demand, but also to improve the economic feasibility
of algal biofuels that will save nutrients and energy (Depraetere et al.
2015; Farroq et al. 2015; Farroq et al. 2015). An analysis of the reuse of
the medium with the optimum DC concentration (75% DC) in
successive N. gaditana cultivation cycles (Matos et al. 2015) found that
the medium could support three successive cultivation cycles of N.
gaditana (each cycle involved 7 days of cultivation, 21 days in total). It
was also observed that biomass productivity of N. gaditana is directly
correlated with lipid productivity, but does not necessarily correlate
with lipid content. For example, biomass (151 mg L-1 day-1) and lipid
(14.3 mg L-1 day-1) productivities were higher in the first cultivation
cycle (7 days of cultivation). The second cultivation cycle presented
lipid content (13.6%) higher than the first cultivation cycle (9.5%) while
the highest lipid content (14.4%) was reached in the third cultivation
cycle. In the first cultivation cycle, N. gaditana cells consumed 60% of
the total N (initial concentration 415 mg L-1) and 71.1% of the total P
(initial concentration 22.5 mg L-1) available in the culture medium.
After three cultivation cycles N. gaditana cells had consumed 80% and
86%, of total N and total P, respectively. Because of N and P were
totally depleted over the course of the cultivation cycles, the lipid
content tends to increase with consecutive reuses of the recycled 75%
DC medium. A similar pattern was reported by Matos et al. (2015) in
studies on the algal biomass productivity and lipid content of Chlorella
sp. in the recycled medium with an optimal DC concentration of 25%.
These findings are in good agreement with reports by Griffiths and
Harrison (2009), who also found that high lipid content in microalgae
does not necessarily represent high lipid productivity. Lipid content
alone is therefore not a good indicator of the suitability of microalgae
for biodiesel production. In order to exploit water recycling to maximize
algal biomass, lipid content and lipid productivity, we need to better
understand the relationship between these three parameters.
90

Concluding remarks and future directions

In our recent study on extracting lipids from dried Chlorella sp.

cultured in optimal 25% DC (Morioka et al. 2014), we applied a single
pre-treatment step (sonication) and compared the performance of
dynamic hexane extraction (Soxhlet apparatus) with that of dynamic
Supercritical-CO2 (SCCO2) extraction. The efficiency of microalgal
lipid extraction was higher using Soxhlet (23.3%) than SCCO2
extraction (4.5%). In terms of fatty acid composition, crude lipids
extracted from Chlorella sp. by the Soxhlet method contained a
substantially higher quantity of C16:0 than the corresponding crude
lipids extracted by SCCO2.
Most studies on the use of DC as a potential substrate for
microalgae cultivation in Brazil have analyzed microalgal biomass and
lipid/protein contents under laboratory conditions (cultivation in batch
culture flasks or photobioreactors). The microalgae strains (C. vulgaris,
S. platensis and N. gaditana) are able to grow in DC, but require
different optimum DC concentrations. In general, when algae are
exposed to optimum DC concentration, which is a ―stress‖ condition,
algae decrease the production of protein and increase the synthesis of
lipids and saturated fatty acids. We suggest that DC represents a good
medium for marine N. gaditana, because it has the ability to tolerate
high extent of DC concentration. Nevertheless, if 100% DC is used as
the culture medium for N. gaditana, algae cannot maintain a satisfactory
growth with only the mineral composition presented in DC. As such,
DC needs to be supplemented with conventional media.
While considerable knowledge has been gained under laboratory
conditions that have demonstrated the potential of DC as a promising
alternative culture medium, the use of DC for algal mass cultivation in
semiarid regions of Brazil needs to be validated in further
investigations. This region of Brazil has exceptional qualities to produce
algae in open raceway ponds on a pilot-scale due to its ample solar
irradiance, low seasonal variation, extensive arid lands, and abundant
brackish groundwater where algae production is proposed. According to
Rogers et al. (2014), utilizing saline or brackish water will be necessary
for the sustainable growth of algae in New Mexico, United States.
Likewise, efforts must focus on growing algae in raceway ponds,
especially using available DC in the semiarid region of Brazil. Clearly,
technoeconomic analyses must be conducted in order to address the
major cost effectiveness needed to produce biofuel from algae in Brazil.
 91

CHAPTER 4

BIOMASS, LIPID PRODUCTIVITIES AND FATTY ACIDS

COMPOSITION OF MARINE Nannochloropsis gaditana
CULTURED IN DESALINATION CONCENTRATE

Abstract

In this study the feasibility of growing marine Nannochloropsis

gaditana in desalination concentrate (DC) was explored and the
influence of the DC concentration on the biomass growth, lipid
productivities and fatty acids composition was assessed. The reuse of
the medium with the optimum DC concentration in successive algal
cultivation cycles and the additional of a carbon source to the optimized
medium were also evaluated. On varying the DC concentration, the
maximum biomass concentration (0.96 g L-1) and lipid content (12.6%)
were obtained for N. gaditana in the medium with the optimum DC
concentration (75%). Over the course of the reuse of the optimum DC
medium, three cultivation cycles were performed, observing that the
biomass productivity is directly correlated to lipid productivity. Palmitic
acid was the major fatty acid found in N. gaditana cells. The saturated
fatty acids content of the algae enhanced significantly on increasing the
DC concentration.

Keywords: Microalgae; desalination wastewater; reuse of water;

mixotrophic cultivation; biomass; lipids.

Graphical Abstract
Desalination Concentrate

Nannochloropsis gaditana
culture

Golden lipids
 93

Introduction

Microalgae have emerged as very promising candidates as a source

material for the sustainable production of energy, offering significant
advantages such as rapid growth, low demand for land area, and high
yield of lipid- and carbohydrate-rich biomass per acre (Baeyens et al.
2015; Foley et al. 2011; Stephens et al. 2010). Autotrophic growth is the
most common approach to microalgae cultivation to provide algal
biomass. However, to complement the commonly explored autotrophic
activity, heterotrophic and mixotrophic algae systems have been
considered as a viable alternative for supporting innovative
bioprocesses (Ji et al. 2014; Perez-Garcia et al. 2011).
Algal growth requires the availability of primary nutrients and
micronutrients, which can be costly if they need to be added in large
amounts. In this regard, nutrients from wastewater can be transferred to
algal biomass, achieving simultaneously an economic microalgae
production and efficient wastewater treatment (Pittman et al. 2011). For
example, desalination through reverse osmosis (RO) is the most suitable
procedure for obtaining fresh water in the semi arid region of Brazil, but
this is associated with the problem of brine disposal. This residue, if not
properly managed, can cause environmental problems such as the
salination of agricultural land, which can render it infertile (Greenlee et
al. 2009; Menezes et al. 2011). The brine discharge, known as
desalination concentrate (DC), is predominately comprises mineral salts
which include the cations Na+ (sodium), Ca+2 (calcium), Mg+2
(magnesium). With a growing need for the inland desalination of
brackish water in Brazil, many solutions have been proposed for the use
of DC as a source of nutrients, such as in aquaculture, the irrigation of
halophyte plants, hydrophonic crops and, agalculture (Soares et al.
2006; Dias et al. 2010; Matos et al. 2014). Additionally, an integrated
scheme using DC for agricultural purposes (Tilapia + Spirulina +
Atriplex) has been suggested by Sánchez and co-authors (2015), aimed
at the production of human food and feed with high protein content for
animals.
Many species of microalgae are able to grow efficiently in
wastewater conditions due to their ability to utilize the abundant organic
and inorganic N and P in the wastewater. In addition, considering that
microalgae require large amounts of water, the recycling of the water
after harvesting can result in savings of up to 84% for water and 55%
for essential nutrients such as nitrate and phosphate (Yang et al. 2011).
Microalgae cultivation using desalination concentrate as a source of
94

water and nutrients is of special interest in the valorization and

recycling of brine waste loaded with valuable compounds (Moheimani
et al. 2015). For example, Volkmann et al. (2008) reported the use of
DC mixed with Paoletti medium for Arthrospira platensis cultivation
aimed at food and feed production. Matos and co-authors (2014)
optimized a culture medium based on DC combined with Bold Basal
Medium for freshwater Chlorella vulgaris cultivation. To complement
these studies, it would be of great interest to grow marine algae with DC
as a culture medium. Nannochloropsis is as a commonly cultivated
organism which has been used to produce biofuel (Vooren et al. 2012),
pharmaceutical compounds (Goiris et al. 2012; Yen et al. 2015), and
fish feed (Camacho-Rodríguez et al. 2015). Nannochloropsis gaditana
was selected for this study because of its high growth rate, high lipid
productivity, and wide environmental tolerance (Griffiths and Harrison
2009).
In this study, the use of desalination concentrate (DC) was
investigated considering two main goals: (1) to evaluate the best DC
concentration (in terms of percentage) for autotrophic growth of N.
gaditana; and (2) to assess the influence of DC on biomass and, lipid
productivities and fatty acids composition. Moreover, the reuse of DC
wastewater, based on the optimum DC concentration for further
autotrophic algal growth was tested and the effect of an additional
carbon source on the biomass and lipid productivities was evaluated
using the medium with the optimum DC concentration (Fig. 4.1). We
hypothesis that marine algae N. gaditana can support high DC
concentration than any other algae studied with higher biomass
productivity and lipid content.
 95

Fig. 4-1 - Flow diagram for the cultivation of marine N. gaditana in

desalination concentrate (DC) and subsequent studies using the DC
percentage selected.

F/2 DC

Experimental cultures

0-, 25-, 50-, 75-, 100% DC

Optimum medium
75% DC

Mixotrophic cultures Cultures in reused medium

Glucose/Glycerol/Glycerin Three cycles performed

Micrographs images

Methods

Algal strain and culture

The marine species Nannochloropsis gaditana (clone 130) was

kindly supplied by Banco de Micro-organismos Marinhos Aidar &
Kutner (BMA&K) of the Oceanographic Institute at the University of
São Paulo. The alga was maintained in autoclaved F/2 seawater medium
(Table 4.1) suitable for marine algae (Guillard 1975).
96

Table 4-1 - Compositions of desalination concentrate (DC) and F/2

medium.
DC Value F/2 medium Value
pH 8.5 ± 0.5 pH 8.0
Cond. (mS cm-1) 4.5 ± 0.5 Conductivity (mS cm-1) 35
TDS (mg L-1) 2190 ± 15 Stock solution (1.0 mL L-1)
Cl (mg L-1) 1691 ± 10 NaNO3 (g L-1) 75
-1
Na (mg L ) 987 ± 99 NaH2PO4 H2O (g L-1) 5
CaCO3 (mg L-1) 985 ± 68 Na2SiO3 9H2O (g L-1) 30
SO42- (mg L-1) 138 ± 3 Trace metal solution (1.0 mL L-1)
Ca (mg L-1) 126 ± 12 FeCl3 6H2O (g L-1) 3.15
K (mg L-1) 47 ± 2 Na2EDTA 2H2O (g L-1) 4.36
NO3- - N (mg L-1) 30 ± 2 CuSO4 5H2O (g L-1) 9.8
-1
Mg (mg L ) 4.7 ± 0.8 Na2MoO4 2H2O (g L-1) 6.3
NH4+ (mg L-1) 1.3 ± 0.5 ZnSO4 7H2O (g L-1) 22.0
PO43- - P (mg L-1) 0.7 ± 0.1 CoCl2 6H2O (g L-1) 10.0
Fe (mg L-1) 0.1 ± 0.05 MnCl2 4H2O (g L-1) 180.0
Vitamin solution (0.5 mL L-1)
Thiamine HCl (mg L-1) 200
Biotin (g L-1) 1.0
Cyanocobalamin (g L-1) 1.0

Desalination concentrate and experimental procedures

Desalination concentrate (DC) was collected from an inland

desalination plant, located in São João do Cariri, Paraíba, Brazil. The
DC samples were collected in a 100-L plastic container and stored at -
20°C in the Laboratory of Food Biotechnology, Federal University of
Santa Catarina, Santa Catarina, Brazil. The chemical composition of the
DC (Table 4.1) was determined according to standard methods (APHA
2005).
The DC mixed with F/2 medium (based on seawater) in different
concentrations (25%, 50%, 75% and 100% of DC) was used as the
experimental media. A control test was conducted simultaneously using
F/2 medium (0% DC). The initial N. gaditana concentration,
approximately 105 cells mL-1, was used for the preparation of the
inoculum. All treatments were performed in 300-mL Erlenmeyer flasks
and incubated at room temperature (25 ± 2 °C), sparing with saturated
air-CO2, photoperiod of 12 h:12 h light/dark provided by fluorescent
lamps, under a light intensity of 80 µmol m-2 s-1. Experimental cultures
 97

were obtained in duplicate over 7 days of cultivation. When algae

reached the stationary phase, samples were harvested by centrifugation
at 4000 rpm for 20 min., washed with ammonium formate (Zhu and Lee
1997) and centrifuged again (Nova Técnica, Piracicaba, Brazil). The
algal pellet was transferred to a dish and dried in a dehydrator at 45 ºC
(Zhejiang, China) for lipid and fatty acid analysis.

Microscopic observation and visualization of lipid bodies

The microalgal growth and intracellular lipid bodies were

investigated by microscopic observation during the algal growth phase
(5 and 7 days of cultivation) conducted with an inverted microscope
(Nikon Eclipse Ti-S, Tokio, Japan), using 20x and 63x objectives. The
intracellular lipid bodies of the algal cells were visualized using Nile
Red stain (9-(diethyl amino) benzo [a]phenoxazin-5(5H)-one, Sigma –
Aldrich), which was prepared as a stock solution of 250 mg L-1 in
acetone. One milliliter of algae (grown for 14 days) was centrifuged at
4000 rpm for 10 min and a pellet was re-suspended in 1 mL of 20%
DMSO. After vortexing for 10 min at room temperature, cells were
centrifuged at 4000 rpm for 10 min. The pellet was further suspended in
1 mL of water and vortexed before adding Nile Red strain (12.5 µL) and
incubating for 5 min in the dark at room temperature (Ahmad et al.
2013). Stained cells were visualized using a Leica TCS – SP5 laser
scanning confocal microscope (Wetzlar, Germany). Images were
acquired using a 100x objective (HCX PL APO CS with a numerical
aperture of 1.44 - oil immersion objective) with a Leica Type F
immersion liquid. Images were taken with a Leica microsystem camera.
The acquisition and processing of data were carried out using the LAS
AF Lite software.

Culture medium recycling experiment

To evaluate the influence of the recycling of the medium with the

optimum DC concentration on the biomass and lipid productivity of N.
gaditana, the medium was reused after harvesting by centrifugation for
the next two cultivation cycles. The cultivation cycles were performed
in duplicate using inverted conical photobioreactors with a working
volume of 3.5 L (Fig. 4.2). In order to maintain satisfactory growth
during three cultivation cycles, 3.0 L of the algal concentration was
harvested by centrifugation for each cultivation cycle, and a small
portion (500 mL) of algal inoculum was kept inside the photobioreactor.
98

Prior to a subsequent cultivation, the spent medium (supernatant) was

re-introduced into the photobioreactors to proceed with the next
cultivation cycle. For each cultivation cycle, the biomass was collected
for lipid/fatty acid determination and 50 mL of the supernatant was
recovered to determine the total nitrogen and total phosphorus content.
The culture conditions were: 25 ± 2 °C, 12 h:12 h light/dark and 80
µmol m-2 s-1. Total nitrogen was determined through the persulfate
digestion method on a Hach spectrophotometer (Loveland, USA) using
Permachem® reagent kits. Total phosphorus was determined by the
phosphorus (4500-P)/vanadomolybdophosphoric acid calorimetric
method described in APHA (2005).

Fig. 4-2 - Scheme showing the procedure to test the potential for the
reuse of the water medium with a DC concentration of 75% in
successive N. gaditana cultivation cycles.

Biomass

Supernatant* Centrifuge 3.0 L

Biomass

1st cultivation 2nd cultivation

Centrifuge cycle Supernatant*
3.0 L cycle

Biomass
Centrifuge 3.5 L

3rd cultivation
Inverted conical photobioreactor cycle
Working volume: 3.5 L

Supernatant* Exhausted medium

discarded

* 50.0 mL was collected for nitrogen and phosphorus determination

Mixotrophic cultivation on optimum desalination concentrate

After the culture medium based on 75% DC concentration

(optimum DC medium) had been established, N. gaditana was cultured
mixotrophically using three different additional carbon sources: glucose
(Vetec®), glycerol (Nuclear) and glycerin (crude glycerin) with three
 99

different concentrations (1.0, 2.0 and 5.0 mL or g L-1 depending on the

substrate). All of the batch experiments were performed in 300-mL
Erlenmeyer flasks applying 7 days of cultivation, under the conditions
(temperature, air, light:dark cycle and light intensity) equal to those
described in previous section.

Determination of microalgal biomass

The N. gaditana biomass concentration was estimated

gravimetrically in terms of ash-free dry weight (AFDW) and also with
the gravimetric method described by Zhu and Lee (1997). The biomass
productivity (BP, g L-1 day-1) during the culture period was calculated
from the equation:

BP = (Xt - X0)/(tx - t0)(1)

where, Xt is the biomass production (g L-1) at the end of the exponential

growth phase (tx) and X0 is the initial biomass production (g L-1) at t0
(day).

The cellular lipid productivity was calculated as the product of BP

and the fractional content (w/w) of the macromolecular pool in the
biomass according to Dickinson et al. (2013):

Lipid productivity (LP, mg L-1 d-1) = Biomass productivity (BP) x

lipid/biomass (w/w)(2)

Lipid extraction and fatty acids analysis

Total lipids were extracted by the Soxhlet method with petroleum

ether for 6 h, after acid digestion with 4 N HCl, followed by
concentration in a rotary evaporator, drying in an oven and weighting
(AOAC 996.06 2005). The fatty acids composition was determined after
conversion of the fatty acids to their corresponding methyl esters. The
fatty acid methyl esters (FAME) were characterized on a gas
chromatograph, model GC-2014 (Shimadzu, Kyoto, Japan), equipped
with split-injection port, flame-ionization detector, a Restek a 105 m-
long capillary column (ID = 0,25 mm) filled with 0.25 μm of 10%
cyanopropylphenyl and 90% biscyanopropylsiloxane. Injector and
detector temperatures were both 260°C. The oven temperature was
initially set at 140°C for 5 min, and then programmed at 2.5°C min-1.
100

The qualitative fatty acids composition was determined by comparing

the retention times of the peaks with the respective fatty acids standards
(Sigma, St. Louis, USA). The quantitative composition was obtained by
area normalization and expressed as mass percent.

Statistical analysis

Statistical analysis was performed applying one-way analysis of

variance (ANOVA), using STATISTICA Software (version 7.0) from
StatSoft Inc. (2004). For all of the data analysis, a p-value of <0.05 was
considered statistically significant. Where significant differences were
observed, treatment means were differentiated using pairwise
comparisons applying the Tukey test.

Results and Discussion

Desalination concentrate

Desalination concentrate (DC) is often rich in chlorides, sodium

and calcium. In addition, other nutrients (nitrogen and phosphorus) and
trace elements necessary for microalgae growth, including potassium,
magnesium, and iron were all detected in DC (Table 4.1). Since DC has
abundant mineral salts, the components existing in the DC were tested
for advantageous microalgae growth. To assess the applicability of DC
as a culture medium, the desalination waste was mixed with the F/2
medium before the algal cultivation (Fig. 4.1). In some cases,
microalgae cannot grow normally in DC due to its high mineral salts
strength. Similarly, C. vulgaris cannot grow normally in DC. For
example, in a previous study by Matos et al. (2015), the freshwater
species C. vulgaris was highly inhibited using DC as culture medium.
That was because the DC is abundant in sodium and chlorides
associated with high salinity. In addition, it was detected in DC a small
quantity of ammonia (1.35 mg L-1) and sulphate (138.0 mg L-1), some of
which are toxic to aquatic life (Greenlee et al. 2009). On the other hand,
marine microalgae Nannochloropsis gaditana performs well when
exposure to a high DC concentration with higher biomass concentration
and lipid content. These characteristics of DC suggested that it
represents a good medium for marine N. gaditana growth in which can
be replaced by conventional nutrients.
 101

Effect of DC concentration on biomass and lipid content of N. gaditana

The biomass concentration and lipid content of the experimental

cultures with 0%, 25%, 50%, 75% and 100% of DC concentration are
shown in Fig. 4.3. The microalgae cultured in 25% and 50% DC
reached biomass concentrations of 0.63 and 0.81 g L-1, respectively. In
the control culture (0% DC), the biomass concentration was around 0.71
g L-1. When N. gaditana was cultured in the 75% DC medium, the
microalgae presented the maximum biomass concentration (0.96 g L-1).
However, algal growth was inhibited when the DC concentration was
increased to 100%, and the biomass yield decreased considerably to
about 0.33 g L-1, but the lipid content remained relatively high, reaching
11.4% (w/w). Lipid accumulation to counterbalance the osmotic
pressure has been previously reported (An et al. 2013), while lower N.
gaditana biomass under 100% DC may be associated with an inability
to maintain a satisfactory growth with only the mineral composition
presented in DC (Table 4.1).

Fig. 4-3 - Effect of concentration of desalination concentrate (DC) on

biomass concentration (g L-1) and lipid content (%).
1,2 16
Biomass
Lipid content 14
1,0
12

Lipid content (%)

Biomass (g L-1)

0,8
10

0,6 8

6
0,4
4
0,2
2

0,0 0
0% 25% 50% 75% 100%
Desalination concentrate (DC)

The lipid content was observed to increase as the DC concentration

increased from 25 to 75%, although a high lipid content was observed at
under 75% DC (12.6% w/w), which is approximately treble that
obtained for the control culture (4.7%) (as shown in Fig. 4.3). This
102

result is consistent with previous reports that Chlorella vulgaris has a

higher lipid content when grown in desalination concentrate mixed with
BBM medium (12.5%) than in 100% standard BBM medium (8.5%)
(Matos et al. 2015). However, the same authors observed that for
optimum C. vulgaris growth, a maximum of 25% DC should be used. In
contrast to the freshwater alga C. vulgaris, the marine alga N. gaditana
can easily adapt to exposure to a high DC concentration. Notably, the
increases in the biomass concentration and lipid content of N. gaditana
are much greater on exposure to high DC concentration. For this reason,
and based on our observations (biomass concentration and lipid content)
during the experimental procedures employing DC in the culture
medium, we conducted the subsequent studies using a DC concentration
of 75%.
To visualize the N. gaditana cells under microscopic observation
in the optimum medium containing a 75% DC concentration, the initial
cultures (5 days) of algal cells were viewed under phase-contrast
observation, as shown in Fig. 4.4 (a). After 7 days of cultivation, the
algal cells appeared to be well adapted to the new proposed medium. As
shown in Fig. 4.4 (b), the marine N. gaditana cells were ellipsoidal with
a diameter of 3-5 µm and, rich in chlorophyll. After staining with Nile
Red, the bright yellow fluorescence from intracellular lipid globules and
red fluorescence from chlorophyll could be observed (Fig. 4.4 (c)).
Fig. 4-4 - Images of N. gaditana cultured in medium with optimum DC concentration of 75%. (a) Initial culture of N.
gaditana viewed under phase-contrast observation using 20x objective; (b) Algal cells viewed under conventional
light observation using 63x objective, showing green appearance of the chlorophyll; (c) Stationary phase of N.
gaditana showing oil droplets with gold color after Nile Red staining. Red chlorophyll auto-fluorescence. Algal cells
viewed under scanning confocal microscopy observation using 100x objective with 530 nm excitation wavelength
and 575 nm emission filters. Scale bars = 5 µm.

a b c
103
104

Effect of the medium reuse on biomass and lipid productivities

To study the effect of medium recycling on the performance of

autotrophic N. gaditana culture, the optimum DC medium was reused
until the total depletion of nutrients. The results for the biomass and
lipid productivities of N. gaditana cultured autotrophically in the reused
optimum DC medium are shown in Table 4.2.

Table 4-2 - Biomass and lipid productivities of N. gaditana cultured in

medium with optimum DC concentration reused in successive cycles.
Cultivation Biomass BP Lipid LP
Cycle (g L-1) (mg L-1 d-1) (%) (mg L-1 d-1)
st a a a
1 1.06 ± 0.2 151 ± 29 9.5 ± 0.8 14.3 ± 2.2a
nd st ab ab b
2 (1 reuse) 0.62 ± 0.1 88 ± 14 13.6 ± 0.7 11.9 ± 1.7ab
rd nd b b b
3 (2 reuse) 0.14 ± 0.02 20 ± 2.5 14.4 ± 1.0 2.8 ± 0.4c
Data are means ± SD (n = 2). Each cycle represents 7 days of cultivation.
Different letters in the same column correspond to significant differences
(p<0.05).

Nannochloropsis gaditana cells supported the spent medium for

three cultivation cycles. The biomass accumulation during three
cultivation cycles decreased as follows: 1st cycle = 1.06, 2nd cycle = 0.62
and 3rd cycle = 0.14 g L-1. A significant difference (p<0.05) in the
biomass concentrations for the three cultivation cycles was noted, since
the total biomass concentration decreased considerably. On the other
hand, the lipid contents of the microalgae cultivated in the recycled
optimum DC medium showed a tendency toward an increasing with
consecutive reuses of the recycled optimum DC medium. In addition,
Fig. 4.5a shows the correlations between the lipid productivity (LP),
biomass productivity (BP) and lipid content of N. gaditana for the three
cultivation cycles performed. The biomass productivity appears to be
directly correlated with the lipid productivity, but does not necessarily
correlate with lipid content. Similar observations have also been
reported by Griffiths and Harrison (2009), who noted that a high lipid
content in microalgae does not necessarily represent high lipid
productivity. In fact, the first cultivation cycle showed the highest
biomass productivity (BP = 151 mg L-1 day-1) and lipid productivity
(14.3 mg L-1 day-1). The second cultivation cycle presented lipid content
(13.6%) higher than the first cultivation cycle (9.5%) while the highest
lipid content (14.4%) was reached in the third cultivation cycle (Table
4.2). The result obtained in the third cultivation cycle is comparable
 105

with the study of Zhao et al. (2015) in Nannochloropsis gaditana Q6,

but in disagreement with the results of Camacho-Rodríguez et al. (2015)
and Mayers et al. (2014) involving Nannochloropsis sp.
Considering that microalgae generally consume the majority of the
nutrients, notably nitrogen and phosphorus, in the growth medium
(Pittman et al. 2011), the concentrations of N and P in the reused
optimum DC medium were measured. Park et al. (2011) mentioned that
the N/P ratio in wastewater can ranged from 4/1 to 40/1 - N/P. The
initial N and P ratio in optimum DC medium was 18.4 N/P (415.0 mg N
/ 22.5 mg P), which was marginally higher than the theoretical ratio
required for the growth of algae (16 N/P) (Redfield 1958), illustrating
that the N/P ratio in DC for N. gaditana growth is suitable.
As can be seen in Fig. 4.5b, the initial N and P concentrations in
the optimum DC medium were around 415 mg L-1 total N and ~ 22.5
mg L-1 total P. In the first cultivation cycle, N. gaditana cells consumed
60% of the total N and 71.1% of total P available in the culture medium
and after three cultivation cycles the corresponding values were 80%
and 86%, respectively. Because of N and P was totally depleted, over
the course of the cultivation cycles, the lipid content trend toward an
enhancement with consecutive reuses of the recycled optimum DC
medium. These results show that the reused optimum DC medium does
not only support algal growth, but also enhances the lipid content.
Furthermore, the reused water and the mineral salts (N and P) present in
the optimum DC medium were suitable for N. gaditana culture even
after 3 cultivation cycles.
106

Fig. 4-5 - Results for N. gaditana cultured in medium with the optimum
DC concentration (75%) applied in three cultivation cycles. Each cycle
involved 7 days of cultivation – 21 days in total. a) Correlations between
lipid productivity (LP), biomass productivity (BP) and lipid content of N.
gaditana for successive cultivation cycles; b) Total nitrogen and total
phosphate consumption by N. gaditana during the cultivation cycles.
a 16,0 16,0
Lipid productivity (mg L-1 day-1)

14,0 14,0

Lipid content (%)

12,0 12,0
3rd cycle

10,0 10,0
2nd cycle

8,0 8,0

1st cycle
6,0 6,0

4,0 4,0

2,0 Lipid productivity Lipids 2,0

0,0 0,0
0 30 60 90 120 150 180
Biomass productivity (mg L-1 day-1)
b 450 30
Nitrate (mg Lˉ¹)
400
Phosphate (mg Lˉ¹) 25
350
N (mg L-1)

P (mg L-1)

300 20

250
15
200

150 10

100
5
50

0 0
Initial culture 1st cycle 2nd cycle 3rd cycle

Cultivation cycles

Effect of mixotrophic cultures on the biomass and lipid productivities

Mixotrophic cultures of N. gaditana were conducted to investigate

the biomass and lipid productivities under optimum DC medium.
Glucose, glycerol and glycerin were used as substrates for microalgae
cultivation. In this study, 2 g L-1 of glucose supplementation produced
 107

1.10 g L-1 of biomass concentration and 8.7 mg L-1 day-1 of lipid

productivity. This concentration of glucose was observed to be the most
favorable carbon substrate and concentration for N. gaditana
mixotrophic cultivation (Table 4.3). On using glycerol as the substrate,
N. gaditana cells showed lower biomass and lipid productivity
compared to glucose supplementation. When N. gaditana cells were
grown on a glycerin substrate, high lipid content (8.5-12%) and low
biomass concentration (0.19-0.25 g L-1) were noted. Far higher rates of
growth were obtained with the glucose substrate than the others tested,
which included sugars, sugar alcohols and organic acids (Perez-Garcia
et al. 2011). In our experiments with an additional carbon source,
glucose could be considered a ―preferred substrate‖ for mixotrophic N.
gaditana cultivation because higher biomass and lipid productivities
were noted. This could be because glucose possesses a high energy
content per mol compared with other substrates (Boyle and Morgan
2009).

Table 4-3 - Comparison of biomass and lipid productivities of N.

gaditana cultured in medium with optimum DC concentration, with
additional carbon source in different concentrations (1.0, 2.0 and 5.0 g
or mL L-1) with 7 days of mixotrophic cultivation.
Glucose Biomass BP Lipid LP
(g L-1) (g L-1) (mg L-1 day-1) (%) (mg L-1 day-1)
a a a
1.0 0.72 ± 0.2 102 ± 28 4.8 ± 0.6 5.0 ± 1.9a
2.0 1.10 ± 0.1a 156 ± 14a 5.5 ± 0.7a 8.7 ± 1.9a
a a a
5.0 0.95 ± 0.1 135 ± 15 1.0 ± 0.1 1.3 ± 0.2a
-1
Glycerol (mL L )
1.0 0.42 ± 0.2a 60 ± 28a 6.2 ± 0.9a 3.9 ± 2.3a
a a a
2.0 0.15 ± 0.05 21 ± 7 3.8 ± 0.3 0.8 ± 0.3a
a a
5.0 0.10 ± 0.01 2.5 ± 1 11.0 ± 1.3 0.3 ± 0.2a
a
-1
Glycerin (mL L )
1.0 0.19 ± 0.05a 27 ± 7a 9.0 ± 1.2a 2.4 ± 0.9a
a a
2.0 0.25 ± 0.1 35 ± 14 12.0 ± 0.8 4.3 ± 2.0a
a
a a
5.0 0.22 ± 0.1 31 ± 14 8.5 ±0.5a 2.6 ± 1.3a
Data are means ± SD (n = 3). Different letters in the same column
correspond to significant differences (p<0.05).
108

Effect of experimental cultures on fatty acids composition of N. gaditana

The fatty acids (FAs) composition of marine N. gaditana after

applying the proposed experimental conditions is shown in Table 4.4.
As a percentage of the total FA in N. gaditana, the predominant FAs for
all DC concentrations in the medium were palmitic acid (C16:0; 29.4-
53.4%), palmitoleic acid (C16:1, 22.2-30.7%) and oleic acid (C18:1,
4.6-14.0%), representing ~ 81.1% of total FA content. Fatty acids
present at moderate levels were eicosapentaenoic acid (C20:5n3, 0.2-
12.2%) and myristic acid (C14:0, 4.4-6.5%), representing ~ 15.5% of
the total FA content. Fatty acids present at trace levels were linoleic acid
(C18:2n, 0.5-3.3%) and linolenic acid (C18:3n, 0.4-0.8%), representing
~ 3.4% of total FA contents. Algal FAs were highest in saturates (SFAs;
39.0-62.3%) followed by monounsaturates (MUFAs; 34.6-41.5%) and
lowest polyunsaturates (PUFAs; 1.2-17.0%), when N. gaditana cells
were cultivated media with increasing DC concentrations. It is evident
that there is a tendency for N. gaditana cells to produce higher SFAs
and lower PUFAs with increasing DC concentration in the culture
medium (Table 4.4). For example, on increasing the DC concentration
from 0% to 100%, the percentage of C16:0 increased from 29.4 to
53.4%, and the percentage of C20:5n3 dropped from 12.2 to 0.2%.
In relation to the total FA content of the N. gaditana biomass after
three cultivation cycles performed with the optimum DC medium,
palmitic acid (40.2-50.6%) was predominant in all cycles (Table 4.4).
During the first cultivation cycle the N. gaditana cells produced more
PUFAs (16.3%), especially C20:5n3 acid (5.7% of total PUFAs).
However, after the second cultivation cycle the algal cells were rich in
SFAs (60.1%) and after the third cultivation cycle the biomass was
abundant in MUFAs (38.3%). The data for the FAs obtained after three
cultivation cycles of N. gaditana shown in Table 4.4, indicate that the
reuse of the water, based on the optimum DC medium, could improve
the diversity of the fatty acids composition of N. gaditana. Over the
course of the reuse cycles with the optimum DC medium, PUFAs
(C18:2, C18:3 and C20:5n3) have a tendency to decrease whereas the
SFAs (C14:0 and C16:0) and MUFA (C18:1) have a tendency to
increase.
Regarding the additional of a carbon source to the optimum DC
medium, SFAs (mostly C16:0), accounted for approximately 39.5-
56.8% of the total FA content. Interestingly, on adding an extra carbon
source (glucose, glycerol and glycerin) to the optimum DC medium, the
production of SFAs decreased, while the percentage of PUFAs
 109

increased. In contrast, when N. gaditana cells were cultured in media

containing increasing DC concentration and with limited nutrients
(reuse of optimum DC water medium), the increase in SFAs may play a
critical role in providing an appropriate degree of membrane fluidity for
growth under ―stress‖ condition. The additional of a carbon source to
the optimum DC medium allows a shift in the metabolism of the algae
resulting in more unsaturated (MUFAs + PUFAs) fatty acids being
produced.

Conclusions

Peaks in the biomass concentration (0.96 g L-1) and lipid prod.

(16.8 mg L-1 day-1) were observed using a DC concentration of 75%,
indicating that this is the ideal concentration for the autotrophic growth
of N. gaditana. The reuse of the optimum DC medium was supported by
N. gaditana for three cultivation cycles. Increasing the DC concentration
weakens the dominance of C20:5n3 (PUFA) and increases the
percentage of C16:0 (SFA). These results indicate that DC is a suitable
medium for N. gaditana cultivation and also demonstrate the
valorization of desalination wastewater thought its application to algal
mass production.
 110

Table 4-4 - Main fatty acids found in N. gaditana under different experimental conditions.
Fatty acids (% TFAs) according to percentage of desalination concentrate (DC) in the medium
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:5n3 SFA MUFA PUFA
0% DC 6.5 29.4 30.7 0.5 4.8 3.3 0.8 12.2 39.0 41.5 17.0
25% DC 6.5 44.5 24.1 1.7 9.3 2.6 0.5 3.6 56.6 35.5 7.5
50% DC 5.5 46.6 24.4 1.4 9.4 2.0 0.6 4.2 55.5 34.6 7.4
75% DC 4.4 48.7 23.7 1.7 12.5 1.7 0.6 3.4 56.9 36.7 7.0
100% DC 5.5 53.4 22.2 2.4 14.0 0.5 0.4 0.2 62.3 36.4 1.2
Fatty acids (% TFAs) obtained from cultures in the reuse of medium with optimum DC concentration
1st cycle 3.8 40.2 18.7 2.7 9.5 5.5 5.1 5.7 51.2 30.8 16
2nd cycle 4.2 50.6 12.9 2.4 13.7 4.8 4.2 0.8 60.1 28.3 10
3rd cycle 4.7 45.6 17.6 1.9 20.0 2.7 1.5 1.2 54.4 38.3 6.0
Fatty acids (% TFAs) profile under mixotrophic cultivation in medium with optimum DC concentration
Glucose
(1.0 g L-1) 10.1 50.5 22.1 1.8 8.0 0.6 0.7 0.8 64.0 9.7 3.6
(2.0 g L-1) 10.4 43.7 23.2 1.7 7.9 2.8 1.0 3.2 59.8 35.5 7.0
(5.0 g L-1) 12.2 41.5 25.8 1.5 10.1 2.2 0.8 3.3 60.4 35.9 7.3
Glycerol
(1.0 mL L-1) 4.8 56.8 14.2 3.1 9.6 3.2 0.8 0.5 63.3 27.3 5.9
(2.0 mL L-1) 7.0 54.0 10.9 5.0 14.8 2.2 0.4 0.8 68.5 26.8 4.7
(5.0 mL L-1) 6.3 44.4 11.1 3.6 22.0 3.4 0.5 2.3 67.5 34.2 9.3
 111

CHAPTER 5

EFFECTS OF DIFFERENT PHOTOPERIOD AND TROPHIC

CONDITIONS ON BIOMASS, PROTEIN AND LIPID
PRODUCTION BY THE MARINE ALGA Nannochloropsis
gaditana AT OPTIMAL CONCENTRATION
OF DESALINATION CONCENTRATE

Abstract

This study investigated the cultivation of the marine alga

Nannochloropsis gaditana in a medium based on desalination
concentrate (DC) with an optimal concentration of 75% DC, under three
trophic conditions and four photoperiod schedules. N. gaditana
produced a peak biomass concentration (1.25 g L-1) under mixotrophic
culture condition and a photoperiod of 16L:08D. N. gaditana cells
compensate to different light-dark regimes producing different amounts
of protein (17.9-44.8%). The intracellular lipid content in N. gaditana
cells increased both under autotrophic conditions with a 16L:08D cycle
(16.7%), and under mixotrophic conditions with a 08L:16D cycle
(15.7%). In heterotrophic culture, N. gaditana cells were rich in
polyunsaturated fatty acids (46.0%). This study demonstrates an
alternative approach to enhancing intracellular lipid content of the
marine alga N. gaditana by modifying the photoperiod, trophic
conditions and stress-salinity-conductivity with the use of a DC-based
medium.

Keywords: Inland desalination wastewater; microalgae; photo-osmotic;

lipid enhancement; saturated fatty acids.
 112

Graphical Abstract

160 40 AUTOTROPHIC MIXOTROPHIC HETEROTROPHIC

Biomass prod. Lipid prod. Conductivity
140 35
120 30
100 25
80 20
60 15
40 10

Biomass and lipid

20 5

Conductivity (mS cm-1)

productivities (mg L-1 day-1)

0 0
0% 25% 50% 75% 100%
Desalination concentrate (DC)
 113

Introduction

Algal biodiesel is a third generation biofuel which shows potential

for the sustainable production of fuel with a small footprint, and it offers
reduced competition with food crops (Carioca et al. 2009). Although
microalgal cultivation does not require arable land, it is associated with
a high demand for large quantities of water, fertilizers and solar
radiation, which considerably limits the availability of suitable locations
(Varshney et al. 2015). A series of strategies is being developed to
circumvent these issues, such as the use of marine species (Simionato et
al. 2011), cultivation coupled with wastewater treatment plants
(Dickinson et al. 2013; Mahapatra et al. 2014; Pittman et al. 2011), and
mixotrophic cultivation using readily available low cost or industrial
waste-derived carbon sources (Perez-Garcia et al. 2011; Selvaratnam et
al. 2015). Different carbon sources have been evaluated for mixotrophic
or heterotrophic microalgal cultivation, including, glucose (Mallick et
al. 2011), acetate (Isleten-Hosoglu et al. 2012), glycerol (Abad and
Turon 2012), xylose (Leite et al. 2016) and urban wastewater
(Henkanatte-Gedera et al. 2015), and all have generally been shown to
increase intracellular lipid content.
The most common procedure for the cultivation of microalgae is
autotrophic growth. Under autotrophic cultivation the cells harvest light
energy and use CO2 as a carbon source. A feasible alternative for
autotrophic cultures, although restricted to a few microalgal species, is
the use of their heterotrophic growth capacity in the absence of light,
replacing the fixation of atmospheric CO2 by autotrophic cultures with
organic carbon sources dissolved in the culture medium (Perez-Garcia et
al. 2011). The mixotrophic growth regime is a variant of the
heterotrophic growth regime, where CO2 and organic carbon sources are
simultaneously assimilated and both respiratory and photosynthetic
metabolism operate concurrently (Ji et al. 2014). Considering that
microalgal species are not truly mixotrophs, although they have the
ability to switch between phototrophic and heterotrophic metabolisms,
Nannochloropsis is commonly cultivated under either mixotrophic
(Cheirsilp and Torpee 2012) or heterotrophic conditions (Perez-Garcia
et al. 2011).
In order to exploit microalgae for biofuel production, a better
understanding of the parameters influencing the growth rate and the
biomass and lipids accumulation is critical to optimize the productivity
(Griffiths and Harrison 2009). As in the case for all photosynthetic
organisms, light is a major factor to be considered, since it provides all
114

the energy that supports metabolism. The capacity for an effective and
prompt acclimation is therefore an important parameter to be considered
in the characterization of an algal species for biomass production
(Simionato et al. 2011). The marine species Eustigmatophyceae
Nannochloropsis gaditana was selected for this study because of its
high growth rate and lipid productivity, as well as its wide
environmental tolerance (Selvakumar & Umadevi 2014; Mitra et al.
2015). Furthermore, the marine algae of the Nannochloropsis genus are
able to synthesize high value chemicals such as proteins and fatty acids
with medium (C16 and C18) and long (C20:5ɷ3) chain carbons, which are
ideal lipid sources for the production of the nutraceutically valuable
eicosapentaenoic acid (EPA) and biodiesel (Matos et al. 2016; Mitra et
al. 2015; Tibbetts et al. 2015).
Several recent studies have analyzed the growth of microalgae
under a variety of wastewater conditions, in particular growth in
multiple municipal, industrial and agricultural wastewaters (Pittman et
al. 2011; Dickinson et al. 2013). These studies have mainly been
focused on evaluating the ability of algae to remove N and P, and in
some instances metals, from wastewater. However, more recently
studies have extended the feasibility of algal systems in combination
with desalination concentrate (DC) for simultaneous N and P removal
with biomass and lipid production (Volkmann et al. 2008; Sánchez et al.
2015; Matos et al. 2016b). Furthermore, DC is abundant in inorganic
minerals, such as Cl-, Na+ and Ca+2, which can lead to salt stress in
microalgae cultivation, especially due to the effect of salinity on
biochemical composition (Volkmann et al. 2008). For example, Matos
et al. (2014) cultivated freshwater Chlorella vulgaris in dilutions of
25%, 35%, 45% and 55% DC mixed with Bold Basal Medium (BBM)
and demonstrated that incubating C. vulgaris with the higher DC
concentrations of 35-55% resulted in growth-salt stress accompanied
with lower biomass concentration and lipid production in comparison to
control (BBM). According to Kumar et al. (2015), the salt concentration
influences algae via effects on osmotic stress, salt stress, and cellular
ionic ratios. For marine algae, salinity is expected to have some effect
on the metabolism, especially on important cell membrane constituents,
such as lipids (Takagi & Karseno 2006) and fatty acids composition (An
et al. 2013). In this context, cells of the marine alga N. gaditana were
successfully cultured in an optimized 75% DC medium under two-stage
photoautotrophic-mixotrophic conditions using glucose (2.0 g L-1) as a
carbon source (Matos et al. 2015). In addition, N. gaditana has a
tendency toward the synthesis of higher saturated fatty acids (C16:0
 115

palmitic and C18:0 stearic acids) when increasing DC concentrations in

the culture medium. Hence, the choice of marine N. gaditana in this
study was motivated by its metabolic versatility that includes the ability
to cope with osmotic changes and alterations in the salt ratios presented
in DC as well as the capability to tolerate a broader range of DC-
salinities with a satisfactory growth (Matos et al. 2016).
This investigation is an extension of previous study on the use of
DC as a potential substrate for N. gaditana cultivation (Matos et al.
2015), which advanced our understating of the effect of optimal DC
concentration on algal growth. The aims of the present study are: (1) to
confirm the optimal DC concentration for N. gaditana growth and
subsequently evaluate the effect of DC concentrations on the production
of biomass and lipid productivities; (2) to evaluate the performance of
N. gaditana under autotrophic, mixotrophic and heterotrophic culture
conditions; and (3) to determine the effect of different photoperiod
regimes on cell growth, biomass concentration, protein/lipids production
and fatty acids composition by exposure to four different light/dark
(L/D) cycles, 24L:00D, 16L:08D, 12L:12D, and 08L:16D.

Material and Methods

Algal strain and culture
The marine species Eustigmatophyceae Nannochloropsis gaditana
(clone 130) was kindly supplied by Banco de Micro-organismos
Marinhos Aidar & Kutner (BMA&K) of the Oceanographic Institute at
the University of São Paulo and maintained in autoclaved F/2 seawater
medium (see Table 4.1) suitable for marine algae (Guillard 1975).

Inland desalination concentrate

Desalination concentrate (DC) was collected from an inland

desalination plant, located in São João do Cariri, Paraíba, Brazil. The
DC samples were collected in a 100-L plastic container and stored at -20
°C in the Laboratory of Food Biotechnology, Federal University of
Santa Catarina, Santa Catarina, Brazil. The chemical composition of the
DC was determined according to standard methods (APHA 2005). The
Cl, Fe, ammonium (NH4+), total phosphorus (PO43-) and total hardness
(CaCO3) values were determined using a Hach Spectrophotometer
(Loveland, USA). Total nitrogen (TN) and sulfate were measured
photometrically using Permachem® Reagent kits. A Shimadzu atomic
absorption spectrophotometer (Kyoto, Japan) was used to determine Ca
116

and Mg and a flame photometer (Diadema, Brazil) to determine K and

Na. The conductivity and total dissolved solids were measured using a
Tecnal conductivity meter (Piracicaba, Brazil) and the pH was measured
using a Digimed pHmeter (Campinas, Brazil).
The DC is rich in Cl-, Na+ and Ca2+ and contains other nutrients (N
and P) and trace elements necessary for microalgae growth, including
Si, K+, Mg+2, Fe+3. The average electrical conductivity of the DC is 4.5
± 0.5 mS cm-1 (see Table 4.1).

Experimental procedures

Optimal DC concentration for N. gaditana growth

The experimental medium contained DC concentrations of 25%,

50%, 75% and 100% in F/2 medium (based on seawater), while F/2
medium with 0% DC was used to conduct control tests. An initial N.
gaditana concentration of 105 cells mL-1 was used for the preparation of
the inoculum. Autotrophic cultures were performed in 500-mL
Erlenmeyer flasks with incubation at room temperature (25 ± 2 °C),
sparged with atmospheric saturated air-CO2 at concentration of 2-3%
vol/vol and a flow rate of 1.5 L min-1, using a photoperiod regime of
12L:12D, under a light intensity of 80 µmol photon m-2 s-1 provided by
cool white fluorescent lamps (LUMILUX, OSRAM, Brazil). The
incubations were done in duplicate over 7 days of cultivation. When the
N. gaditana reached the stationary phase, samples were collected and
centrifuged (Nova Técnica, Piracicaba, Brazil) at 4000 rpm for 20 min.,
washed with ammonium formate (Zhu and Lee 1997) and centrifuged
again. The algal pellet was transferred to a dish and dried in a
dehydrator at 45 ºC (Zhejiang, China) for further analysis.

Effect of trophic conditions and photoperiod on N. gaditana cultures at

optimal DC concentration

Experiments were carried out using a in a 4.0 L working volume in

a 6.0 L photobioreactor (New Brunswick Bioflo 2000 Fermenter, New
Jersey, US) operating in batch mode. The photobioreactor was sterilized
by autoclaving at 121°C for 15 min. prior to the algal cultivation. An F/2
culture medium with DC concentration of 75% and a conductivity of
8,200 ± 500 µS cm-1, was used in all experiments. Each experiment
started with a pre-culture grown at 105 cells mL-1 in the exponential
phase, which was diluted to 10% and cultivated for 9 days. The airflow
 117

in the photobioreactor was provided via filtered saturated air-CO2. The

temperature was kept at 25 ± 1°C and the culture was continuously
agitated (75 rpm).
In order to identify the growth medium for N. gaditana which
would provide the highest biomass and lipid productivity, three different
trophic conditions (autotrophic, mixotrophic and heterotrophic) and four
different photoperiod regimes, i.e., light/dark (L/D) cycles of 24L:00D,
16L:08D, 12L:12D, and 08L:16D were studied. For autotrophic
cultivation, the cultures were illuminated with a light intensity of 100
µmol photon m-2 s-1 by four 20 W cool white fluorescent lamps directed
to the surface of the bioreactor at different photoperiods. For
mixotrophic and heterotrophic cultivations, glucose was used as a
carbon source at a concentration of 2.0 g L-1, with and without light
illumination, respectively. When the N. gaditana cells reached the
stationary growth phase, the culture was harvested by centrifugation
using a Heinz Janetzki SD600 centrifuge (Engelsdorf-Leipzig,
Germany) at 3500 rpm for 25 min., washed with ammonium formate
(Zhu and Lee, 1997) and then centrifuged again. The algal pellet was
transferred to a porcelain crucible and dried in an oven at 40 ºC prior to
further analysis.

Determination of algal growth and biomass

Cell counting was performed every day using a hemocytometer

coupled to a microscope (Olympus, Germany). The growth rate of each
culture was calculated based on the following relationship:

ln = µt, (1)

where X0 = initial biomass concentration, at time; X = final

biomass concentration and µ = specific growth rate (day-1).
The biomass concentration was measured, every two days of
cultivation, gravimetrically in terms of ash-free dry weight (AFDW) and
also with the gravimetric method described by Zhu and Lee (1997). The
biomass productivity (BP, g L-1 day-1) during the culture period was
calculated using equation (2):

BP = (Xt - X0)/(tx - t0) (2)

118

where, Xt is the biomass production (g L-1) at the end of the exponential

growth phase (tx) and X0 is the initial biomass production (g L-1) at t0
(day-1).
Cellular protein and lipid productivities were calculated as the
product of BP and the fractional content (w/w) of the macromolecular
pool in the biomass according to Dickinson et al. (2013) as follows:

Lipid productivity (LP, mg Lip. L-1 d-1) = BP x lipid/biomass (w/w)

(3)

Protein productivity (PP, mg Prot. L-1 d-1) = BP x protein/biomass

(w/w) (4)

Biochemical analysis

Total nitrogen was determined by the Kjeldahl method after acid

digestion (AOAC 991.20), ammonium addition, steam distillation and
titration with 0.1 N HCl (AOAC 2005). Protein content was calculated
using a nitrogen-to-protein conversion factor of N x 4.78 (Lourenço et
al. 2004).
After acid digestion with 4.0 N HCl for 6 h, intracellular lipids
were extracted with petroleum ether by the Soxhlet method (AOAC
963.15), concentrated in a rotary evaporator, dried in an oven and
weighed (AOAC 2005).
The fatty acids composition was determined after converting the
fatty acids to their corresponding fatty acid methyl esters (FAME),
which were determined by gas chromatography using a Shimadzu GC-
2014 chromatograph (Kyoto, Japan), equipped with a split-injection
port, flame-ionization detector and Restek capillary column (length =
105 m; ID = 0.25 mm) coated with 0.25 μm of 10% cyanopropylphenyl
and 90% biscyanopropylsiloxane. Injector and detector temperatures
were 260°C, the oven temperature was initially set at 140°C for 5 min
and, programmed to increase at 2.5°C min-1 to 260ºC, which was held
for 30 min. The injection volume was 1µL, and the split ratio was 10:1.
Nitrogen was used as the carrier gas (flow rate 2.2 mL min-1) at a
constant pressure of 130.3 pKa. FAMEs were identified by comparison
with the retention time of individual standards (Sigma, St. Louis, USA).
The proportions of each individual acid was calculated from the ratio its
peak area and total area for all acids and expressed as a mass percentage.

Statistical analysis
 119

Statistical analysis was performed by one-way analysis of variance

(ANOVA) using STATISTICA Software (version 7.0; StatSoft Inc). A
p-value of <0.05 was considered to indicate statistical significance using
the Tukey test.

Principal component analysis (PCA)

The biomass, lipid/protein contents, main fatty acids (C14:0,

C16:0, C18:1, C18:2, C18:3 and C20:5ɷ3) and fatty acids properties
(SFA, MUFA and PUFA) were selected for PCA using the Statistica 7.0
software (Statsoft Inc. 2004). All variables were normalized using (n –
1), and Kaiser normalization was performed prior to construction of the
PCA biplot. In the resulting PCA biplot, the A, B, C, and D groups were
circled according to their distinct cluster formation.

Results and Discussion

Effect of DC concentration on biomass and lipid productivities of N.

gaditana

To assess the applicability of DC as a culture medium, it was

mixed with the F/2 medium before the algal cultivation. The
compositions of DC and F/2 are shown in Table 4.1. Different DC
concentrations were tested to determine their effect on microalgal
growth, measured as biomass and lipid productivities. Productivity
results, as well as the conductivity for each DC concentration, are shown
in Table 5.1. Briefly, biomass productivity (BP) in control culture (0%
DC) was 101 mg L-1 day-1, increased slightly at 25% and 50% DC and
was maximal (126 mg L-1 day-1) at 75% DC. At 100% DC the BP
decreased to 46 mg L-1 day-1, likely because of the inability to maintain
satisfactory growth at the mineral composition of DC only, indicating
that DC needs to supplemented with conventional media. These results
are in excellent agreement with previous data that N. gaditana produced
more biomass when grown in DC mixed with F/2 medium than in F/2
medium only (Matos et al. 2015).
The lipid productivity (LP) showed a comparable trend and
increased proportionally with increasing DC concentration from 5.4 mg
L-1 day-1 in control F/2 medium to a maximum of ~14.5 mg L-1 day-1 at
75% DC. At 100% DC the LP decreased close to control levels (5.4 mg
lip. L-1 day-1) (Table 5.1). Functionally, increased intracellular lipid
120

productivity in N. gaditana at 50-75% DC can be explained by an

adaptive osmoregulatory mechanism to cope with either rapid or gradual
changes in salinity/conductivity caused by the DC-medium, which are
associated with the algal cell membrane permeability (Bartley et al.
2013; Lawton et al. 2015). Our results with marine alga N. gaditana
confirm that cells produce more lipids when exposed to hypotonic
salinity/conductivity, which was also observed by An et al. (2013) for
the lipid content of Chlamydomonas ICE-L exposed to hypotonic
conditions. More importantly, these data indicate that the mineral
composition of the conventional F/2 medium can be partially replaced
by the nutrients present in DC to create favorable conditions for
microalgal cultivation. For this reason, we conducted the subsequent
studies using the optimum DC concentration of 75%.

Table 5-1 - Effect of desalination concentrate (DC) concentrations on

biomass productivity (BP) and lipid productivity (LP) of N. gaditana.
BP (mg L-1 LP (mg L-1 Conductivity (mS cm-1)
-1 -1
Cultivation day ) day ) Initial Final
ab
0% DC* 101 ± 9 5.0 ± 2.6c 35.1 ± 0.4 38.2 ± 0.5
25% DC 90 ± 8c 5.4 ± 2.1c 15.5 ± 1.3 16.8 ± 0.6
b
50% DC 115 ± 4 10.5 ± 2.2b 10.2 ± 1.0 12.8 ± 1.0
75% DC 126 ± 8a 14.5 ± 5.1a 8.0 ± 1.2 10.2 ± 0.8
100% DC 46 ± 3d 5.4 ± 2.1c 4.5 ± 1.1 5.6 ± 0.4
* Control test (F/2 medium based on seawater). Different letters in the same
column correspond to significant differences (p<0.05).

Effect of trophic conditions and photoperiod regimes on algal growth

and biomass

The aim of this experiment was to evaluate the effects of different

trophic conditions and light/dark cycles on culturing N. gaditana in a
medium with the optimum DC concentration of 75% by measuring
growth rate and biomass productivity.
Several microalgal species have the ability to switch between
phototrophic and heterotrophic metabolisms. Heterotrophic growth
eliminates the need for illumination, which is required for cultivation
under autotrophic conditions. However, light has multiple effects on
photosynthetic organisms: it provides not only energy to support
metabolism, but is also a fundamental signal that influences many
intracellular components (Simionato et al. 2011).
 121

The alga N. gaditana was grown in batch cultures exposed to four

different light/dark cycles (24L:00D, 16L:08D, 12L:12D, 08L:16D)
under three trophic conditions: autotrophic, mixotrophic and
heterotrophic. For all cultures the growth kinetics was monitored
through the increase in cell number. Fig. 5.1A shows that growth rates
differ significant for most trophic conditions and light/dark cycles.
Autotrophic conditions coupled with a 16L:08D cycle resulted in a
maximum cell density (MCD) of 2.9 x 107 cells mL-1, while mixotrophic
and heterotrophic conditions resulted in cell densities of ~1.3 x 107 cells
mL-1 and 6.6 x 106 cells mL-1, respectively. N. gaditana cultures grew
fastest under autotrophic culture conditions (µ ranging from 0.40 to 0.62
day-1), followed by mixotrophic (µ ranging from 0.43 to 0.56 day-1), and
heterotrophic (0.38 day-1) conditions (Fig. 5.1B). Biomass
concentrations differ significantly (p < 0.05) under the three trophic
conditions, and were maximal at mixotrophic (0.60 to 1.25 g L-1),
intermediate at heterotrophic (0.85 g L-1) and minimal at autotrophic
(0.30 to 0.77 g L-1) conditions (Table 4.2). In addition, N. gaditana
showed a maximum BP of 142 mg L-1 day-1 under mixotrophic
conditions and a 16L:08D cycle. Higher cell densities under
mixotrophic conditions could be due to higher amounts of available
energy contributing to aerobic respiration coupled with catabolism of
the glucose present in the medium along with photosynthesis (Mitra et
al. 2012). The synergetic effect between light and organic substrates in
terms of algal growth has been reported before (Cheirsilp and Torpee
2012). Under complete darkness (i.e., heterotrophic condition), N.
gaditana cells produced intermediate amounts of biomass (BP = 85 mg
L-1 day-1), while cultures exposed to continuous illumination (24L:00D)
reached the stationary phase earlier with a lower BP (~29 mg L-1 day-1),
indicating photo-inhibition caused by excess illumination (Table 5.2).
Moreover, N. gaditana cells grown under continuous illumination
(24L:00D) produce less lipids (LP, 5.8-9.9 mg lip. L-1 day-1) in
comparison with cultures exposed to light/dark cycles of 16L:08D,
12L:12D and 08L:16D, with LP ranging from 13.7 to 15.3 mg lip. L-1
day-1 (Table 4.3). Comparable LP values of 8.5-10.0 mg lip. L-1 day-1
were reported for euryhaline N. gaditana cells exposed to photoperiods
of 24L:00D, 16L:08D, 12L:12D, and 08L:16D (Mitra et al. 2015). In
summary, a maximum BP value was observed for marine N. gaditana
cultured using a DC concentration of 75%, under mixotrophic
conditions and a light/dark cycle of 16L:08D, which is thus the most
appropriate treatment for this species.
122

Fig. 5-1 – N. gaditana growth under different trophic conditions and

light regimes of 24L:00D, 16L:08D, 12L:12D and 08L:16D. (A)
Increase in cell number according to cultivation time (days), (B)
Specific growth rate during exponential phase (µ) calculated for all
growth curves.

10000

A
Cells mL-1 (x104)

1000

100

Aut. 24:0 h Aut. 16:8 h Aut. 12:12 h

Aut. 8:16 h Mix. 24:0 h Mix. 16:8 h
Mix. 12:12 h Mix. 8:16 h Het. (dark)
10
0 2 4 6 8 10
Cultivation time (days)
0,8
B
Duplication rate (days-1)

0,6

0,4

0,2

0,0

Trophic and light variation

Table 5-2 - Growth parameters of N. gaditana cultured with optimal DC concentration of 75% under different
experimental conditions.
Maximum cell density Specific growth rate Biomass concentration Biomass productivity
(cell mL-1 x 104) (day-1) (g L-1) (BP, mg L-1 day-1)
Autotrophic
(light/dark)
24L:00D 400 ± 9a 0.50 0.30 ± 0.10a 28 ± 4a
d ab
16L:08D 2900 ± 14 0.62 0.45 ± 0.05 42 ± 6abc
b abc
12L:12D 1200 ± 20 0.50 0.77 ± 0.10 88 ± 10cd
b a
08L:16D 1180 ± 15 0.40 0.35 ± 0.05 35 ± 7ab
Mixotrophic
(light/dark)
24L:00D 1020 ± 17b 0.43 0.60 ± 0.15ab 30 ± 7a
c c
16L:08D 1700 ± 11 0.45 1.25 ± 0.10 142 ± 12e
c bc
12L:12D 1654 ± 16 0.53 0.98 ± 0.14 112 ± 8e
b abc
08L:16D 1010 ± 13 0.56 0.82 ± 0.12 80 ± 8bcd
ab abc
Heterotrophic 665 ± 10 0.38 0.85 ± 0.10 85 ± 13cd
(dark)
Values in the same column with different superscript letters are significantly different (p < 0.05).
123
124

Effect of experimental conditions on protein and lipid productivities

This experiment examined the effect of different experimental

conditions of the intracellular compositions, i.e. protein and lipid
productivities of marine N. gaditana. Using a nitrogen-to-protein (N-to-
P) conversion factor of N x 4.78 (Lourenço et al. 2004), protein contents
varied from 17.9 to 44.8% (Table 5.3), with statistical different protein
contents obtained for the different treatments (p < 0.05). The protein
content was highest for the mixotrophic culture conditions, with an
average of 30.1% (range 17.9-44.8), followed by autotrophic, with an
average of ~23.4% (range 20.2-27.5%), and heterotrophic (23.2%)
condition, the latter two average values being statistically equal (p <
0.05). These results are comparable to data, for instance by Tibbetts et
al. (2015), who found a protein content of 35.0% for N. granulata, using
the same conversion factor of N x 4.78.
With regard to the algal lipids, total lipid contents vary from 10.5
to 16.7%, which in some cases translated to a relatively high lipid
productivity when coupled to high biomass productivity. Interestingly,
N. gaditana has a remarkable ability to accumulate protein and lipids
under quite different culture conditions. For example, N. gaditana
cultures show optimal protein synthesis (37.3 to 44.8%) under
mixotrophic conditions and a photoperiod of 16L:08D or 12L:12D. In
contrast, comparable lipid accumulation occurred in N. gaditana cells
under autotrophic conditions and a 16L:08D cycle (16.7%) and under
mixotrophic conditions and a 08L:16D cycle (15.7%). Under
heterotrophic condition, a lipid content of 12.1% was noted (Table 5.3).
It appears that under autotrophic conditions, N. gaditana requires a
longer light period, i.e., a 16L:08D cycle for a high lipid productivity
(15.9 mg L-1 day-1). With a shorter light period as in a 08L:16D cycle,
high lipid productivity (15.3 mg L-1 day-1) requires mixotrophic
cultivation (Fig. 5.2). According to Mitra et al. (2012), shorter light
periods during mixotrophic cultivation may act as a stress signal,
prompting the cells to convert the excess glucose that is available in the
medium into storage lipids and minimize cell division. This stress-
adaptation phenomenon is similar to changes in response to salinity or
nitrogen limitation and increased lipid production in other algae (Matos
et al. 2014; Selvaratnam et al. 2015).
Simionato et al. (2011) found that nutrient starvation during the
stationary phase is the major factor that induces lipid accumulation in N.
gaditana cells during acclimation to different illumination regimes, and
that the light intensity per se does not influence lipid accumulation.
 125

Nevertheless, the illumination conditions can play an indirect but

important role when combined with other factors that influence
photosynthesis and growth, such as salinity and nutrient
supplementation or depletion. In this study, we hypothesized that algae
cultured autotrophically (with only air-saturated CO2 as an inorganic
carbon supply), accumulate lipids to counterbalance the gradual changes
in salinity/conductivity triggered by the DC-medium, representing a
‗stress condition‘. Furthermore, algae probably accumulate lipids under
mixotrophic cultivation, due to their ability to assimilate glucose as an
extra carbon source, suggesting that increased availability of carbon
leads to an increased proportion of stored lipids. The light/dark cycle
was found, in this study, to have a crucial impact on biomass
productivity of N. gaditana, which is directly correlated with lipid
productivity, indicating that the illumination photoperiod is an important
factor that need to be taken into consideration.

Fig. 5-2 - Summary scheme showing how to enhance lipid productivity

(LP, mg L-1 day-1) in marine N. gaditana through the manipulation of
growth parameters. Step 1 = optimization of culture medium based on
desalination concentrate (DC) which affects alga through the change in
salinity/conductivity; Step 2a = enhancing LP of alga after establishing
the optimal photoperiod under autotrophic culture conditions; Step 2b =
enhancing LP of alga after adding an extra carbon source (glucose, 2.0 g
L-1) with optimal photoperiod under mixotrophic culture conditions.

N. gaditana
(75% DC medium)
Step 2a
Autotrophic/16L:08D
N. gaditana Step 1 LP = 15.9 mg L-1 day-1
N. gaditana
(F/2 medium) (75% DC medium)
LP = 5.0 mg L-1 day-1 LP = 14.5 mg L-1 day-1
N. gaditana
Step 2b (75% DC medium)
Mixotrophic/08L:16LD
LP = 15.3 mg L-1 day-1
 126

Table 5-3 - Protein and lipid productivities of N. gaditana cultured with optimal DC concentration of 75% under
different experimental conditions.
Protein Lipid Protein prod. Lipid prod.
(%) (%) (PP, mg prot. L-1 day-1) (LP, mg lip. L-1 day-1)
Autotrophic
(light/dark)
24L:00D 20.2 ± 0.2ab 10.7 ± 0.6a 18.8 ± 1.2bc 9.9 ± 0.5b
b c bc
16L:08D 23.5 ± 0.3 16.7 ± 0.5 21.9 ± 0.8 15.9 ± 0.7e
c ab cd
12L:12D 27.5 ± 0.5 12.0 ± 0.8 31.4 ± 2.3 13.7 ± 0.7d
b a b
08L:16D 22.5 ± 0.4 10.5 ± 1.0 22.5 ± 0.7 10.5 ± 0.2b
Mixotrophic
(light/dark)
24L:00D 20.5 ± 0.9ab 11.6 ± 1.2ab 10.2 ± 0.8a 5.8 ± 0.1a
d ab d
16L:08D 37.3 ± 0.3 11.8 ± 0.8 42.3 ± 1.6 12.8 ± 0.5c
e a e
12L:12D 44.8 ± 1.3 11.3 ± 0.7 51.2 ± 2.1 12.9 ± 0.3c
a bc ab
08L:16D 17.9 ± 0.1 15.7 ± 0.3 17.4 ± 0.9 15.3 ± 0.6e
b ab bc
Heterotrophic 23.2 ± 0.2 12.1 ± 0.4 23.2 ± 1.0 12.1 ± 0.4bc
(dark)
Values in the same column with different superscript letters are significantly different (p < 0.05).
 127

Effect of trophic conditions and photoperiod on fatty acids composition

The fatty acids (FAs) composition of marine N. gaditana cultured

in DC at the optimum concentration is shown in Table 5.4. Under all test
conditions the predominant FAs in marine N. gaditana, are (expressed
as percentage of total FAs) palmitic acid (C16:0; 25.1-54.5%) and
margaric acid (C17:0; 4.4-28.6%), representing 56.0% of the total FA
content. FAs present at intermediate levels are oleic acid (C18:1; 2.2-
12.3%), linoleic acid (C18:2; 3.7-22.7%) and linolenic (C18:3; 0.5-
13.3%), representing 27.3% of the total FA contents. The FAs myristic
acid (C14:0; 0.8-12.0%) and eicosapentaenoic acid (C20:5ɷ3; 0.1-5.6%)
were present at trace levels and represent 9.2% of the total FA content.
In general, autotrophic, heterotrophic and mixotrophic cultivation
conditions the main FAs are saturated fatty acids (SFAs; 33.8-84.7%),
polyunsaturated fatty acids (PUFAs; 10.2-46.0%) and monounsaturated
fatty acids (MUFAs; 5.0-24.1%).
As shown in Table 5.4, N. gaditana cells that were cultured
autotrophically tend to produce higher levels of SFAs (mainly C14:0
and C16:0) at an average of ~70.0% of total FAs, especially when a
16L:08D cycle photoperiod was applied (SFA ~85.0% of total FAs),
consistent with previous data on this saturated fatty acid (Matos et al.
2015). On the other hand, when adding glucose to the optimal DC
medium, more PUFAs are formed. For example, in cultures grown in
complete darkness (i.e. heterotrophic condition), N. gaditana
synthesized PUFAs at high concentrations (46.0% of the total FAs),
which were mainly linoleic (C18:2, 26.6% of total FAs), linolenic
(C18:3, 13.8% of total FAs) and eicosapentaenoic (C20:5ɷ3, 5.6% of
total FAs) acids. The cultures grown under mixotrophic culture
conditions resulted in the production of a balanced ratio of saturated
(SFA) and unsaturated (MUFA + PUFA) fatty acids. Overall, for the
marine N. gaditana cells cultured in optimal DC-medium, the microalga
was virtually devoid of long-chain fatty acids (e.g. C20, and C22) and
showed a trend toward an increased formation of medium-chain (C16
and C18) fatty acid methyl esters, which is an ideal lipid source for
biodiesel production (Cheirsilp and Torpee 2012; Ji et al. 2014).
According to Fernandes et al. (2016), SFAs and MUFAs in
microalgae are produced by de novo FA synthesis in the chloroplast and
provide the substrates needed for PUFA biosynthesis in the endoplasmic
reticulum. In addition, algal diets with low (SFA + MUFA)/PUFA ratios
(i.e. < 2.00) are optimal for the feeding of larval and juvenile oysters.
Our data indicate that the (SFA + MUFA/PUFA) ratio decreases in the
128

order of autotrophic (6.15) > mixotrophic (3.35) > heterotrophic (1.15)

culture conditions (Table 5.4), suggesting that heterotrophic cultivation
is most suitable to produce this microalga for aquaculture. Although the
lipid profiles of the marine N. gaditana biomass varied, the microalga
mainly accumulated the following fatty acids (C): 14:0; 16:0; 17:0;
18:1; 18:2; 18:3 and 20:5. Therefore, the microalga study herein is
industrially important for pharmaceutical and nutritional supplements
for humans and animal feed (polyunsaturated fatty acids), and the
production of biofuels, mainly biodiesel (Mitra et al. 2015; Tibbetts et
al. 2015; Varshney et al. 2015).

Principal Component Analysis

Given the large number of variables, the amounts of biomass,

proteins, lipids and main fatty acids produced under different trophic
and photoperiod regimes were selected for principal component analysis
(PCA). The PCA biplot (Fig. 5.3) revealed four important clusters based
on the PC 1 and PC 2 with variabilities of 44.86% and 18.52%,
respectively. Cluster A was encircled with the variables C18:2, C18:3,
C20:5ɷ3, PUFA and heterotrophic culture condition, while cluster B
consisted of variables C18:1 and MUFA. Cluster C was encircled of the
variables photoperiod (12L:12D, 16L:08D and 24L:00D), biomass,
protein and mixotrophic culture conditions. Cluster D was encircled
with variables photoperiod (08L:16D, 12L:12D, 16L:08D), lipids,
C14:0, C16:0, SFAs and autotrophic culture conditions.
The autotrophic culture condition with 16L:08D photoperiod in
cluster D were positively associated with lipids and SFAs along with
C14:0 and C16:0 (Fig. 5.3), indicating that a 16L:08D photoperiod is
optimal for the maximal production of SFAs. In addition, higher SFA
production is caused mainly due to the synthesis of dominant C16:0 that
contributes to the highest SFA + MUFA)/PUFA index of 8.79 at this
photoperiod regime (Table 5.4). Interestingly, at the same 16L:08D
photoperiod PUFA content was the lowest (10.2% of total FAs) of all
treatments and is thus negatively associated with PUFA.
 129

Fig. 5-3 - Principal component analysis (PCA) of the biomass, protein,

lipid and main fatty acids composition under variable trophic and light
photoperiod conditions, based on a culture medium with optimal
desalination concentrate (DC) of 75%.

1.0
Cluster A Cluster D

12L:12DAut
24L:00DAut
Lipid
C18:3 C20:5ɷ3 SFA
PUFA 16L:08DAut C16:0
C14:0
PC 2 18.52%

C18:2 08L:16DMix 08L:16DAut

00L:00DHet
0.0

24L:00DMix

C18:1 16L:08DMix
Biomass

MUFA Protein

12L:12DMix

-1.0 Cluster B Cluster C

-1.0 0.0 1.0
PC 1 44.86%

It should be noted that under mixotrophic culture conditions the

16L:08D and 12L:12D cycles increased the protein levels (cluster C),
whereas the C18:1 and MUFA were lonely located in the cluster B. with
regard to variables that are located in cluster A, the unsaturated fatty
acids (i.e. C18:2, C18:3, C20:5ɷ3) and PUFA were strongly correlated
with heterotrophic culture condition, indicating that N. gaditana cells
have a tendency to synthesize more PUFA in complete darkness. The
PCA biplot clearly demonstrates that the 16L:08D cycle during
autotrophic cultivation is the best condition for optimal SFA production,
which is an important finding, since ΣSFA and ΣMUFA are very
important for biodiesel production (Cheirsilp and Torpee 2012).
 130

Table 5-4 - Fatty acids composition (% of total fatty acid content) of N. gaditana cultured with optimal DC
concentration of 75% under different experimental conditions.
C16:0 C18:1 C18:2 C20:5ɷ3 SFA MUFA PUFA
Autotrophic
(light/dark)
24L:00D 33.5 ± 0.7 12.3 ± 0.7 12.9 ± 0.9 0.7 ± 0.1 46.5 ± 2.5 24.1 ± 1.6 17.3 ± 0.8
16L:08D 42.9 ± 1.6 9.1 ± 0.6 4.3 ± 0.2 1.7 ± 0.2 84.7 ± 3.6 5.0 ± 0.9 10.2 ± 0.7
12L:12D 54.5 ± 1.8 9.7 ± 0.8 3.7 ± 0.3 0.3 ± 0.1 69.5 ± 2.7 14.5 ± 1.1 15.9 ± 1.1
08L:16D 50.5 ± 1.5 2.2 ± 0.1 11.8 ± 0.7 0.1 ± 0.1 79.4 ± 2.2 5.8 ± 0.7 14.7 ± 1.2
Mixotrophic
(light/dark)
24L:00D 35.1 ± 1.1 4.9 ± 0.4 22.7 ± 0.4 1.4 ± 0.1 62.0 ± 2.0 11.2 ± 0.9 26.7 ± 0.7
16L:08D 36.0 ± 1.3 2.8 ± 0.2 9.6 ± 0.6 3.3 ± 0.2 67.5 ± 2.4 10.3 ± 0.7 14.3 ± 0.7
12L:12D 25.1 ± 1.3 5.1 ± 0.3 19.2 ± 0.7 0.5 ± 0.1 46.3 ± 2.5 11.2 ± 1.1 20.0 ± 1.4
08L:16D 39.0 ± 1.2 5.5 ± 0.3 16.8 ± 0.7 0.2 ± 0.1 67.3 ± 2.3 11.9 ± 1.1 20.8 ± 0.8
Heterotrophic 25.5 ± 1.2 12.2 ± 0.3 26.6 ± 0.7 5.6 ± 0.1 33.8 ± 1.0 20.1 ± 0.7 46.0 ± 1.4
(dark)
Values in the same column with different superscript letters are significantly different (p < 0.05).
 131

Conclusions

Nannochloropsis gaditana was shown to be capable of higher lipid

production in culture medium with optimal DC concentration compared
to control medium. By varying the growth conditions (trophic mode,
photoperiod and salinity based on DC conductivity) the chemical
composition (protein, lipids and fatty acids) of N. gaditana can be
manipulated. Under autotrophic conditions, the algae synthesize more
SFAs, while by adding an addtional carbon source to the DC medium,
the percentage of PUFAs is increased. The present study has identified
an effective strategy for the optimization of culture medium based on
DC for enhancing algal lipid contents.
 133

CHAPTER 6

CHEMICAL CHARACTERIZATION OF SIX

MICROALGAE WITH POTENTIAL UTILITY FOR
FOOD APPLICATION

Abstract

Microalgae contain high levels of proteins, carbohydrates and

lipids, and have found a useful application in enhancing the nutritional
value of foods. These organisms can also synthesize long chain fatty
acids in the form of triacylglycerols, such as α-linolenic acid (ALA),
eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linolenic
acid (LA), γ-linolenic acid (GLA) and arachidonic acid (AA). The aim
of this study was to determine the chemical composition and measure
protein, carbohydrates, fibers, lipids as well as the fatty acids
composition of six microalgae species with potential application in the
food industry. Two freshwater species Chlorella vulgaris and Spirulina
platensis and four marine species Nannochloropsis oculata,
Nannochloropsis gaditana, Porphyridium cruentum and Phaeodactylum
tricornutum were used in the experiments. Intracellular protein was the
most algal component (42.8-35.4%), followed by carbohydrate + fiber
(32.3-28.6%) and lipids (15.6-5.3%). N. gaditana is rich in saturated
fatty acids, mainly palmitic acid (5.1 g/100g), while the cells of S.
platensis and C. vulgaris algae are abundant in GLA (1.9 g/100g) and
ALA (2.8 g/100g) acids, respectively. P. cruentum differs from other
algae, because it contains a large amount of AA (3.7 g/100g). The
marine microalgae N. oculata and P. tricornutum are also a source of
essential long chain polyunsaturated fatty acids (LC-PUFA-ɷ3), mainly
composed of EPA and DHA. Our results suggest that the freshwater
species C. vulgaris and S. platensis are attractive nutritional
supplements because of their low fiber and high protein/carbohydrate
contents, while the marine species P. tricornutum and N. oculata can
enrich foods with LC-PUFA-ɷ3, because of their favorable ɷ3/ɷ6 ratio.

Keywords: Microalgae; biomass composition; lipids, fatty acids;

omega-3; nutraceuticals.
134

Introduction

Microalgae are a group of eukaryotic organisms and photosynthetic

cyanobacteria able to accumulate sugars, carbohydrates, proteins, lipids
and other valuable organic substances by the efficient use of solar
energy, CO2, and nutrients. These microorganisms convert inorganic
substances such as carbon, nitrogen, phosphorus, sulfur, iron, and trace
elements into organic matter (green, blue-green, red, brown, and other
colored biomass) (Batista et al. 2013).
Microalgae are considered one of the most promising feedstock
materials for developing a sustainable supply of commodities, including
both food and non-food products. Microalgae have also great potential
as they produce natural compounds, that could be used as functional
food ingredients (Draaisma et al. 2013; Ryckebosch et al. 2014).
Currently, edible oils, proteins and carbohydrates are consumed in a
variety of food products, which contain ingredients from both plant and
animal origin. In this regard, microalgae can be used to enhance the
nutritional value of foods. When using screening methodologies to
identify valuable compounds (pigments, antioxidants, polyunsaturated
fatty acids, etc.) in microalgae, knowledge of the chemical composition
is a first requirement (Batista et al. 2013; Henrikson 2009). The
microalgae are an extremely diverse collection of organisms with a large
variation in chemical compositions, but this diversity is not yet fully
explored (Borowitzka 2013). For this reason, the nutritional content of
algal biomass is sometimes poorly defined and for most species,
including well-studied species like Spirulina, there is little consensus on
their biochemical composition of different algal species.
Several fatty acids are synthesized by humans, but there is a group
of essential fatty acids, the polyunsaturated fatty acids (PUFAs), which
the human body cannot produce: omega-3 (ɷ-3) and omega-6 (ɷ-6).
Therefore, both ɷ-3 and ɷ-6, which are necessary for human health, are
entirely derived from the diet (Fernandes et al. 2014) and nutrition
experts have recommended that an ɷ3/ɷ6 fatty acids ratio of ≤ 1:5 is
desirable (Armenta & Valentine 2013). Since the Western diet contains
massive quantities of ɷ-6, ɷ3/ɷ6 ratios of up 1:25 have been reported in
the literature (FAO 2008), which has been recognized to be undesirable
(Martin et al. 2006), and most people consume thus a PUFA-deficient
diet. Therefore, nutritionists emphasize the need to consume seafood
(notably fish) and green vegetables to prevent an array of disorders,
especially cardiovascular diseases (FAO 2008; Kleiner et al. 2014). As
reported by Armenta and Valentine (2013), single cell oils (SCO)
 135

containing long chain polyunsaturated fatty acids (LC-PUFA) such as

EPA/DHA acids derived from algae are considered a promising oil
alternative to oils from fish and land based plant sources.
The development of novel foods based on microalgal biomass is an
exciting tool for providing nutritional supplements with biologically
active compounds (e.g., antioxidants, PUFAs-ɷ3) (Lemahieu et al.
2013). Depending on species/strain, environmental conditions and
harvesting/processing methods, algal biomass after oil extraction may be
a highly attractive source of essential dietary amino acids, fatty acids,
sugars, vitamins, minerals, carotenoids and other health-promoting
nutrients that are well suited as human food or feed additives for
terrestrial livestock and aquatic animals (Tibbetts et al. 2015; Tibbetts et
al. 2015). Although the potential for algal products/co-products for
nutritional applications has long been recognized, so far it has had
limited commercial success with only a few species (e.g. Spirulina,
Chlorella) occupying niche markets (Brennan & Owende 2010).
Advancing our knowledge of the biochemical composition of algae is a
key requirement for realizing the potential of algal products/co-products.
The main objective of the present study was to compile information
on the biochemical composition of microalgae, which is needed for
selecting the optimal species for specific food applications (rich in
protein, carbohydrates, PUFA, etc.). The microalgae species Chlorella
vulgaris, Spirulina platensis, Nannochloropsis gaditana,
Nannochloropsis oculata, Phaeodactylum tricornutum and
Porphyridium cruentum were mass cultivated in artificially illuminated
photobioreactors to produce sufficient biomass quantities to be able to
determine their chemical composition and nutritional value.

Experimental Procedures

Acquisition of algal biomass

Freshwater species Chlorella vulgaris and Spirulina platensis were

obtained from the Laboratory of Biochemistry Engineering, Federal
University of Rio Grande. Marine species Nannochloropsis gaditana
(clone 130) was kindly supplied by Banco de Micro-organismos
Marinhos Aidar & Kutner (BMA&K), Oceanographic Institute at the
University of São Paulo. Microalgae C. vulgaris, S. platensis and N.
gaditana were cultured in the Laboratory of Food Biotechnology,
Federal University of Santa Catarina. Microalgae were cultivated
through the inoculation of microalgal cultures (monospecific algal
136

cultures) in appropriate growth media: for C. vulgaris, Bold Basal

Medium (BBM) was used (Nichols 1973), S. platensis cells were grown
in Paoletti Synthetic Medium (PSM) (Ferraz et al. 1985), and N.
gaditana cells were cultured in previously autoclaved (121°C/15 min)
seawater enriched with F/2 media (Guillard 1975) with salinity of about
34.0‰. Cultures of C. vulgaris, S. platensis and N. gaditana were
developed in inverted conical photobioreactors (5 L capacity) with the
respective growth media and scaled-up to 100-L capacity fiber
photobioreactors (0.50 m diameter, 0.90 m length). The
photobioreactors were placed under a photoperiod of 12:12 h light/dark
at room temperature (25 ± 2°C), at a light intensity of 216 µmol photons
m-2 s-1 provided by cool-white fluorescent lamps. Air saturated with CO2
was continuously pumped into the photobioreactors. When the
microalgal culture reached stationary growth phase, the entire culture
broth (100 L) was harvested by continuous centrifugation at 4,000 rpm
for approximately 1 h. The concentrated microalgal pellet was then
transferred to a dish and dried in a dehydrator at 45 ± 5ºC for 24 h
before analysis.
Marine algal species N. oculata, P. tricornutum and P. cruentum
were cultured in the Laboratory of Algae Cultivation, Federal University
of Santa Catarina. The cultures were developed in two open tanks and a
glass fiber cylinder that we custom designed and built. After cultures
were grown in 5 L capacity Erlenmeyer flasks, mass cultures of N.
oculata and P. tricornutum were scaled-up in 500-L capacity open tanks
(1.60 m diameter and 2.75 m length) containing F/2 media (Guillard
1975). Cool-white fluorescent lamps were placed over the tanks under a
constant light intensity of 100 µmol photons m-2 s-1. The alga P.
cruentum mass culture was scale-up in 180-L capacity glass fiber
cylinder (0.50 m inner diameter and 1.00 m length) containing F/2
media [17]. The surface of the cylinder was exposed continuously to 10
cool-white light lamps (100 µmol photons m-2 s-1). The tanks and
cylinder were constantly CO2-aerated (40 L min-1) and maintained at
room temperature (22 ± 1ºC) using air conditioners. Salinity in the
experiments was 35.0‰. As soon as the cultures reached the stationary
growth phase, the entire culture broth was harvested by continuous
centrifugation and concentrated to a biomass slurry, which was washed
with isotonic ammonium formate (Zhu & Lee 1997) and centrifuged at
3500 rpm for 35 min. Finally, the wet biomass was transferred to a dish
and dried in a dehydrator (45 ± 5ºC, 24 h) before further analysis.
 137

Biomass composition analysis

For each of the six microalgae species, triplicate samples of dried

biomass were analyzed to determine moisture, ash, dietary fiber,
carbohydrate, protein, lipid contents and fatty acids composition.

Moisture

Moisture was determined by drying the sample in an oven at 105°C

for 3-4 h (until constant weight) (AOAC 2005). The average moisture
values were used to calculate the chemical composition as a percentage
of total dry matter.

Mineral content

Total ash content was determined by heating the samples to 550ºC

for 5 h using a carbolite muffle furnace (IAL 2005).

Dietary fiber

Total dietary fiber (TDF) content was determined with a total

dietary fiber analysis kit (Megazyme International Ireland Ltd,
Wicklow, Ireland), which includes enzymatic hydrolysis with α-
amylase, protease and amyloglucosidade and is approved by the AACC
(Method 32-05-01) and the AOAC (Official Method 985.29). Duplicate
samples (approximately 1 g) were suspended in 50 mL phosphate buffer
and submitted to enzymatic hydrolysis by incubating with 50 μL of α–
amylase at 100°C for 30 min. The pH was adjusted to 7.5, 100 μL of
protease was added and samples were incubated at 60°C for 30 min.
Next, the pH was adjusted to 4.5, 200 μL of amyloglucosidade was
added and the samples were incubated at 60°C for 30 min. Finally, fiber
was precipitated with 95% ethanol at 60°C, filtered through fritted glass
crucibles with a Celite filter and the residue in the crucible was dried in
an oven at 105°C, cooled in a desiccator and weighed.

Protein content

Total nitrogen was determined by the Kjeldahl method after acid

digestion (AOAC 991.20), ammonium addition, steam distillation and
titration with 0.1 N HCl (AOAC 2005). Protein content was calculated
138

using a nitrogen-to-protein conversion factor of N x 4.78 (Lourenço et

al. 2004).

Lipid content

After acid digestion with 4.0 N HCl for 6 h, intracellular lipids

were extracted with petroleum ether by the Soxhlet method (AOAC
963.15), concentrated in a rotary evaporator, dried in an oven and
weighed (AOAC 2005).

Carbohydrates

The total carbohydrate content of each sample was calculated

according to: (100% – (moisture + ash + protein + lipid + fiber))
(ANVISA 2003).

Fatty acids composition

Fatty acids composition was determined after converting the fatty

acids to their corresponding fatty acids methyl esters (FAME), which
were determined by gas chromatograph using a GC-2014 (Shimadzu,
Kyoto, Japan), equipped with split-injection port, flame-ionization
detector and 105 m-long Restek capillary column (ID = 0.25 mm)
coated with 0,25 μm of 10% cyanopropylphenyl and 90%
biscyanopropylsiloxane. The injector and detector temperatures were
both 260°C. The oven temperature was initially set at 140°C for 5 min,
programmed to increase at 2.5°C min-1, and held at 260ºC for 30 min.
The injection volume was 1µL, and the split ratio was 10:1. Nitrogen
was used as the carrier gas (flow rate was 2.2 mL min-1) at a constant
pressure of 130.3 pKa. Fatty acid methyl esters were identified by
comparison with the retention time of individual standards (Sigma, St.
Louis, USA). For long-chain fatty acids (>C19) a correction factor for
the quantification was used (AOCS 1995). The proportions of the
individual acids were calculated by the ratio of their peak area to the
total area of all observed acids and expressed as mass percentage.

Lipids nutritional quality indexes (IQN)

The nutritional quality of the lipid fraction can be assessed by three

separate indexes that are calculated based on the concentration of
saturated fatty acids (lauric C12:0, myristic C14:0, palmitic C16:0 and
 139

stearic C18:0), monounsaturated fatty acids (MUFA, oleic C18:1ɷ9),

and polyunsaturated fatty acids (linoleic C18:2ɷ6, linolenic C18:3ɷ3,
arachidonic C20:4ɷ6 and eicosapentaenoic C20:5ɷ3) according to:

(1) Atherogenicity index (AI) = [(C12:0 + (4 x C14:0) + C16:0)] /

(ƩMUFA + Ʃɷ6 + Ʃɷ3) (Ulbricht & Southgate 1991).
(2) Thrombogenicity index (TI) = (C14:0 + C16:0 + C18:0) / [(0.5 x
ƩMUFA) + (0.5 x Ʃɷ6 + (3 x Ʃɷ3) + (Ʃɷ3/Ʃɷ6)] (Santos-Silva et
al. 2002).
(3) Fatty acids hypocholesterolemic/hypercholesterolemic ratios (H/H)
= (C18:1ɷ9 + C18:2ɷ6 + C20:4ɷ6 + C18:3ɷ3 + C20:5ɷ3) / (C14:0
+ C16:0) (Santos-Silva et al. 2002).

Statistical analysis

Statistical analysis was performed by one-way analysis of variance

(ANOVA) using STATISTICA Software (version 7.0) from StatSoft
Inc. (2004). A P-value of <0.05 was considered statistically significant,
and if significant differences were observed, treatment means were
pairwise compaired with the Tukey test.

Results and Discussion

Composition of algal biomass

The chemical compositions of the six microalgae species are

shown in Table 6.1. The moisture content in dried microalgal ranged
from 1.4 to 12.6%, with P. cruentum and S. platensis having relatively
high moisture values (12.6% and 10.0%, respectively). Ash contents
were higher in biomass of the marine species P. tricornutum and P.
cruentum (16.1% and 15.9%, respectively). Zhu and Lee (1997) have
shown that washing the wet biomass with a 0.5 M ammonium formate
solution greatly reduces the salt content of dry matter (DM) of these
marine algal species. In fact, ash levels in non-washed biomass of
marine P. tricornutum and P. cruentum were 40.7% and 33.2%,
respectively. The diatom P. tricornutum has a high inorganic content
due to the fact that its cell wall is covered by silica. The species N.
140

gaditana and S. platensis had intermediate total ash contents (12.3-

11.6%), while N. oculata and C. vulgaris were found to have the lowest
ash contents (8.5-7.3%, respectively).
Dry matter (protein, carbohydrate + fiber and lipids) is the major
component in all algae studied and differences among species were
small. Protein is the most abundant component followed by
carbohydrate + fiber, and lipids. Algal biomass from the species studied
contains on average 40 g protein (range 35.4-42.8%), 18 g carbohydrate
(range 12.5-26.7%), 12 g fiber (range 5.6-18.3%) and 10 g lipid (range
5.3-15.6%) per 100 g of biomass DM. The total dry matter accounted
for an average of 93.3% of total DM (range 87.4-98.6%), in good
agreement with Matos et al. (2014), who reported a total biomass DM of
81.3-94.5% for C. vulgaris, using similar methods. In general it is
common that total dry matter of microalgae is found to be less than
100% (Tibbetts et al. 2015).
Table 6-1 - Chemical composition of six microalgal biomass (%).
Chlorella Spirulina Nannochloropsis Nannochloropsis Phaeodactylum Porphyridium
vulgaris platensis gaditana oculata tricornutum cruentum
Dry matter 93.8 ± 0.3a 89.9 ± 0.5d 94.7 ± 0.4ab 95.9 ± 0.1b 98.6 ± 0.2e 87.4 ± 0.3c
b d ab
Moisture 6.2 ± 0.5 10.1 ± 0.4 5.3 ± 0.3 4.1 ± 0.2a 1.4 ± 0.3c 12.6 ± 0.4e
a b ac
Total ash 7.3 ± 0.2 11.6 ± 0.3 12.3 ± 0.2 8.5 ± 1.7bc 16.1 ± 0.2c 15.9 ± 0.3c
a b c
Total fiber 5.6 ± 0.4 8.5 ± 0.5 14.1 ± 0.3 13.0 ± 0.4c 13.2 ± 0.3c 18.3 ± 0.2d
a a a
Total protein 41.4 ± 0.4 42.8 ± 0.1 41.6 ± 0.3 42.1 ± 0.1a 39.0 ± 0.1b 35.4 ± 0.9c
a b b
Total lipid 12.8 ± 0.1 5.5 ± 1.2 8.1 ± 0.1 15.6 ± 1.1c 14.9 ± 0.4c 5.3 ± 0.3b
a b bc
Carbohydrates 26.7 ± 1.2 21.5 ± 0.5 18.6 ± 0.3 16.7 ± 0.6bc 15.4 ± 0.5cd 12.5 ± 0.6d
Values in the same row with different superscript letters are significantly different (p < 0.05)
141
142

Using a nitrogen-to-protein (N-to-P) conversion factor of N x 4.78

(Lourenço et al. 2004), the protein content in S. platensis (42.8%) was
calculated to be significantly higher than in N. oculata (42.1%), N.
gaditana (41.6%) and C. vulgaris (41.4%), whereas the lowest levels
were found in P. tricornutum and P. cruentum (39.0% and 35.4%,
respectively, P<0.05). Among the six microalgae, Spirulina has been
most extensively used as a source of single cell protein (SCP) and was
even carried by astronauts during space travel. Many microorganisms
(algae, bacteria, fungi yeast/filamentous) can be used as a source of
SCP, but due to their low nucleic acid content and high level of essential
amino acids, algae are preferred over fungi and bacteria as a source of
SCP for human consumption (Anupama & Ravindra 2000). The non-
protein nitrogen (NPN) content in microalgae has been reported to range
from 4 to 40% depending upon species, season and growth phase
(Tibbetts et al. 2015; Lourenço et al. 2004). The N-to-P factor assumes
that the protein source contains 16% N and does not take into account
the often high content of NPN found in microalgae. Our findings on
protein content in the microalgal biomass are in accordance with data
from Tibbetts et al. (2015), who reported that the N-to-P factor of N x
6.25, which has been historically applied for microalgae, is incorrect and
should be avoided (Tibbetts et al. 2015; Lourenço et al. 2002). It should
be noted that the estimates for the crude protein include other nitrogen
compounds, e.g., nucleic acids, amines, glucosamides, and cell wall
materials, which in general are expected to account for around 10% of
the total nitrogen found in microalgae (Vonshak 2002).
The carbohydrate and dietary fiber contents in the algal biomass
samples were found to be very diverse, varying between 12.5-26.7% and
5.6-18.3%, respectively. P. cruentum has the highest and well-balanced,
carbohydrate (12.5%) and dietary fiber (18.3%) content (Table 6.1).
Actually, P. cruentum showed the highest carbohydrate + fiber content
(30.8%) which could be associated with its composition in sulfated
polysaccharides (exopolysaccharide) (Cohen 1990; Rebolloso et al.
2000). Of the species studied, Chlorella and Spirulina were found to
contain the lowest amounts of fiber (5.6% and 8.5%, respectively),
which is important for human use, since low fiber values suggest an
easily digestible biomass (Rebolloso et al. 2000; Vonshak 2002). It is
important to note that, while the fiber fraction in most terrestrial plants
is generally comprised of cellulose, hemicellulose and lignin, the fiber
in microalgae contain no lignin and has low hemicellulose levels. This
makes it easier to digest, and Chlorella and Spirulina are in fact widely
 143

used as a dietary supplement for human consumption. Moreover,

Chlorella is recognized as safe food ingredient, with GRAS (Generally
Recognized as Safe) status by the US FDA (Food and Drug
Administration) (Anupama & Ravindra 2000). The other microalgae (N.
gaditana, N. oculata, P. cruentum, and P. tricornutum) have high fiber
contents, which has been an argument against the use of marine
microalgae in human nutrition, such as SCP (Anupama & Ravindra
2000; Vonshak A 2002). While all microalgae produce hydrocarbons as
energy and carbon stores, some microalgae have a preference for
carbohydrate rather than lipid accumulation and these species are
gaining attention as potential feedstocks for bioethanol production (li et
al. 2014). We found that C. vulgaris biomass contains the highest
carbohydrate content (~26.7%), in agreement with Tibbetts et al. (2015).
These authors reported that Chlorella sp. (and similar species like
Scenedesmus, Chlamydomonas and Tetraselmis) typically produce large
amounts of carbohydrate as energy and carbon reserves. As a result, it
has been proposed that the use of carbohydrate-rich algal biomass (for
example C. vulgaris) as feedstock for bioethanol production may be
advantageous over conventional feedstocks by providing increased
hydrolysis efficiency, higher fermentable yields and reduced production
costs (Baeyens et al. 2015; Lee et al. 2015).
With regard to the algal intracellular lipids, total lipid contents vary
from 5.3% to 15.6% of dry matter. Although this can be considered as
narrow range, statistical differences were observed between lipid
contents among species (P<0.05). The marine algae P. cruentum and S.
platensis have relatively low, statistically equal values for lipid contents
(5.3% and 5.5%, respectively) and similar lipid contents values were
reported by Rebolloso-Fuentes et al. (2000) for P. cruentum (6.3%) and
by Henrikson et al. (2009) for S. platensis (4.5-7.0%). The algae N.
gaditana and C. vulgaris had low to moderate lipid content (8.1% and
12.8%, respectively), while P. tricornutum and N. oculata showed the
highest lipid content (14.9% and 15.6%, respectively) (Table 6.1), with
an interesting composition in terms of PUFA-ɷ3 (Table 6.2). Our
findings are consistent with those of Franz et al. (2013), who reported a
lipid content for P. tricornutum and N. oculata of about 15.6% and
16.1%, respectively. Since the algal cultures studied were not subjected
to nutrient starvation (N-deficient) or any other mechanism that can
induce high lipid productivity, it is not surprising that the lipid content
was relatively low compared to protein and carbohydrate + fiber. If the
fatty acids profile of that lipid would be nutritionally attractive, these
products could potentially be marketed as single cell oil (SCO).
144

However, modified cultivation/processing protocols could easily be

employed to enhance lipid accumulation (Neto et al. 2013; Wong &
Franz 2013).

Fatty acids composition

The microalgal lipid fraction was analyzed in terms of its fatty

acids composition by identifying the main fatty acids, as well as the
proportion of total saturated (SFA), monounsaturated (MUFA), and
polyunsaturated (PUFA) ω3 and ω6 fatty acids. Fifteen fatty acids,
ranging from of C12:0 to C22:6 ɷ-3, were identified and quantified as
percentage of the total fatty acid content of the algal samples (Table
6.2).
The results showed that the marine species (P. tricornutum and N.
oculata) contain high concentrations of PUFAs-ɷ3, predominantly
C20:5 ɷ3 (EPA) and C22:6 ɷ3 (DHA) along with substantial amounts
of C16:1 (monounsaturated fatty acid) and C16:0 (saturated fatty acid).
In contrast, marine P. cruentum has relatively high concentrations of
PUFAs-ɷ6, predominantly C20:4 ɷ6 (AA), whereas the freshwater
algae species C. vulgaris and S. platensis contain high concentrations of
C18:3 ɷ3 (ALA) and C18 ɷ6 (GLA) PUFAs, respectively. N. gaditana
contains a high concentration of saturated fatty acids (SFA),
predominantly C16:0 with a low level of PUFAs. These results indicate
that freshwater algae C. vulgaris and marine species N. oculata, P.
tricornutum and P. cruentum followed the same PUFA > SFA relative
pattern, while S. platensis and N. gaditana show the opposite pattern,
SFA > PUFA.
Table 6-2 - Fatty acids composition of biomass of six microalgae (mg/100g).
C. vulgaris S. platensis N. gaditana N. oculata P. cruentum P. tricornutum
C14:0 34 ± 2 95 ± 5 890 ± 11 540 ± 12 22 ± 1 560 ± 23
C16:0 1930 ± 28 3420 ± 34 5130 ± 35 1940 ± 36 2950 ± 21 1360 ± 41
C18:0 67 ± 10 35 ± 5 160 ± 8 26 ± 1 78 ± 2 24 ± 2
Other SFA 125 ± 17 340 ± 13 344 ± 12 227 ± 9 344 ± 12 400 ± 11
Ʃ SFA 2156 ± 54 3930 ± 47 6613 ± 41 2840 ± 15 3437 ± 26 2420 ± 25
C16:1 150 ± 4 560 ± 12 1900 ± 11 2240 ± 18 140 ± 4 1550 ± 13
C18:1 128 ± 6 45 ± 1 445 ± 8 280 ± 11 210 ± 10 129 ± 5
Other MUFA 131 ± 2 110 ± 5 192 ± 7 600 ± 6 17 ± 2 260 ± 6
Ʃ MUFA 690 ± 16 1105 ± 16 2606 ± 21 3205 ± 37 517 ± 11 2219 ± 17
C18:3 ω3 (ALA) 2820 ± 41 130 ± 8 34 ± 1 15 ± 2 20 ± 1 92 ± 2
C20:5 ω3 (EPA) - 38 ± 1 - 2973 ± 24 697 ± 21 2753 ± 16
C22:6 ω3 (DHA) - 62 ± 1 - 43 ± 5 - 80 ± 5
Ʃ PUFA-ω3 2820 ± 41 260 ± 7 34 ± 1 3031 ± 10 767 ± 12 2945 ± 34
C18:2 ω6 (LA) 1030 ± 22 150 ± 14 300 ± 10 290 ± 7 1040 ± 54 250 ± 8
C18:3 ω6 (GLA) 280 ± 10 1920 ± 42 120 ± 4 150 ± 3 18 ± 2 34 ± 2
C20:4 ω6 (AA) 28 ± 2 38 ± 2 - - 3705 ± 26 83 ± 4
C22:5 ω6 - 38 ± 3 - - 182 ± 7 115 ± 7
Ʃ PUFA-ω6 1338 ± 36 2146 ± 41 420 ± 8 440 ± 10 4945 ± 21 482 ± 12
SFA+MUFA+PUFAs 7004 ± 55 7441 ± 64 9673 ± 78 9516 ± 102 9666 ± 89 8066 ± 82
145
146

Saturated fatty acids

Palmitic acid (C16:0) is the most abundant SFA (1.3-5.1 g/100g)

followed by myristic acid (C14:0, 22-890 mg/100g) and stearic acid
(C18:0, 24-160 mg/100g) (Table 6.2). The sum of all identified SFAs
ranged from 29% to 68% of the total fatty acid content. We found that
N. gaditana contains 68% SFA, mainly comprised of palmitic acid
(C16:0), followed by S. platensis with 52% SFA, also mainly C16:0,
which is in agreement with values reported in the literature for N.
gaditana (Selvakumar & Umadevi 2014) and S. platensis (Henrikson
2009). P. cruentum and C. vulgaris have intermediate SFA contents
(35% and 30%, respectively), N. oculata and P. tricornutum the lowest
SFA content.

Monounsaturated fatty acids

The highest monounsaturated fatty acids (MUFA) content was

found in N. oculata, representing ~34% of total fatty acid content,
followed by P. tricornutum (28%) and N. gaditana (27%), whereas S.
platensis, C. vulgaris and P. cruentum contain very low concentrations
of MUFAs, on average about 10%. Palmitoleic acid (C16:1) is the main
MUFA found in all marine species studied, ranging from 1.9 to 2.2
g/100g, except for P. cruentum that has a very low content of 0.14
g/100g (Table 6.2). These results are consistent with those obtained by
Oh et al. (2009), who demonstrated that P. cruentum contains low levels
of palmitoleic acid and higher levels of arachidonic acid (AA,
C20:4ɷ6). It has been reported that the marine Nannochloropsis species
(Matos et al. 2015; Mitra et al. 2015; Zhu & Dunford 2013) and P.
tricornutum (Ryckebosch et al. 2015) have a high concentration of
palmitoleic acid suggesting that these algae have a tendency to produce
unsaturated fatty acids.
Regarding the WHO/FAO recommendation about the amount of
MUFAs in the human diet, the Expert Consultation (2008) stated that
there is convincing evidence that replacing SFA (C12:0-C16:0) as well
as carbohydrates with MUFA reduces LDL and increases HDL
cholesterol concentrations. Our data suggest that the marine species N.
oculata and P. tricornutum have optimal compositions for the above
purposes because these two species have the lowest SFA content (28-
29%), a low carbohydrate content (15.4-16.7%) and a high MUFA
content (28-34%).
 147

Polyunsaturated fatty acids

Among the polyunsaturated ɷ-3 and ɷ-6 fatty acids (PUFAs), α-

linolenic (ALA, C18:3 ɷ3) and eicosapentaenoic (EPA, C20:5 ɷ3) acids
are the predominant PUFAs-ɷ3, while γ-linolenic (GLA C18:3 ɷ6) and
arachidonic (AA, C20:4 ɷ6) acids made up most of PUFAs-ɷ6. High
concentrations of PUFAs-ɷ3 were observed in C. vulgaris (40%), P.
tricornutum (~37%) and N. oculata (~32%) of the total fatty acids,
while high concentrations of PUFAs-ɷ6 were found in S. platensis
(29%) and P. cruentum (51%) of the total fatty acids. N. gaditana cells
contain very low concentrations of PUFA (5%) and has the least
favorable ɷ3/ɷ6 ratio (0.08) among all algae (Table 6.3).
Chlorella vulgaris contained 60% PUFA, with a high proportion of
ɷ3 acids. In fact, except for S. platensis, P. cruentum and N. gaditana,
all other microalgae showed a ɷ3/ɷ6 ratio of ≥ 2.0 (Table 6.3). Indeed,
in cyanobacteria (e.g., S. platensis) the unsaturated double bonds are
preferentially in the ɷ6 position while in Chlorophyceae they are
mainly in the ɷ3 position (Batidsta et al. 2013). S. platensis is rich in γ-
linolenic acid (GLA, C18:3 ɷ6) (1.9 g/100g). Gamma linolenic acid is
as a precursor of C20 eicosanoids (prostaglandins, leukotrienes and
thromboxanes) and has been associated with beneficial health effects,
such as a reduction in LDL (low-density lipoproteins), anti-
inflammatory effects, stimulation of the apoptosis of cancer cells, and
reduction in pain and inflammation associated with rheumatoid arthritis
(Martin et al. 2006). The species S. platensis is a well-known source of
GLA, since in cyanobacteria this fatty acid plays the same role as α-
linolenic acid (ALA, C18:3 ɷ3) in algae and higher plants (Henrikson
2009). Although PUFA levels are relatively high for all species (>25%
of total fatty acid), except for N. gaditana cells, it consists of medium-
chain PUFA (e.g., C16 and C18) and is devoid of long-chain (LC) PUFA
(e.g., C20 and C22). This is generally typical for freshwater
microalgae/cyanobacteria and make them poor sources of nutritionally-
essential LC-PUFA, arachidonic acid (AA, C20:4 ɷ6), eicosapentaenoic
acid (EPA, C20:5 ɷ3) and docosahexaenoic acid (DHA, C22:6 ɷ3). For
this reason, N. oculata and P. tricornutum are more interesting for
nutrition applications, because they are able to synthesize high amounts
of EPA and DHA acids, and thus can be used to enrich functional foods
with ɷ-3 fatty acids. Algal oils and single cell oil (SCO) sources of LC-
PUFAs are now becoming available (to provide EPA+DHA+AA)
(Armenta & Valentine 2013; Ryckebosch et al. 2014). In addition, an
148

advantage of a SCO from algae is that it usually contains a significant

amount of natural antioxidants (e.g. carotenoids and tocopherols), which
can protect ɷ-3 fatty acids from oxidation, hence making this oil less
prone to oxidation than oil derived from plants and marine animals (Li
et al. 2007).
As shown in Table 2, C. vulgaris and P. cruentum have the same
PUFA content (~60%). However, α-linolenic acid (ALA, C18:3 ɷ3) in
C. vulgaris contributes to an increased ɷ3/ɷ6 ratio of 2.10, while
arachidonic acid (AA, C20:4 ɷ6) in P. cruentum causes a low ɷ3/ɷ6
ratio of 0.15. Consequently, C. vulgaris has a favorable ɷ3/ɷ6 ratio that
is about 15-fold higher than that of P. cruentum, but the large amount of
AA (3.7 g/100g) in P. cruentum makes this marine alga a potential
source for the production of arachidonic acid. Since the metabolic
breakdown of AA leads to an increased production of prostaglandin E2,
which belongs to a class of hormone-like substances that participate in a
wide range of bodily functions, thromboxane and leukotriene (Raposo et
al. 2014), the importance of this fatty acid to human-cell functioning is
evident.
The marine microalgae N. oculata and P. tricornutum contain 37%
and 43% PUFA, respectively, with a highly ɷ3/ɷ6 ratio of 6.50. These
microalgae are also, rich in EPA and contain a small quantity of DHA.
The species N. oculata contains 3.0 g EPA and 43 mg DHA, and P.
tricornutum 2.7 g EPA and 80 mg DHA per 100 g microalgal biomass.
According to Borowitzka (2013) the main market of these oils is infant
formula and an oil rich in both DHA and EPA from a strain of
Schizochytrium has recently reached the market. Currently, there is no
other commercial production of EPA-rich oils from microalgae, but
Aurora Algae has announced a product from marine eustigmatophyte
Nannochloropsis. According to American Heart Association a daily
intake of 500 mg EPA + DHA and 800-1000 mg of ALA per day are
recommended for the primary prevention of coronary heart disease
(ISSFAL 2015). Our results indicate that the marine species N. oculata
and P. tricornutum are potential sources of EPA, with an average of 2.8
g/100g whereas C. vulgaris has a robust ALA (2.8 g/100g) producing
profile. These microalgae have therefore an enormous potential for
application in the development of health food products, such as single
cell oil (SCO).
 149

Lipids nutritional quality indexes (IQN)

The nutritional quality of lipid profiles observed in the algae

species was evaluated by different indexes as shown in Table 6.3.
Foods with polyunsaturated and saturated fatty acids (P/S) ratios
below 0.45 are considered by the FAO/WHO to be undesirable in the
human diet (FAO 2008), because of their potential to induce increases in
blood cholesterol. The P/S ratios in all six algae are above 0.45, ranging
from 0.46 in N. gaditana to 2.35 in P. tricornutum.

Table 6-3 - Nutritional quality indexes of the lipid fraction in the

biomass of six microalgae.
Species P/S ɷ3/ɷ6 H/H AI TI
C. vulgaris 2.25 2.10 2.04 0.42 0.21
S. platensis 0.89 0.12 0.66 1.10 1.46
N. gaditana 0.46 0.08 0.12 1.70 3.82
N. oculata 2.33 6.88 1.44 0.63 0.22
P. cruentum 1.79 0.15 1.90 0.49 0.60
P. tricornutum 2.35 6.10 1.72 0.65 0.19
P/S = polyunsaturated/saturated; ɷ3/ɷ6 = Ʃ of the Omega 3 series/Ʃ of the
Omega 3 series; H/H = Ʃ hypercholesterolemic/Ʃ hypocholesterolemic; AI
= atherogenicity index; and TI = thrombogenicity index.

An additional approach to the nutritional evaluation of lipid

profiles is the calculation of an index based on functional effects of fatty
acids, e.g. the ratio hypocholesterolemic fatty acids
/hypercholesterolemic fatty acids (H/H) index, which is based on current
knowledge of the effects of individual fatty acids on cholesterol
metabolism (Santos-Silva et al. 2002; Simat et al. 2015). Nutritionally,
higher H/H values are considered more beneficial for human health,
because a higher H/H ratio is directly proportional to a high PUFA
content. Fatty acids from microalgae that are highly polyunsaturated are
thought to have beneficial effects on cholesterol. The highest H/H value
(2.04) was found in C. vulgaris, followed by P. cruentum species (H/H
= 1.90). These results are in excellent agreement with H/H values for
marine fish such as sardine and mackerel (H/H = 2.46) reported by
Fernandes et al. (2014). In addition, Testi et al. (2006) reported H/H
values for fish fillets of sea bass and rainbow trout of 2.18 to 2.40.
Two other indexes are used to evaluate the potential for stimulating
platelet aggregation, the atherogenicity index (AI) and thrombogenicity
index (TI) according to Turan et al. (2007). Lower AI and TI values
150

indicate a greater potential to protect against coronary artery disease. In

our study, AI values ranged between from 0.42 to 1.70, with C. vulgaris
(0.42) and P. cruentum (0.49) showing the lowest values (Table 6.3). It
is noteworthy that these two species also have the highest PUFA content
(~60%). Simat et al. (2015) reported values of 0.59 to 0.92 for the
omnivorous fish bogue (Boops boops Linnaeus). The lowest
thrombogenicity index (TI) value of 0.19 was observed in the marine P.
tricornutum, which is comparable to TI values for the marine fish
sardine of 0.20 reported by Fernandes et al. (2014).
With regard to the ɷ3/ɷ6 ratio, typical Western diets have ɷ3/ɷ6
ratios that are profoundly skewed toward omega-6, which is believed to
promote or cause several diseases (ISSFAL 2015). This is mainly due to
the disproportionately greater consumption of ɷ-6 rich vegetable oils
(e.g., sunflower, peanut, corn) in comparison with the intake of ɷ-3 rich
food sources, such as seafood, nuts, etc. Our data indicate that ɷ3/ɷ6
ratios of the algae species decreased in the order of N. oculata (6.88) >
P. tricornutum (6.10) > C. vulgaris (2.10) > P. cruentum (0.15) > S.
platensis (0.12) > N. gaditana (0.08), (Table 6.3). These results are in
accordance with findings of other authors, for example Batista et al.
(2013), who reported higher ɷ3/ɷ6 ratios in marine algae species than
freshwater algae species, suggesting that the marine species studied here
(i.e. N. oculata and P. tricornutum) could be categorized as beneficial to
human health consumption.

Conclusions

This study determined the biochemical compositions of the six

microalgae strains. Algal biomass from these species contain on average
40 g protein, 18 g carbohydrate, 12 g fiber and 10 g lipid per 100 g of
biomass DM. The species C. vulgaris and S. platensis are rich in ALA
(2.8 g/100 g) and GLA (1.9 g/100g), respectively. The marine algae P.
tricornutum and N. oculata contain 42% and 37% PUFA, respectively,
with a favorable ɷ3/ɷ6 ratio of around 6.5, and are rich in EPA and
DHA acids. The alga P. cruentum contains high PUFA-ɷ6 levels, due
its high concentration of AA (3.7 g/100 g). Taken together, the results
show that microalgae are excellent candidates as sources of high protein
(Spirulina), high carbohydrate/low fiber (Chlorella) and high LC-
PUFA-ɷ3 (N. oculata and P. tricornutum) contents with a high
nutritional value, similar to sardine fish oil.
 151

CHAPTER 7

ESSENTIAL FATTY ACIDS FROM MICROALGAE

Background

Microalgae are considered to be one of the most promising

feedstock materials for developing a sustainable supply of commodities,
including food and non-food products (Batista et al. 2013). They also
produce natural compounds that could be used as functional food
ingredients to enhance the nutritional value of foods (Borowitzka 2013).
Microalgae are a more sustainable and efficient source of lipids and
long-chain fatty acids (LCFA) than conventional livestock. Their
sustainability is based on the efficient use of solar energy, CO2, and
nutrients, offering rapid growth and high yields of lipid- and
carbohydrate-rich biomass (Mitra et al. 2015). Moreover, these
microorganisms convert inorganic substances such as carbon, nitrogen,
phosphorus, sulfur, iron, and trace elements into organic matter (green,
blue-green, red, brown, and other-colored biomass) (Batista et al. 2013).
Several fatty acids (FAs) are synthesized by humans, but the
human body cannot produce omega-3 and omega-6 polyunsaturated
fatty acids (PUFAs). Therefore, both of these PUFAs, which are
necessary for human health, are entirely derived from the diet.
Typical Western diets have omega-3/omega-6 ratios that are
profoundly skewed toward omega 6, with omega-3/omega-6 ratios as
high as 1:25 reported in the literature (Kleiner et al. 2014). This is
mainly due to a disproportionately greater consumption of omega-6-rich
vegetable oils (e.g., sunflower, peanut, corn) than omega-3-rich food
sources, such as seafood and nuts. High omega-6 ratios are believed to
promote or cause cardiovascular disease and other chronic conditions
(Kleiner et al. 2014); consequently, nutrition experts have recommended
omega-3/omega-6 fatty acids ratios of ≤ 1:5 (FAO 2008), and emphasize
the need to consume seafood (notably fish), green vegetables, and other
foods that are rich in omega-3s (FAO 2008).
Our research group at the Federal University of Santa Catarina in
Brazil, recently evaluated the lipid content, fatty acids composition, and
omega-3/omega-6 ratios of six microalgae species: Chlorella vulgaris,
Spirulina platensis, Nannochloropsis gaditana, Nannochloropsis
oculata, Phaeodactylum tricornutum, and Porphyridium cruentum
(Chapter 6).
152

Table 7.1 shows the intracellular lipid contents of the six species,
which vary from 5.3%—15.6% of dry matter. The marine alga P.
cruentum and freshwater species S. platensis have relatively low lipid
contents (5.3% and 5.5%, respectively). The algae N. gaditana and C.
vulgaris have low to moderate lipid contents (8.1% and 12.8%,
respectively), while P. tricornutum and N. oculata have the highest lipid
contents (14.9% and 15.6%, respectively), with an interesting
composition in terms of PUFA-omega 3.
The data from Table 7.1 indicate that the omega-3/omega 6-ratios
of the algae species decreased in the order of N. oculata (6.88) > P.
tricornutum (6.10) > C. vulgaris (2.10) > P. cruentum (0.15) > S.
platensis (0.12) > N. gaditana (0.08). These results are in accordance
with findings of other authors. For example, Batista et al. (2013)
reported that the omega-3/omega-6 ratios in marine algae species were
higher than those in freshwater algae species, suggesting that the marine
species N. oculata and P. tricornutum could be categorized as beneficial
to human health consumption.
Since fish and humans do not synthesize PUFAs or readily convert
omega-6 to omega-3 PUFAs, the levels of these biomolecules are
largely determined by dietary intake (Kleiner et al. 2014). Therefore,
algal diets with low (SFA + MUFA)/PUFA ratios and omega-3/omega-6
ratios higher than 2 are optimal for the feeding of larval and juvenile
oysters (Mitra et al. 2015). For all microalgae studied, C. vulgaris
appears to be the most suited for use as feed in aquaculture (Table 7.1).
The microalgal lipid fraction was analyzed in terms of fatty acid
composition. The main fatty acids were identified as well as the
proportion SFA, MUFA and ω-3 and ω-6 PUFA (Fig. 7.1). We observed
that N. gaditana contains 68.0% SFA—mainly comprised of palmitic
acid (C16:0)—and a low content of PUFA (5.0%). Similarly, S.
platensis contains high content of SFA (52%) and PUFA (33%), with a
much larger proportion of ω-6s in relation to ω-3s (Table 7.1). S.
platensis is also rich in γ-linolenic acid (GLA, 1.9 g/100g biomass).
GLA has been associated with several beneficial health effects,
including a reduction in low-density lipoproteins (LDL). It also serves
as a precursor to C20 eicosanoids (prostaglandins, leukotrienes and
thromboxanes), which have anti-inflammatory effects (Kleiner et al.
2014). The species S. platensis is a well-known source of GLA, since in
cyanobacteria this fatty acid plays a role similar to that of α-linolenic
acid (ALA, C18:3 omega 3) in algae and higher plants.
 153

The green alga C. vulgaris contains 30% SFA (mainly C16:0), 10%
MUFA (mainly C16:1), and 60% PUFA, with a higher proportion of ω-
3s. In fact, except for S. platensis, N. gaditana, and P. cruentum, all
other microalgae studied present ω-3/ω-6 ratios of ≥ 2, as can be
observed in Table 7.1. Indeed, in cyanobacteria such as S. platensis, the
unsaturated double bonds are preferentially in the ω-6 position, while in
Chlorophyceae they are mainly in the ω-3 position (Batista et al. 2013).
As shown in Fig. 7.1, C. vulgaris and P. cruentum have the same PUFA
content (60%). However, in C. vulgaris, α-linolenic acid (ALA, C18:3
omega 3) contributed most to this species‘ higher omega-3/omega 6-
ratio, while in P. cruentum arachidonic acid (AA, C20:4 omega 6) was
primarily responsible for this alga‘s lower omega- 3/omega 6-ratio,
which is 10 times lower than that of C. vulgaris.
On the other hand, P. cruentum is rich in AA (3.8 g/100g) and
could serve as a source material for producing this fatty acid which, as a
result of its metabolic breakdown, leads to an increased production of
prostaglandin E2 (a group of hormone-like substances which participate
in a wide range of bodily functions), thromboxane, and leukotriene
(Kleiner et al. 2014).
In the marine alga N. oculata and the diatom P. tricornutum, the
main SFA is palmitic acid (C16:0), and the main MUFA is palmitoleic
acid (C16:1). These two microalgae also contain 36% and 44% PUFA,
respectively, with favorable omega- 3/omega-6 ratios of about 6.5. They
are also rich in EPA and have a small fraction of DHA. N. oculata
contains 3.1 g EPA and 45 mg DHA per 100 g microalgal biomass,
while P. tricornutum has 2.8 g EPA and 84 mg DHA per 100 g
microalgal biomass.
The main market of an oil rich in both DHA and EPA is infant
formula, which is mostly derived from a strain of Schizochytrium that
recently entered the market (Borowitzka (2013). Currently, there is no
other commercial production of EPA-rich oils from microalgae, but
Aurora Algae has announced a product from the marine
eustigmatophyte Nannochloropsis. According to the American Heart
Association, a daily intake of 500 mg EPA + DHA and 800–1000 mg of
ALA per day are recommended for the primary prevention of coronary
heart disease (FAO, 2008). The results of our evaluations indicate that
the marine species N. oculata and P. tricornutum are potential sources
of EPA, with an average of 2.8 g/100g, whereas C. vulgaris has a robust
ALA (2.8 g/100g) producing profile. Therefore, such microalgae have
an enormous potential for applications in the development of health-
enhancing products, such as single cell oils (SCO).
 154

Table 7-1 - Lipid content (%), (saturated + monounsaturated/polyunsaturated fatty acids) and ɷ-3/ɷ-6 ratios from six
microalgae species.
C. vulgaris S. platensis N. gaditana N. oculata P. tricornutum P. cruentum
Lipids 12.8 ± 0.1 5.5 ± 1.2 8.1 ± 0.1 15.6 ± 1.1 14.9 ± 0.4 5.3 ± 0.3
ɷ-3/ɷ-6 2.10 0.12 0.08 6.88 6.10 0.15
(SFA + MUFA)/PUFA 0.66 2.03 19.0 1.77 1.33 0.66

Fig. 7-1 - Chart showing fatty acids composition from six microalgae biomass.
P. tricornutum 28 28 38 6 %

N. oculata 29 35 31 5 %

C. vulgaris 30 10 40 20 %

P. cruentum 35 5 8 52 %

S. platensis 52 15 4 29 %

N. gaditana 68 27 1 4 %

Fatty acid content normalized to 100 percent

Saturated Monounsaturated Polyunsaturated ω-3 Polyunsaturated ω-6

 155

Principal component analysis (PCA) was applied to determine

which variables best define and differentiate the six microalgae species.
Fig. 7.2 shows the projection of the microalgae samples, which are
distributed in planes according to their fatty acid compositions. In the
upper-right quadrant, N. oculata and P. tricornutum are positively
correlated because of their EPA and DHA contents. In the lower-left
quadrant, the red alga P. cruentum is distinguished from all other algae
due to the high levels of AA and LA. The alga C. vulgaris and blue-
green alga S. platensis, which are both producers of ALA and GLA, are
located in the upper-left quadrant, while the species N. gaditana is
positioned in the upper-right quadrant associated with the major SFAs,
especially the acid C16:0. In the microalgae we studied, it was observed
that higher total omega-3 PUFA values are associated with higher
C16:1, EPA, and DHA levels, and that there are positive correlations
between total omega-6 PUFA and ALA or AA.

Fig. 7-2 - Principal component analysis (PCA) projection and

distribution of the six microalgae species and their major fatty acid
component.
1.0

N. gaditana
GLA

S. platensis N. oculata
ALA

C. vulgaris
EPA/DHA
0.0

P. tricornutum

LA
P. cruentum

AA

-1.0
-1.0 0.0 1.0

C18:2 ɷ6 (LA linoleic acid), C18:3 ɷ3 (ALA α-linolenic acid), C18:3 ɷ6

(GLA γ-linolenic acid), C20:4 ɷ6 (AA arachidonic acid), C20:5 ɷ3 (EPA
eicosapentaenoic acid), C22:6 ɷ3 (DHA docosahexaenoic acid)
 157

CHAPTER 8

NON-THERMAL PLASMA ASSISTED EXTRACTION OF

INTRACELLULAR LIPIDS FROM Nannochloropsis
gaditana FOR BIODIESEL PRODUCTION

Abstract

The effect of non-thermal plasma (NTP) on Nannochloropsis

gaditana cell rupture and subsequently lipid extraction by mixture of
solvents chloroform and methanol was assessed. Descriptions of
disrupted algal cell by NTP were supported by micrograph images of the
damaged cells. Applying 10 min of NTP to the algal biomass
significantly increased the lipid recovery (18.5%) in comparison to
control test (9.5%). Lipids from unruptured cells were mainly composed
of polyunsaturated fatty acids ω-3 (~31.0% of total fatty acids), while
the ones from the ruptured algal cells were predominantly composed in
saturated fatty acids, which may be associated to the effect of high
voltage of the NTP. This study demonstrates and explains an alternative
approach based on NTP-technology for algal cell rupture prior to lipid
extraction, and provides the first-time description of using NTP-assisted
for algal application.

Keywords: Microalgae; plasma-induced chemistry; cell disruption;

lipid recovery; fatty acids, biodiesel properties.

Graphical abstract

Non-thermal plasma
(high voltage) Disrupted algal
Intact algal
cells cells
158

Introduction

As part of the plasma science, the use of non-thermal plasma

(NTP) has been emerging as a potential alternative approach into diverse
applications, including industrial sterilization (Alkawareek et al. 2014),
pollution control (Kim 2004), polymer science (Pandiyaraj et al. 2015),
food safety (Ma et al. 2015; Ragni et al. 2010), biomedicine (Zhang et
al. 2014) and nanoscience (Graham and Stalder 2011). NTP-technology
has been extensively employed for processing organic materials and also
been considered very promising for catalyst modifications or treatments
because of its non-equilibrium properties and its capacity to induce
physical/chemical reactions at relatively low temperatures (Fridman
2008). In the context of food processing, a NTP is specifically an
antimicrobial treatment being investigated for applications to fruits,
eggs, vegetables and other foods with fragile surfaces (Liao et al. 2017).
By definition, NTP is an ionized gas that can be obtained by applying a
high difference of potential between two electrodes isolated each other
by a dielectric. Under a gaseous atmosphere, electrons are accelerated
by an electric field and collide with neutral molecules in the gas phase
which generate highly active species (atoms, ions, electrons, free
radicals, photons, etc) (Gaunt et al. 2006; Van Durme et al. 2014).
Non-thermal technologies can be defined as technologies that do
not need any external source of heating, the chemical reaction being
active either by (i) the technology input via the action of a pressure,
electric or magnetic field, waves, light to mention a few or (ii) by the
heat generated in situ. With the emergence of biomass in chemical
processes, much effort has been paid to these non-thermal technologies
such as ultrasound, atmospheric plasma, photochemistry, microwaves,
mechanocatalysis, etc (Jérôme 2016). Very recently, this technology has
emerged as a promising route to synthesize biofuels, such as bioethanol
(Benoit et al. 2012; Delaux et al. 2016) and biodiesel (Cubas et al.
2016). In this regard, our group has investigated the contribution of NTP
based on corona discharge technology for the production of biodiesel
from waste frying oil containing free fatty acids (Cubas et al. 2016). Use
of NTP for the synthesis of biodiesel has never been deployed and it has
recently emerged as a promising technology in biorefinery. According
to latter authors, the major advantage of applying NTP for biodiesel
purposes is that the methyl ester can be obtained in the absence of
chemical catalysts and without the formation of the co-product
(glycerin). Furthermore, NTP offers advantages on faster reaction time
and easy separation of the final product (Istadi et al. 2011). Despite
 159

some benefits of NTP for biofuel production in terms of short reaction

times (few minutes) and no need of a solvent or catalyst offering a
convenient downstream processing, the energy consumption of NTP is
the main cost driver and its long-term reliability, efficiency and
effectiveness are still unknown for biofuel production (Jérôme 2016;
Zhang et al. 2017).
Microalgae have been identified by many researchers as a
promising feedstock for biofuel production (Chisti 2007; Franz et al.
2013; Jazzar et al. 2015; Phukan et al. 2011). In fact, several species of
microalgae are able to accumulate carbohydrates and lipids which can
be chemically converted into biofuels (Jazzar et al. 2015). Among them,
the ones belonging to the Eustigmatophyceae genus is Nannochloropsis
gaditana species which is particularly interesting because of its high
growth rate and lipid productivity as well as the ability to synthesize
high value chemicals such as fatty acids with medium (C16 and C18) and
long (C20:5ɷ3) chain carbons, which are ideal lipid sources for the
production of the nutraceutically valuable eicosapentaenoic acid (EPA)
and biodiesel (Matos et al. 2015; Matos et al. 2016; Mitra et al. 2015).
Production and extraction of intracellular metabolites from
microalgae involve extensive processing steps starting with algal
cultivation, algal biomass recovery or harvesting through separation of
the biomass from the carrier media, followed by further processing such
as dewatering, drying, cell disruption, extraction and product
purification (Show et al. 2014). The complete disruption of the algal cell
wall prior to product recovery has been shown to be critical for
successful process-scale lipid extraction from wet algal biomass
(Mendes Pinto et al. 2001; Gunerken et al. 2015). Feasible ways of
extracting intracellular products is through disruption by physical,
chemical or enzymatic methods to release the intracellular oil into the
external medium (Yap et al. 2015). A range of cell rupture techniques
including high pressure homogenization (Samarasinghe et al. 2012; Yap
et al. 2015), ultrasonication (Halim et al. 2012; Lee et al. 2010),
microwave heating (Lee et al. 2010; McMillan et al. 2013), bead milling
(Postma et al. 2015) and osmotic shock (Lee et al. 2010) have been
tested on algal suspensions and all have generally been shown to have a
positive effect on lipid extraction. Alternatively, we are proposing the
use NTP as a method to disrupt algal cell wall prior to lipid extraction,
which is included as an emerging technology.
The algal cell wall is a robust structure that completely encloses the
cytoplasm and allows the cell to increase its turgor pressure without
bursting. The multiple variations observed in algal cell wall,
160

ultrastructures and compositions distinguish them from each other

(Borowitzka and Moheimani 2013). The marine N. gaditana is
distinguished basically by its rigid cell wall, and its high amount of
membrane-bound eicosapentaenoic acid (Yap et al. 2015). Furthermore,
the spherical shape and small size (1-2 µm) of Nannochloropsis cells
make them especially difficult to lyse. As such, N. gaditana one of the
most industrially promising yet difficult to mechanically rupture species
of algae, was chosen as the target organism. Here, we report a cross-
disciplinary study at the border of chemistry and physics based on the
utilization of NTP on N. gaditana biomass. The aims of the present
study are: (1) to apply the NTP on algal biomass and subsequently
determine the lipid content as well as the main fatty acids composition;
(2) to examine the algal cell wall subjected to NTP using direct
microscopy techniques such as laser scanning confocal microscope and
scanning electron microscopy; and (3) to estimate the biodiesel physical
properties using empirical formulas.

Materials and methods

Microalgae and cultivation

The marine species N. gaditana (strain BMAK 130) was kindly

supplied by Banco de Micro-organismos Aidar & Kutner (BMA&K) of
the Oceanographic Institute at the University of São Paulo (USP). This
microalga was cultivated in autoclaved F/2 seawater medium suitable
for marine algae (Guillard 1975). The culture was developed using
inverted conical photobioreactors (5.5 L) with a working volume of 3.5
L and incubated at room temperature (26 ± 2 °C), sparing with saturated
air-CO2, photoperiod of 12 h:12 h light/dark provided by fluorescent
lamps (Osram Universal, Brazil), under a light intensity of 80 µmol m-2
s-1. When algae reached the stationary phase (10 days of cultivation),
sample was harvested by centrifugation at 4000 x g for 20 min. washed
with ammonium formate and centrifuged again (Nova Técnica
centrifuge, Piracicaba, Brazil) in order to remove salts from the algal
biomass. In addition, NTP-treatment of marine microalgae requires pre-
washing and deionization to increase the electrical resistance of the
medium surrounding the cells (Gunerken et al. 2015).
 161

Non-thermal plasma reactor

The in-house-built kHz-driven plasma source used in this study is

shown in Fig. 8.1. A cylindrical discharge reactor with point-to-plate
geometry operated at atmospheric pressure was used in this work. The
cylinder is made of borosilicate glass, has a radius of 22 mm and a
height of 70 mm. The active electrode is a hollow needle made of
stainless steel, the tip of which is placed 10 mm above the solution
surface (volume of the solution: 40 mL). Through this hollow needle,
argon gas is fed into the reactor at a flow rate of 1 L min-1. The ground
electrode is made of stainless steel wire with a diameter of 1.0 mm. The
top of the glass cylinder is closed by a teflon cover equipped with a gas
outlet. The reactor is fed by a direct current (DC) impedance voltage
transformer (60 Hz, 220 V/12,000V) purchased from Variac (model
ATV-215-MP, São Paulo, Brazil). A tripler voltage equipment
composed of 4 diodes and 8 capacitors was used to convert the alternate
current (AC) into direct current (DC). An iCEL Manual MD-1000
multimeter was used to monitor the voltage, electrical resistance and
current between the discharge gap. The electrical parameters applied for
all experiments were: 17 kV and 30 mA. Under these conditions, an
intense core plasma was formed between the two electrodes, and a
luminous plume extended out of the tube end reaching the treated
sample. For NTP-treatments, a mixture of 1 g of algal sample and 40
mL of water was put in the glass container and placed in the plasma
setup.

Fig. 8-1 - Schematic diagram and a photograph of the non-thermal

plasma (NTP) reactor.

Ground electrode
Voltage
Tungsten rod tripler
Teflon cap

Borosilicate
glass
Power supply
Algal sample (High voltage)
Argon

Sonication
162

A sample of ~1.0 g of the wet algal biomass was mixed to 40 mL

of distilled water and transferred to Falcon tube and subjected to
sonication treatment using an Ultrasonic Cleaner processor (Indaiatuba,
São Paulo, Brazil) at a resonance of 20 kHz. Algal separation was
conducted by centrifugation at 4,000 rpm for 20 min. and the biomass
was analyzed in terms of lipid content.

Cell breakage quantification via hemocytometry

Quantification of cell rupture was performed by cell counting due to

its accuracy and reproducibility (Yap et al. 2014; Samarasinghe et al.
2012). The extent of cell disruption was assessed by the number of
intact cells remaining after NTP using a Neubauer hemocytometer
(Labotoptik Ldt., Lancing, United Kingdom) with a 100 μm chamber
depth. All imaging was performed using an Olympus BX40F4 (Japan)
light microscope with 40x object resolution and with an Opticam®
digital camera attachment.

Micrographs images

Scanning electron microscopy

A scanning electron microscopy (SEM) (JEOL JSM-6460LA,

Tokyo, Japan) with a tungsten filament electron gun was used to
characterize the algal powder surface at 10-15 kV. Algal powder was
mounted onto SEM stubs by placing or sputtering them on a carbon
double-sided adhesive tape. Excess particle were removed with gentle
tapping. Coating was done with gold using a Xenosput Sputter Coater
(Xenosput XE200, Edwards High Vacuum International, Cambridge,
U.K.) for 2 min (~10 nm thick).

Laser scanning confocal microscope

A detail examination of the algal cells by using direct optical laser

scanning confocal microscope was verified. In parallel, intracellular
lipid bodies of the algal cells were visualized using Nile Red stain (9-
(diethyl amino) benzo [a]phenoxazin-5(5H)-one, Sigma – Aldrich),
which was prepared as a stock solution of 250 mg L-1 in acetone. One
milliliter of algae was centrifuged at 4000 rpm for 10 min and a pellet
was re-suspended in 1 mL of 20% DMSO. After vortexing for 10 min at
room temperature, cells were centrifuged at 4000 rpm for 10 min. The
 163

pellet was further suspended in 1 mL of water and vortexed before

adding Nile Red strain (12.5 µL) and incubating for 5 min in the dark at
room temperature (Ahmad et al., 2013). Stained cells were visualized
using a Leica TCS – SP5 laser scanning confocal microscope (Wetzlar,
Germany). Images were acquired using a 100x objective (HCX PL APO
CS with a numerical aperture of 1.44 - oil immersion objective) with a
Leica Type F immersion liquid. Images were taken with a Leica
microsystem camera. The acquisition and processing of data were
carried out using the LAS AF Lite software.

Lipid extraction and fatty acids analysis

Intracellular lipids were extracted from dried biomass by

maceration of the biomass in a mixture of methanol:chloroform:water
(2:1:0.8 v/v/v) (Bligh and Dyer, 1959) and quantified gravimetrically.
Fatty acids composition was determined after conversion of the
fatty acids to their corresponding methyl esters. The fatty acid methyl
esters (FAME) were characterized on a gas chromatograph, model GC-
2014 (Shimadzu, Kyoto, Japan), equipped with split-injection port,
flame-ionization detector, a Restek a 105 m-long capillary column (ID =
0,25 mm) filled with 0.25 μm of 10% cyanopropylphenyl and 90%
biscyanopropylsiloxane. Injector and detector temperatures were both
260°C. The oven temperature was initially set at 140°C for 5 min, and
then programmed at 2.5°C min-1. The qualitative fatty acids composition
was determined by comparing the retention times of the peaks with the
respective fatty acids standards (Sigma, St. Louis, USA). The
quantitative composition was obtained by area normalization and
expressed as mass percent.

Estimation of physical properties of biodiesel

The key physical properties of biodiesel such as saponification

value (SV, mg KOH), iodine value (IV, gI2 per 100g), cetane number
(CN), long-chain saturation factor (LCFS, % wt), cold filter plugging
point (CFFP, °C), degree of unsaturation (DU, % wt), higher heating
value (HHV, MJ kg-1) and oxidative stability (OS, hr) were calculated
by using following empirical formulas (Aurora et al. 2016):

SV = Σ560 (%FA)/Mi
IV = Σ254 DB x (%FA)/Mi
CN = 46.3 + 5458/SV – (0.255 x IV)
164

DU = MUFA + (2 x PUFAs)
LCFS = (0.1 x C16) + (0.5 x C18)
CFFP = (3.147 x LCFS) – 16.477
HHV = (49.43 – 0.041 (SV) – 0.015 (IV)
OS = 117.9295 / (% wt C18:2 + % wt C18:3) + 2.5905

where FA = content of each fatty acid component, Mi = molecular

mass of each fatty acid component, DB = number of double bonds,
MUFA = content of monounsaturated fatty acids (percent by weight),
PUFAs = content of polyunsaturated fatty acids (percent by weight) and
C18:2 (linoleic acid) and C18:3 (linolenic acid) represent the 18 carbon
chain having two and three double bonds, respectively.
Fig. 8-2 - Flow diagram for the usage of sonication and NTP technology as a tool for microalgal lipid extraction.

Algal culture (photobioreactor)

Centrifugation

Wet biomass (~1 g)

Centrifugation + ammonium formate

N. gaditana cells (1-2 μm)

Sonication Non-thermal plasma Micrograph images

Laser scanning confocal

Experimental time:
microscopy (LSCM)
1, 2, 5, 10, 15 and 20 min.

Untreated (control treatment)

Scanning electron
microscopy (SEM)
Lipid content

Fatty acid composition/Biodiesel analysis

(time = 10 min.)
165
166

Results and Discussion

Effect of non-thermal plasma on lipid and fatty acids

Since NTP has been proposed as a potential pretreatment for algal-

lipid extraction, the aim of this experiment was to compare the lipid
content of N. gaditana after NTP and sonication treatments (Fig. 8.2). N.
gaditana cells were passed through the NTP and sonication once at
times of 1, 2, 5, 10, 15 and 20 min. The amount of lipids that could be
extracted with chloroform and methanol mixture (Bligh & Dyer 1959)
was then determined for each sample and related to the measured degree
of cell rupture. Lipid content in N. gaditana cells ranged from 14.7 to
18.7% using NTP-assisted method, while testing with sonication, lipid
content varied from 15.5 to 21.7% (Fig. 8.3). The data show that the
maximum lipid content (~19.0%) for NTP-assisted was observed at 10
min., which is similar and comparable to 10 min. of sonication (19.0%).
Both lipid content (an average of 19.0%) 2-fold higher than untreatment
algal cells (9.5%).

Fig. 8-3 - Comparison of lipid content of N. gaditana biomass after

different time of application of NTP-assisted and sonication treatments.

25
Sonication Non-thermal plasma

20
Lipid content (%)

15

10

0
1 2 5 10 15 20
Time (min.)
 167

Several forces are behind the mechanism of sonication cell

disruption. Sonication vibrations from the emitting tip result in acoustic
cavitation that can disrupt cells, but cavitation also results in thermolysis
of water around the bubbles forming highly reactive free radicals (H●,
HO● and HOO●) that react with the substances in water. Bubble
implosion and fragmentation during acoustic cavitation produce micro-
regions of extreme conditions with estimated temperatures as high as
5000 °C and pressures up to 100 MPa (Gunerken et al. 2015). Similarly,
we hypothesized that NTP promotes a disruption of the rigid algal cell
wall and probably release the intracellular lipid content to the
extracellular matrix, thereby increasing lipid content. In fact, the amount
of lipids extracted was approximately consistent with the extent of cell
rupture (9.5% lipid from 0% cell rupture; 16.7% lipid (2 min. NTP)
from 25-30% cell rupture and 18.7% lipids (10 min. NTP) from 65-70%
cell rupture). However, after 15 and 20 min. of NTP, lipids remain
constant, 18.5 and 18.6% (Fig. 8.3), respectively, with a significant
population cells probably incapable of resisting breakage by NTP.
These findings on the use of NTP-assisted on N. gaditana biomass
for lipid extraction can be comparable to the similar observations by
Goettel et al. (2013) when 40 min. after pulse electric field (PEF-
assisted) was applied on the Auxenochlorella protothecoides biomass
with higher lipid-yields than control treatment coupled with algal cell
disintegration by PEF treatment. In addition, the enhancement of the
extraction efficiency of pigments such as C-phycocyanin from PEF
electroporated microalgae (S. platensis) has been observed by other
authors (Martínez et al. 2016). According to Gunerken et al. (2015),
PEF uses an external electric field to induce a critical electrical potential
across the cell membrane/wall. Cell disruption by PEF is caused by
electromechanical compression and electric field-induced tension
inducing pore formation in the membrane/wall. The same authors also
mentioned that PEF does not only destroy the cell wall, but also affects
the molecules inside the cells. Though temperature increase is not the
mechanism of cell disruption, the increase in bulk temperature during
treatment leads to a reduced nutritional value and protein digestibility
(Janczyk et al. 2005) and an increased extraction of lipids (Puértolas et
al. 2016).
168

Table 8-1 - Main fatty acids methyl esters (FAMEs) presented in N.

gaditana biomass (%).
Treatment
Control Sonication Non-thermal plasma
(10 min.) (10 min.)
C12:0 1.1 ± 0.1 0.9 ± 0.1 0.5 ± 0.1
C14:0 5.4 ± 0.2 7.2 ± 2 5.2 ± 0.2
C16:0 19.4 ± 5 20.0 ± 3 36.0 ± 4
C18:0 2.6 ± 0.1 1.4 ± 2 1.4 ± 0.1
Ʃ SFA 26.1 ± 6 31.7 ± 3 44.6 ± 5
C15:1 0.8 ± 0.1 0.7 ± 0.1 0.7 ± 0.1
C16:1 22.4 ± 3 22.5 ± 2 22.8 ± 4
C18:1 2.8 ± 0.1 5.8 ± 0.1 9.7 ± 0.8
C22:1 6.0 ± 0.2 2.9 ± 0.1 2.3 ± 0.1
Ʃ MUFA 32.0 ± 5 33.6 ± 6 36.0 ± 4
C18:3 ω3 (ALA) 0.3 ± 0.1 2.0 ± 0.1 0.7 ± 0.1
C20:5 ω3 (EPA) 31.1 ± 4 27.8 ± 4 11.0 ± 2
Ʃ PUFA-ω3 31.4 ± 3 28.0 ± 2 11.7 ± 1
C18:2 ω6 2.9 ± 0.1 3.2 ± 0.1 0.3 ± 0.1
C18:3 ω6 (GLA) 0.5 ± 0.1 0.1 ± 0.1 0.1 ± 0.1
C20:3 ω6 1.1 ± 0.1 0.2 ± 0.1 0.6 ± 0.1
C20:4 ω6 - 0.3 ± 0.1 0.2 ± 0.1
Ʃ PUFA-ω6 4.5 ± 0.5 4.3 ± 0.2 1.2 ± 0.2
Note: ALA (alpha-linolenic-acid); EPA (eicosapentaenoic acid); GLA
(gamma-linolenic-acid); SFA (saturated fatty acids); MUFA
(monounsaturated fatty acids); PUFA (polyunsaturated fatty acids).

With regard to the fatty acid compositions of N. gaditana

biomass (Table 8.1), control test (without any pretreatment) showed an
expressive content in PUFAs of around 31.4% of the total fatty acids,
which was comparable trend to the sonication pretreatment (PUFAs =
28.0% of the total fatty acids), mainly composed by eicosapentaenoic
acid (EPA, C20:5 ɷ3). Interestingly that the PUFAs ɷ-3, especially
C20:5 ɷ3 decreased from 31.4% (control test) to 11.0% when NTP was
applied. In addition, a noticeable formation of SFAs, mainly palmitic
acid (C16:0, 36.0% of the total fatty acids) has been observed when
NTP-assisted was employed. It appears that when NTP-treatment is
applied on N. gaditana biomass, there is a tendency toward the
formation of SFAs. These changes in fatty acid composition, i.e. an
increase formation of SFAs, can be comparable with the study
performed by Yepez et al. (2016), who observed an increase formation
 169

of SFAs when high-voltage atmospheric cold plasma (HVACP-assisted)

was applied in soybean oil hydrogenation. The same authors also
observed that the unsaturated fatty acid contents can be chemically
modified without the formation of trans-fatty acids.
The application of NTP to algal biomass and the effect of NTP on
fatty acid composition hampers the description of the exact mechanism
of plasma fatty acid reaction, but studies of possible reactions of the
NTP (Cubas et al. 2016) suggest that the high-energy atmosphere of
plasma facilitates the breakage of the double bonds and hydroxyl
groups, which leads to the conclusion that there is the formation of
single bounds when NTP-assisted treatment is applied. In addition, this
phenomenon could be attributed to the presence H+ and OH- molecules
that causes dissociation, ionization and vibrational/rotational excitation
of the polyunsaturated fatty acids which leads to numerous electron-
collision-dominated reactions that can fragment molecules into neutral
and ionic products (Graham and Stalder 2011). According to Jiang et al.
(21014), the reaction of plasma with organic compounds is generated by
three basic mechanism of reaction: hydrogen abstraction, the
unsaturated electrophilic addition reaction and electron transfer. In the
case of compounds having aliphatic hydrocarbons such as lipids, the
hydrogen abstraction is the primary mechanism for producing SFAs.

Micrograph images of algal biomass after non-thermal plasma

Microscope observations represent a qualitative approach to the

success of the different cell disruption techniques. The aim of this
experiment was to visualize by microscopy techniques the degree of
disruption of the rigid microalgal cell wall. N. gaditana cells were
passed through the NTP after applying 10 min. of exposure. Microscope
images of the cells after NTP-assisted showed the effect of the extent of
the cell rupture. Scanning electron microscopy (SEM) and laser
scanning confocal microscopy (LSCM) images supported that the NTP
was indeed very potent, giving rise to effective cell disruption (Fig. 8.4
and 8.5). There was a noticeable difference between the intact cells and
cells that were exposed to NTP. The surfaces of the original N. gaditana
cells appeared smooth and had no apparent holes (Fig. 8.5a), whereas
the cells disrupted by NTP, showed a non-uniform surface, apparently
fractured (Fig. 8.4a), and many pores and large splits (Fig. 8.4b and
8.5b).
170

Fig. 8-4 - Scanning electron micrographs (SEM) of N. gaditana cells

that were exposed to non-thermal plasma; (a) The image shows a non-
uniform surface of algal cells apparently fractured; (b) N. gaditana cells
showing large splits.

bb
 171

Fig. 8-5 - Laser scanning confocal micrographs (LSCM) of N. gaditana

cells; (a) original cells showing red fluorescence from chlorophyll after
UV excitation; (b) and (c) micrographs of cells after exposure to non-
thermal plasma. N. gaditana cells disrupted showing large splits (b) and
algal cells showing oil droplets with yellow color after Nile Red stained
(c). Algal cells viewed under 100x objective with 530 nm excitation
wavelength and 575 nm emission filters. Scale bars = 5 µm.

a c

The intracellular lipid bodies of the microalgal cells, after NTP-

assisted and stained with Nile Red, appear to be more prompt to lipid
extraction (Fig. 8.5c). Interestingly in Fig. 8.5(c) that there are some
microalgal cells without the cell body, i.e. it appears that the matrix is
released in the cytosol, and stained-lipids are kept within the plasma
membrane in which marks the boundary between the cell wall on the
outside and cytoplasm on the inside. From these results, the NTP-
assisted method that had an ability to change the surface of the
microalgal cells as well as modify or lyse the cell could be a potential
pretreatment method on disruption of the microalgal cell wall prior to
target molecules, such as lipids.
172

According to Puértolas et al. (2016) which studied the effect of

NTP based on pulsed electric field (PEF-assisted) on olive oil
extraction, suggest that NTP causes formation of pores in the cell
membranes of plant issues. This process, called electroporation,
enhances the diffusion of solutes through cell membranes, favoring
recovery of intracellular oil. In the same way, the application of NTP-
assisted on algal biomass could improve lipid recovery by a double
mechanism: the improvement of lipid extraction from algal biomass
through disruption of rigid cell wall, and the releasing of lipids trapped
in cell-membrane body.

Estimation of physical properties of biodiesel

In order to assess the acceptability of biodiesel produced from N.

gaditana that was subjected to NTP and sonication pretreatment,
biodiesel physical properties were calculated using empirical formulas.
The physical properties were compared with commercially used plant
oil methyl esters (Jatropha and Palm) and ASTM D6751 in the United
States and EN 14214 in Europe biodiesel fuel quality. Data indicated
that the physical properties complied with the fuel standards and were
comparable with the plant oil methyl esters as shown in Table 8.2.
Iodine value (IV) is often used to determine the amount of
unsaturation in fatty acids. This unsaturation is in the form of double
bonds, which react with iodine compounds. The higher IV, the more
C=C bonds are present in the oil. It can be seen in Table 8.2 that IV
from N. gaditana oil in control test (171.5 g I2/100 g) and sonication
(154.7 g I2/100 g) are higher than IV from NTP-assisted (83.6 g I2/100
g), which is comparable with IV from Jatropha oil (96.5 g I2/100 g), and
in accordance with IV from ASTM D6751 fuel standard set to a
maximum of 120 g I2/100 g oil.
Cetane number (CN) is one of the most significant indicators for
determining combustion behavior of biodiesel (Islam et al. 2013). High
CN ensures less ignition delay, better combustion and cold start
properties with minimum white smoke resulting in efficient engine
performance (Knothe 2006). The highest CN (53.0) was recorded in
NTP treatment due to higher content in SFA (mainly C16:0), followed
by sonication (34.8) and control test (31.6) which are mainly composed
of PUFAs, maily EPA. Most biodiesel fuels from vegetable oil sources
possess CN in the range of high 40s to lower 60. The specifications for
minimum CN of biodiesel are 47 in ASTM D6751 and 51 in EN 14214.
Generally, higher CN are more desirable (Knothe 2013).
Table 8-2 - Comparison of physical properties of biodiesel produced from N. gaditana with vehicular biodiesel
standards (ASTM D6751 and EN 14214), and plant based biodiesel (Jatropha oil methyl esters (JME) and palm oil
methyl esters (PME)).
Quality parameters N. gaditana Plant oil
methyl esters
Biodiesel property ASTM EN Control Sonication NTP PME JME
D6751 14214 test
a a
Saponification value (SV, 187.4 193.0 194.5 - -
mg KOH)
Iodine value (IV, g I2/100 g) 120 109.2 171.5 154.7 83.6 49.5 96.5
(maximum)
Cetane number (CN, 47 51 31.6 34.8 53.0 61 54
minimum)
a a
Degree of unsaturation (DU, 103.8 96.5 61.3 - -
% wt)
a a
Long-chain saturation factor 5.3 7.3 9.7 - -
(LCSF, % wt)
a
Cold filter plugging property ≤5/≤-20 0.2 6.7 14.2 13 -2
(CFPP, °C)
a a
High heating value (HHV, 39.2 39.1 40.2 - -
-1
MJ kg )
Oxidative stability (OS, hr) 3 ≥6 34.6 35.6 101.7 16.5 3.8
a
No limit designated for the physical property
173
174

When possible, oxidation stability (OS) was calculated based on

C18:2 and C18:3 acids content. Most of OS values calculated here
(34.6, 35.6 and 101.6 hr) are above the range reported for EN 14214
biodiesel standard (≥6 hr) and algal methyl esters (8.5-11 hr) (Islam
2013). It must be pointed out that these estimates must be taken with
caution, as values much exceeding the set time frame of 6 hr are likely a
function of low contents of these fatty acids. In addition, this OS
formula was developed by Park et al. (2008) for higher plants, where
other long-chain polyunsaturated acid contents are low, for example
EPA and DHA. Consequently, the reliability of OS for N. gaditana with
high EPA content but low C18:2 and C18:3 contents is questionable.
Biodiesel derived from these oils with high amounts of polyunsaturated
fatty acids would likely display poor OS per the Rancimat test in EN
14112 and not meet the minimum values prescribed in standards besides
not meeting the restrictions on such kinds of acids in the European
biodiesel standard EN 14214 (Knothe 2013).
For comparison purposes, the fatty acid profiles of biodiesel
(methyl esters) derived from two common commodity oils may be noted
here. The fatty acid profile of soybean oil consists of palmitic (usually
10-11%), stearic (4-6%), oleic (21-25%), linoleic (50-55%) and
linolenic (around 8%) acids; the palm oil: palmitic (around 40-45%),
stearic (4-5%), oleic (around 40%) and linoleic (around 10%) with
lesser amounts of other fatty acids. The differences in the fatty acid
profiles are manifested in different fuel properties (Knothe 2013). As N.
gaditana exhibit high amount of unsaturated fatty acid EPA, biodiesel
derived from oils with such fatty acid profile would likely display poor
cold flow and poor OS simultaneously. On the contrary, after applying
NTP in N. gaditana biomass, palmitic acid appears to be the most
common fatty acid suggesting that cold flow and OS properties may be
less problematic for biodiesel production, enhancing the quality of the
biodiesel.

Correlated studies and future research

The cutting-edge NTP-technology provides notable advantages, but

also drawbacks. Determining the exact mechanism taking place in the
NPT reactor is difficult. Particularly, the characterization of active
species in the NTP gas (or in water in the case of algal biomass) is not
trivial due to their short life time and formation of a cocktail of active
species. Real time in-situ FT-IR has been proposed to better understand
the nature and how active species react with the surface of valuable
 175

oligossacharides (depolymerization of cellulose into branched glucans)

exposed to NTP. It has been hypothesized that these radicals react with
the surface of cellulose to form sugar radicals ultimately leading to bond
cleavages (Jérome 2016). Similarly, NTP-induced morphological
alterations of microalgae that cause damage or disruption of cell wall
leading to diffuse lipids within the bulk material.
It has been previously established that, under air, NTP creates a
cocktail of highly excited species and particularly radicals. In the case of
algal biomass for which NTP occurs in water, we cannot be certain,
whether the pronounced reactive radicals species lead to hydrolysis or
possible oxidation reactions between membrane lipids containing
PUFAs and freshly produced O2, N2, O3, H●, HO●, H2O2 radicals.
Although there is limited direct contact between the reactive radicals
and cell surfaces in the aqueous environment of algal cells, we do not
discount a secondary role for such reactions.
In the context of algal biodiesel production by NTP-technology, it
is suspect that adding methanol to the plasma reactor, the
transesterification reaction may occur inside the NTP reactor leading to
the formation of biodiesel, which is in consonance with Cubas et al.
(2016). At the stage of current investigations, three parameters seem
important to convert algal oil into biodiesel by NTP (1) the oil/methanol
molar ratio, (2) water concentration and (3) reaction time. To date, this
aspect remained unclear and much effort is still needed in this direction
to explore the potential of applications of NTP in this field and more
largely in the field of algal biodiesel.
The results presented here highlight key differences in the
lipid/fatty acid fraction and biodiesel properties from N. gaditana that
were subjected to sonication and NTP-treatment. The evaluation of the
relationship between cell disruption and the subsequent intracellular
lipid extraction undertaken in this study precedes a quantitative
description of this process and its dependence on key process variables,
such as degree of cell disruption, the effects of plasma-chemistry and
potential formation of radicals. One the biggest issue is related to
mechanism investigations (Jérome 2016).
Particularly, a better understanding of the nature of produced
radicals and how they diffuse within the bulk of algal matrix is of high
importance to determine the potential of NTP. In addition, further
understating of the interaction among reaction time, algal cell wall
chemistry and unsaturation of fatty acid could provide valuable
information for further optimization of this process. Ultimately, NTP is
indeed closely depending on the access to energy and particularly
176

electricity. Like for many technologies/processes, the sustainability of

NTP will definitely rely on a sustainable source of energy, the aspect
being difficult to predict since chemistry is a dynamic system.

Conclusions

This study has investigated the application of NTP-assisted as a

pretreatment algal cell disruption when applied to N. gaditana at
laboratory scale. Taken together, NTP clearly evidence that a
considerable cell disintegration of this microalgae was noticed. Thus,
NTP may be used as an alternative pretreatment method prior to algal
lipid extraction. Results also suggest that the high voltage of NTP
allows the breakdown of the double bonds of PUFAs, which lead to
converting them in single bonds, and consequently formation of SFAs.
While there is still more to go in the development and utilization of NTP
for algal bioprocess, this emerging technology towards algal cell
disruption prior to lipid extraction is a promising technology for algal
biodiesel production. Finally, defining NTP as a sustainable cell
disruption method is too early at this stage, but to support future
research and to be able to compare cell disruption efficiencies, a generic
method for data collection should be used systematically to produce
comparable data.
 177

4 GENERAL DISCUSSION AND CONCLUSIONS

The main contributions of this thesis were: i) to review microalgae

biotechnology with a focus on food science and technology (Chapters 1
and 2); ii) to review, based on literature data, the state-of-art of
combining desalination concentrate (DC) and microalgae cultivation in
Brazil, especially on experimental studies that assess the maximal
biomass and lipid productivities (Chapter 3); iii) to determine the best
DC concentration for maximal biomass and lipid production of marine
Eustigmatophyceae algae N. gaditana. In addition, the reuse of water
and additional carbon source (glucose, glycerin and glycerol) under
mixotrophic conditions were also studied (Chapter 4); iv) to manipulate
the growth conditions (trophic mode, photoperiod and salinity based on
DC-conductivity) of marine N. gaditana in order to determine the
growth parameters (growth rate, maximum cell density and biomass
concentration) and chemical composition (protein, lipids and fatty acids)
(Chapter 5); v) to determine the chemical composition in terms of
moisture, ash, fiber, carbohydrate, protein, lipid and fatty acid
composition as well as to evaluate the nutritional quality indexes (i.e.,
polyunsaturated/saturated fatty acids (P/S), ω-3/ω-6 ratio,
hypocholesterolemic fatty acids/hypercholesterolemic fatty acids (H/H),
atherogenicity index (AI) and thrombogenicity index (TI)) of six
microalgae (C. vulgaris, S. platensis, N. gaditana, N. oculata, P.
tricornutum and P. cruentum) species (Chapter 6); vi) to complement
the previous work by adding a new table in terms of lipid content,
(saturated + monounsaturated/polyunsaturated fatty acids ratio) and
omega-3/omega 6 ratio, and two figures (fatty acids proportion and
principal component analysis) (Chapter 7); vii) to evaluate the effect of
non-thermal plasma (NTP), based on corona discharge, on algal biomass
and lipid content. In addition, NTP proved to be an effective technique
to rupture or lyse N. gaditana cell wall and release the target molecules,
such as lipids, into the aqueous phase (Chapter 8).
A wide range of studies have analyzed the growth of microalgae
under a variety of wastewater conditions, in particular growth in
multiple municipal, industrial and agricultural wastewaters. However,
combing DC and microalgae cultivation is an original and particular
research work of our research group, with several papers been published
in relevant scientific journals. With a growing need for the inland
desalination of brackish water in Brazil, particularly in the semiarid
region of Brazil, the use of DC for algal mass cultivation in semiarid
regions of Brazil needs to be validated in further investigations. This
178

region of Brazil has exceptional qualities to produce algae in open

raceway ponds on a pilot-scale due to its ample solar irradiance, low
seasonal variation, extensive arid lands, and abundant brackish
groundwater where algae production is proposed.
There is a general agreement that microalgae are considered one of
the most promising feedstock materials for developing a sustainable
supply of commodities, including both food and non-food products. As
a result, the chemical characterization of microalgae biomass is crucial
for food/feed/biofuel applications due to the wide range of microalgae
species existing coupled to a variety natural compounds that can be
extracted from these microorganisms. This approach opens significant
opportunity to isolate and identify new strains from natural
environment. In addition, it allows the possibility of finding the best
suitable species for the target compound, for example for food or feed
application, biofuel production or even for pharmaceutical interest.
Based on this fact, further isolation and characterization of new
microalgae species could be of interesting approach than the use of
known algae strains.
The application of non-thermal plasma (NTP) on algal biomass
was the main novelty of this thesis. There has no similar work in the
literature data during the last five years, open up new opportunity for
continuous research. Several doubts about the exact mechanism of NTP-
assisted method still need to be further investigated. In addition, NTP-
assisted was studied in only one algae specie (N. gaditana) and thus it
could be of interesting to evaluate the effect of NTP in different algae
strains, specially for algae that have rigid cell wall, for example
Chlorophyta Chlorella sp. and Tetraselmis sp.
Non-thermal plasma has also been studied in the area of biofuel
production, such as bio-ethanol and biodiesel. In this sense, NTP may
convert the algal-oil coupled with methanol to methyl esters (biodiesel).
However, further investigation must be conducted as well as to optimize
the variables in the process. If NTP convert algal-oil into biodiesel, the
biodiesel quality standards such as acid value, calorific value, cetane
ratings, boiling point and flash point need be determined.
In summary, the present research work has demonstrated the
feasibility of growing microalgae in desalination concentrate,
determined the chemical composition of microalgae biomass for food
application and evaluated the effect of NTP on algal biomass.
 179

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Table A1 Amino acid composition (mg/g) of three microalgae cultured in different proportion of desalination
concentrate (DC). * Recommendation for children of 3-10 years old, according to FAO/WHO (2013).
C. vulgaris S. platensis N. gaditana FAO/WHO (2013)*
(25% DC) (50% DC) (75% DC)
Essential amino acids
Isoleucine 0.82 2.41 0.72 2.8
Leucine 1.71 3.88 1.40 6.6
Lysine 1.12 2.21 0.90 5.8
Methionine 0.31 0.66 0.30 2.5
Phenylalanine 1.00 1.86 0.81 6.3
Threonine 0.93 2.01 0.68 3.4
Tryptophan 0.19 0.51 0.10 1.1
Valine 1.08 2.55 0.90 3.5
Non-essential amino acids
Alanine 1.46 3.78 1.24
Arginine 1.16 2.93 0.96
Aspartic acid 0.49 4.95 0.52
Cystine 0.11 0.35 0.08
Glutamic acid 1.24 5.87 0.97
Glycine 1.06 2.36 0.87
Histidine 0.33 0.64 0.26
Proline 0.88 1.86 0.76
Serine 0.78 2.15 0.65
Tyrosine 0.74 1.97 0.56
Total amino acids 15.41 42.95 12.68
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ANNEX

Declaration by author

This thesis is composed of my original work. I acknowledge that an

electronic copy of my thesis must be lodged with the University Library
and, subject to the police and procedures of the Federal University of
Santa Catarina, Florianópolis SC Brazil.

Publications during candidature:

Original Research Articles:

Ângelo P. Matos, Marina S. Teixeira, Marina M. Machado, Rhuamm

I.S. Werner, Ana C. Aguiar, Anelise L.V. Cubas, Ernani S. Sant‘Anna,
Elisa H.S. Moecke. Non-thermal plasma assisted extraction of
intracellular lipids from Nannochloropsis gaditana for biodiesel
production. Energy Conversion and Management, 2017 (Submitted).

Ângelo P. Matos. The impact of microalgae in food science and

technology. Journal of the American Oil Chemists’ Society, v. 94,
Issue 11, p. 1333-1350, 2017.
https://link.springer.com/article/10.1007/s11746-017-3050-7

Ângelo P. Matos, Elisa H.S. Moecke, Ernani S. Sant‘Anna. The use of

desalination concentrate as a potential substrate for microalgae
cultivation in Brazil. Algal Research, v. 24, p. 505-508, Part B, June
2017.
http://www.sciencedirect.com/science/article/pii/S2211926416302600

Ângelo P. Matos, Monnik C. Gandin, Elisa H.S. Moecke, Ernani S.

Sant‘Anna. Effects of different photoperiod and trophic conditions on
biomass, protein and lipid production by the marine alga
Nannochloropsis gaditana at optimal concentration of desalination
concentrate. Bioresource Technology, v. 224, p. 490-497, January
2017.
http://www.sciencedirect.com/science/article/pii/S0960852416315097
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Ângelo P. Matos. Essential Fatty Acids from Microalgae. Inform

(Champaign): International News on Fats, Oils and Related
Materials, v. 27, p. 22-26, November/December 2016.
http://www.informmagazine-
digital.org/informmagazine/november_december_2016?pg=24#pg24

Ângelo P. Matos, Rafael Feller, Elisa H.S. Moecke, José V. Oliveira,

Agenor F. Junior, Roberto B. Derner, Ernani S. Sant‘Anna. Chemical
characterization of six microalgae with potential for food application.
Journal of the American Oil Chemists’ Society, v. 93, p. 963-972,
July 2016. https://link.springer.com/article/10.1007/s11746-016-2849-y

Ângelo P. Matos, Rafael Feller, Elisa H.S. Moecke, Ernani S.

Sant‘Anna. Biomass, lipid productivities and fatty acids composition of
marine Nannochloropsis gaditana cultured in desalination concentrate.
Bioresource Technology, v. 197, p. 48-55, December 2015.
http://www.sciencedirect.com/science/article/pii/S0960852415011475

Book Chapters:

Ângelo P. Matos & Ernani S. Sant‘Anna. Avaliação bioquímica da

biomassa de microalgas cultivadas em águas residuais
dessalinizadas: De escala de laboratório à produção de algas em
escala piloto no Semiárido do Nordeste do Brasil. Edição 2015 do
Prêmio MERCOSUL de Ciência e Tecnologia: Inovação e
Empreendedorismo, Categoria Jovem Pesquisador. Brasília: Ministério
da Ciência, Tecnologia, Inovações e Comunicações, p. 115-143, 2017.
http://www.recyt.mincyt.gov.ar/files/PremioMercosul/Premio2015/Livr
o_Premio_Mercosul_2015.pdf

Antônio S. Sánchez & Ângelo P. Matos. Desalination concentrate

management and valorization methods. In.: Sustainable Desalination
Handbook: Process Design and Implementation Strategies. Edited by
Veera Gnaneswar Gude. Elsevier Inc. In Press, 2017.

Conference Papers:

AP Matos, EHS Moecke, ES Sant‘Anna Essential fatty acids from

microalgae for food application. In: AOCS, 106th Annual Meeting and
Industry Showcases. Orlando, Florida, USA, 2015 (Oral presentation).
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AP Matos & ES Sant‘Anna. Composição química e índices nutricionais

de seis espécies de microalgas com aplicação em alimentos funcionais,
suplementos e nutracêuticos. In: Jornadas de Jóvenes Investigadores -
AUGM, São Pedro - São Paulo. UNESP, 2016 (Oral presentation).
http://sinter.ufsc.br/publicacoes/

AP Matos, MM Machado, ALV Cubas, ES Sant‘Anna, EHS Moecke.

Microalgae lipid extraction using corona discharge plasma in the
biodiesel production. In: AOCS, 106th Annual Meeting and Industry
Showcases. Orlando, Florida, USA, 2015 (Poster section).

R Feller, AP Matos EHS Moecke, ES Sant‘Anna, RB Derner, JV

Oliveira, JR Furigo. Comparative study of biochemical composition of
five microalgae for biodiesel/bioproducts application. XX Congresso
Brasileiro de Engenharia Química, Florianópolis/SC, v. 1. p. 1499-1506,
2015. http://www.proceedings.blucher.com.br/article-details/16795

Awards & Recognition:

January 2016 - Researcher Honorable Mention by Algae Industry

Magazine – United States.
http://www.algaeindustrymagazine.com/readers-poll/

May 2016 – Prêmio MERCOSUL de Ciência e Tecnologia. Tema

Inovação e Empreendedorismo. Categoria Jovem Pesquisador.
Ministério da Ciência, Tecnologia e Inovação – MCTI CNPq. Processo
433306/2016-5.
Título do trabalho: Avaliação bioquímica da biomassa de microalgas
cultivadas em águas residuais dessalinizadas: De escala de laboratório à
produção de algas em escala piloto no Semiárido do Nordeste do Brasil.
http://premios.cnpq.br/web/pmct/ganhadores

October 2016 – Trabalho selecionado na UFSC – Composição química

e índices nutricionais de seis espécies de microalgas com aplicação em
alimentos funcionais, suplementos e nutracêuticos - Viagem Prêmio
XXIV Jornadas de Jóvenes Investigadores. Associação das
Universidades do Grupo de Montevidéu (AUGM). Desafios
Contemporâneos dos Jovens Investigadores no Desenvolvimento da
Ciência na América Latina. São Pedro: UNESP, SP.
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